## II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for benfluralin

.

III

. MRID Table

Chemical: Benfluralin PC Code: 084301

Guideline Assay MRID

890.1100 Amphibian Metamorphosis Assay (Frog) 48614901

890.1150 Androgen Receptor Binding (Rat Prostate) 48614902

890.1200 Aromatase Assay (Human Recombinant) 48614903

890.1250 Estrogen Receptor Binding 48614904

890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa

- 9903) 48614905

890.1350 Fish Short

- Term Reproduction 48614906

890.1400 Hershberger (Rat) 48614907

890.1450 Female Pubertal (Rat) 48669301

890.1500 Male Pubertal (Rat) 48669302

890.1550 Steroidogenesis (Human Cell Line

– H295R) 48614908

890.1600 Uterotrophic (Rat) 48614909

Page 2 of 2

### AMY

BLANKINSHIP Digitally signed by AMY BLANKINSHIP
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY
BLANKINSHIP, dnQualifier=0000040917

Date: 2015.06.04 12:13:45 -04'00'

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

EPA MRID Number 48614901

Page 2 of 67

DER Template Version: 22 September 2011 CITATION: Coady, K.K., T.A. Marino, K.L. Hutchinson, E.L. McClymont, and J. Thomas. 2011. Benefin: The

Amphibian Metamorphosis Assay Using the African Clawed Frog, Xenopus laevis . Unpublished study

performed by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland,

Michigan 48674. Laboratory report number 101097. Study sponsored by Dow AgroSciences LLC,

Indianapolis, Indiana 46268. Study completed May 24, 2011.

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

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

the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within

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

should not be considered in isolation but rather should be considered in the context of other assays in the

battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has

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

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

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

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

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

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

EPA MRID Number 48614901

Page 3 of 67

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of benfluralin (97.5% purity) on amphibian metamorphosis of the African clawed frog

( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF)

stage 51 (80/control and treatment group; 20/replicate) were exposed to negative and solvent (0.1 mL/L

dimethylformamide; DMF) controls and benfluralin at nominal concentrations of 0.004, 0.020, and 0.100 mg

a.i./L. Time-weighted average (TWA), measured concentrations were <0.00020 (<LOQ; controls), 0.0027,

0.0141, and 0.0744 mg a.i./L. The test system was maintained at 21.8 to 22.3°C and a pH of 6.9 to 7.9.

There were no significant differences (p>0.05) between the negative and solvent control responses for any

endpoint. Unless otherwise indicated, all effects are reported based on comparison to the negative control.

No mortalities or clinical signs ( i.e. , behavioral and other sublethal effects) were observed. Day 21 wet weight

was significantly increased (p<0.05) by 10% in the low treatment group; the mid and high treatment groups

were not significantly different (p>0.05) from the negative control.

Normalized (for snout-vent length) hind-limb length (HLL) was comparable for both controls and all treatment

groups on Day 7 and Day 21. The median NF developmental stage was the same for both controls and all

treatment groups on both Day 7 (NF stage 54) and Day 21 (NF stage 57); no asynchronous development

was observed throughout the test. No treatment-related effects on thyroid gland histopathology were observed

in any treatment group. Mild follicular cell hypertrophy was detected in both controls and all treatment groups.

All validity criteria were met for this study with the exception that the coefficient of variation (CV) for the

measured concentrations ranged from 27.3 to 31.1%, exceeding the guideline performance criterion of  20%.

The treatment concentrations dropped off from nominal values substantially on Day 1 of measurements to

percent recoveries of 26.4, 59.2, and 68.5% of nominal for the low, mid, and high treatment levels, respectively.

These measurements stabilized back to nominal value by Day 3, but another significant drop off occurred at

Day 14 (percent recoveries of 55.1, 48.2, and 53.3% for the low, mid, and high treatment concentrations,

respectively). These percent recoveries failed to stabilize during the final week of exposure, with all subsequent

measurements indicating a percent recovery below 70%. Additional samples were collected on Day 7 to assess

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

EPA MRID Number 48614901

Page 4 of 67

DER Template Version: 22 September 2011 the variability associated with the analytical method as well as solution homogeneity. However, overall, the

exposure was considered adequate in this assay, and this departure from the guideline performance criterion

did not impact the interpretation of the assay.

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: 51

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

Test type: Flow-through

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

### in the Amphibian

Metamorphosis Assay (AMA) with Benfluralin.

Treatment

(mg a.i./L)

[TWA- 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

0.0027 No No No No No No No

0.0141 No No No No No No No

0.0744 No No No No No No No

Abbreviations: Diff. 

### Difference. NA 

### Not applicable.

1 

### A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless

otherwise specified.

2 

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

the DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a boardcertified pathologist.

3 

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

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

EPA MRID Number 48614901

Page 5 of 67

DER Template Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted based on the following guidelines: United States

Environmental Protection Agency (USEPA) 2009, Endocrine Disruptor

Screening Program Test Guidelines OCSPP 890.1100: Amphibian

Metamorphosis (Frog), EPA 740-C-09-002, October 2009; and

Organization for Economic Cooperation and Development (OECD), 2009,

OECD Guideline for the Testing of Chemicals: The Amphibian Metamorphosis

Assay, OECD 231 Adopted 7 September 2009. The following deviations

from OCSPP 890.1100 were noted:

1. The coefficient of variation for the measured concentrations ranged from 27.3 to 31.1%, exceeding

the guideline performance criterion of  20%.

2. The acclimation period for parental frogs was not specified. Further, no details were provided

regarding parental feeding and pre-test health.

3. It was not specified if the “best single spawn” approach was used to select embryos for use in

the definitive test.

4. Solvent concentration (0.1 mL/L) exceeded EFED recommendation (0.02 mL/L), though was

within acceptable limits specified in OECD guidance (0.1 mL/L).

These deviations do not impact the interpretation of the study.

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

statements were provided. This study was conducted in compliance with

OECD Series on Principles of Good Laboratory Practice and Compliance

Monitoring, Number 1, OECD Principles on Good Laboratory Practice (as

revised in 1997), ENV/MC/CHEM(98)17; and US Environmental Protection

Agency- FIFRA GLPs Title 40 CFR, Part 160- Federal Insecticide, Fungicide,

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

EPA MRID Number 48614901

Page 6 of 67

DER Template Version: 22 September 2011

and Rodenticide Act (FIFRA), Good Laboratory Practice Standards, Final

Rule.

A. Test Material Benfluralin (Benefin; CAS No. 1861-40-1)

Description: yellowish-orange crystalline solid

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

Kow, vapor pressure of test compound, expiration date.



### Lot No./Batch No. : ACD13683 (Lot No.)

Purity: 97.5%

Impurities: Not Reported

Stability of Compound: The TWA concentrations yielded recoveries of 67.6 to 74.4% of nominal.

Coefficient of variation ranged from 27.3 to 31.1%.

Storage Conditions of

Test Chemicals: Stored between 5°C to ambient conditionsData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page7of67 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inc lusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be identified.Species scientific name: Xenopus laevisSpecies strain (if stated):Not stated1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the EndocrineDisruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention(OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page8of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained as inhouse stock?YesBreeding pairs were ordered through

XenopusExpress (Brooksville, Florida). EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?Acclimated under flowthrough conditions withcomparabletemperature. No furtherdetails provided.Acclimation period for parentalfrogs (if applicable):Not specifiedDetails on parental feeding:Not specifiedDetails on parental health:Not specifiedData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page9of67 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Not specifiedMultiple spawns were at times availablefor use in the study. The selection ofthe spawning group used wasdependent upon therebeing a sufficientamount of stage 51 tadpoles availablefrom a single spawn to initiate theexposure. 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):Not specifiedNumber of eggs sampled perspawn:Not specifiedNF stage at test initiation51 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:15 days postfertilization (dpf)Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page10of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMean total length at testinitiation (if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?NoDetails on larval selection:Tadpoles wereindividually stagedwithout anesthesia.Stage 51 tadpoles wereexternally characterizedby a conical-shapedhind limb.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page11of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsLoading rate (rearing density):~4 larvae/LHolding capacity of 5.2 L in testvessels, stocked with 20 larvae. EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow - through systems or 4 tadpoles/L in static - renewal exposure systems.Type of food:Sera Micron® EPA recommends Sera Micron ® throughout

pre - exposure (after NF stage 45/46) and during the entire 21 - d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Source of food:Sera North America(Montgomeryville, PA)Iodide concentration in diet (ifknown):40 mg/kgAnalyzed at A & L Great LakesLaboratories, Inc.Frequency of feeding:2 times/day EPA recommends that feeding occur at least twice per day.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page12of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime:Same as guidelinerecommendations. It is recommended that food rations during

the pre - exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation. EPA and

OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study Day 0 -4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page13of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem (if applicable):Continuous flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):50 mL/min on Days 0-14 and 60 mL/min onDays 15-21.Volume turnover was approximately every1.7 hrs onDays 0-14 and approximatelyevery 1.4 hrs onDays 15-21. The higherflow rates were an attempt to maximizemeasured concentrations ofbenfluralininthe water column and reach concentrationsthat were closer to the nominal targetdoses. Study authors reportedthatincreased flow rates have beendemonstrated to have no deleteriouseffects on the rate of X. laevistadpoledevelopment. Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page14of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Syringe pumps with gastight syringescontinuously delivered aconcentrated stocksolution at a designatedrate to a stainless steelmixing chamber where itwas mixed with dilutionwater, then gravity fed(throughsplitters) toreplicate test vessels.The diluter was calibrated prior to testinitiation and was monitored at least twicedaily throughout the test.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2 ) Aeration is not recommended for mixing;

3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy

is within 10%.Renewal period for staticrenewal (ifapplicable):NA If static renewal is used, EPA recommends 24 -hr renewal; renewal period is recommended not to exceed 72

hours.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page15of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration?Not reportedDO levels remained above 40% saturationthroughout definitive exposure. 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:Natural water from LakeHuron supplied by theCity of Midland WaterTreatment Plant. Waterwas obtained from theupper Saginaw Bay andwas limed andflocculated with ferricchloride. Water waspumped to the laboratoryprior to municipaltreatment. Prior to use in EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the thyroid gland (e.g., fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in

the test guideline.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page16of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsthe test, water wassand-filtered, pHadjusted with CO2,carbon-filtered, and UVirradiated.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesIodide supplementation inwater?NoStudy authors reported thatsupplementation of water was notconsidered necessary since tadpolesdeveloped at theexpected rate and 40mg/kg iodide was measured in the diet. If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 ս g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page17of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel type/materials:Glass aquaria sealedtogether with clearsilicone adhesive.EPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:~30 x 14.5 x 20 cmdeep. Water depthmaintained at 12 cm witha screen-covered drain.Fill volume:5.2 LAdditional details on exposuresystem:Diluter and aquaria werecleaned prior to test andall vessels were cleaneddaily. Mixing cells andtubing were cleaned orreplaced as needed.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page18of67 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsHardness (mg/L asCaCO3)56 68 62aWeekly in replicate tanks from thecontrol and highest treatment group. EPA recommends hardness 40 to 48 mg/L

as CaCO 3.pH6.97.97.4aWeekly in each replicate test vessel EPA recommends pH 7.5 ± 1, inter-replicate

and inter -treatment differentials should not

exceed 0.5.Dissolved oxygen(mg/L)6.9 8.4 7.9aWeekly in each replicate test vessel EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature(°C) 21.822.322aWeekly in all replicate test vessels,and continuous in one replicate tank EPA recommends temperature 22±1 oC; interreplicate and inter -treatment differentials should not exceed 0.5 o C.Iodide<0.01 mg/LTwice per year EPA recommends aquatic iodide range 0.5 –

10 ս g/L (supplemental iodide should not

exceed 2 ս g/L).Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page19of67 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsAmmonia<0.01 mg/LTwice per year 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

ha rdness be measured at least at the beginning and end of the test.Fluoride<0.01 mg/LTwice per yearPerchlorate<0.2սg/LTwice per yearChlorate<10սg/LTwice per yearAlkalinity (mg/L asCaCO3)20 40 33aMeasured on Days 0 and 21 in thecontroland highest treatment group.Conductivity(սmhos/cm)152 206174aacalculated by the reviewer from data reported in the Tables and Appendices of the study report.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page20of67 DER Template Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details orRemarksGuideline RecommendationsWas a range-finder conducted?YesIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?Solubility limit EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever concentration is lowest.Species: Xenopus laevisLife stage:NF stage 49-51Test duration:96 hoursData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page21of67 DER Template Version: 22 September 2011ParameterValue(s)Details orRemarksGuideline RecommendationsAdditional details:A non-GLP range-finding study wasconducted for 96 hours under static-renewalconditions with tadpoles at NF stages 49-51.The NOAEC and LC50values were 200 and>200սg a.i./L, respectively. Since the limitof water solubility (100սg a.i./L) was lowerthan the limit dose of 100 mg a.i./L and thelevel of acute signs of toxicity, the limit ofsolubility was used as the highest test level.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page22of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in thedefinitive test:Range-finder and limitof solubility EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of testconcentrations:0.2 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page23of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Time-weighted averageLimit of quantification (LOQ):0.2սg 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:Concentrations weremeasured on Days 0,1, 3, 7, 10, 14, 17, and21 It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week thereafter.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (ifapplicable):4 EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page24of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of larvae per treatmentat test initiation:8020 larvae/repWas a solvent used?YesSolvent type (if applicable):DMFMaximum solvent concentration(if applicable):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?No3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page25of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12

(light:dark).Light intensity at water’ssurface:0.623-0.890 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:Specific details on the appearance of thetest solution did was observed in thestudy reportData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page26of67 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Benfluralin.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration-TWA(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNAMaximum allowable%CV (i.e., 20%) wasexceeded for alltreated levels. EPA and OECD recommend that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent Control0<LOQNATreatment 10.0030.002731.1Treatment 20.0200.014127.3Treatment 30.1000.074427.4Abbreviations:CVCoefficient of variation.LOQ = 0.00020 mg a.i./LData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page27of67 DER Template Version: 22 September 2011E. ObservationsBiological Endpoints:Days 7 and 21: whole body wet weight, SVL, HLL, normalized HLL, NF stage, and asynchronous developmentDay 21: thyroid gland histopathologyDaily: mortality and sublethal effects (e.g., edema, malformations, abnormal behavior)Were raw (individual) data provided?Yes EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (D ays 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-base d larval selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also incl udes thyroid gross

pathology observations.

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

EPA MRID Number 48614901

Page 28 of 67

DER Template Version: 22 September 2011 II. RESULTS AND DISCUSSION

A. Results

No mortalities were observed in the controls or treatment groups throughout the duration of the 21-

day assay.

Table 9: Larval Mortality in Xenopus laevia

.

Treatment (mg a.i./L)

[TWA-measured] Larval Mortality

Day 7 1 

### Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 20 0 0 15 0 0

Solvent Control 20 0 0 15 0 0

0.0027 20 0 0 15 0 0

0.0141 20 0 0 15 0 0

0.0744 20 0 0 15 0 0

Abbreviations: NA 

### Not applicable.

1 

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

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

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DER Template Version: 22 September 2011 Median NF stage at Day 7 was 54 in both controls and all treatment groups. On Day 21, median NF

stage was 57 in both controls and all treatment groups, which adheres to OCSPP and OECD

guidelines. Further, the 10 th 

### and 90 th 

### percentiles (57 and 59, respectively) did not differ by more than

4 stages. No asynchronous development was observed throughout the test.

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

Treatment ( mg a.i./L) [ TWAmeasured] Developmental Stage

Day 7 Day 21

n Median

Stage # Asynchronous n Median

Stage # Asynchronous

Negative Control 4 54 0 4 57 0

Solvent Control 4 54 0 4 57 0

0.0027 4 54 0 4 57 0

0.0141 4 54 0 4 57 0

0.0744 4 54 0 4 57 0

Abbreviations: NA 

### Not applicable.

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

EPA MRID Number 48614901

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DER Template Version: 22 September 2011 Normalized HLL on Day 7 ranged from 0.12 in the controls and the TWA 2.70 and 14.1 ս g a.i./L

treatment groups to 0.13 in the TWA 74.4 ս g a.i./L treatment group. On Day 21, normalized HLL

ranged from 0.45 in the TWA 2.70 ս g a.i./L treatment group to 0.48 in the solvent control and the

two highest treatment groups.

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

Treatment

(mg a.i./L)

[TWA-measured] Hind Limb Length (HLL)

Day 7 Day 21

n 1 Mean

(mm) ±SD HLL:

SVL 3 n 1 Mean

(mm) ±SD HLL:

SVL 2



### Negative Control 4 2.03 0.10 0.12 4 13.20 0.65 0.47

Solvent Control 4 2.03 0.15 0.12 4 14.13 1.13 0.48

0.0027 4 1.95 0.06 0.12 4 13.15 0.45 0.45

0.0141 4 2.13 0.17 0.12 4 13.73 1.30 0.48

0.0744 4 2.08 0.15 0.13 4 13.50 0.60 0.48

Abbreviations: NA 

### Not applicable. SD 

### Standard deviation.

1 

### Sample size (n) represents the number independent replicates

2 

### Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page31of67 DER Template Version: 22 September 2011Day 7 SVL ranged from 16.18 mm in the TWA 0.0744սg a.i./L treatment group to 17.30 mm in solvent control. Day 21 SVL ranged form 28.30 mm inthe TWA 0.0744սg a.i./L treatment group to 29.23 mm in the solvent control. Day 7 body weight ranged from 0.41 g in the TWA 0.0027սg a.i./Ltreatment group to 0.48 g in the TWA 0.0414սg a.i./L treatment group. Day 21 body weight ranged from 1.96 g in the negative control to 2.16 g in theTWA 0.0027սg a.i./L treatment group.Table 12: Larval Growth in Xenopus laevis.Treatment(mg a.i./L)[TWA-measured]Snout-Vent Length (SVL)Body Weight1Day 7Day 21Day 7Day 21n2Mean(mm)±SDn2Mean(mm)±SDn2Mean(g)±SDn2Mean(g)±SDNegative Control416.580.59428.230.3040.430.0341.960.03Solvent Control417.300.71429.230.8040.440.0842.100.120.0027416.430.13429.050.3740.410.0242.160.040.0141417.580.74428.600.8240.480.0542.030.110.0744416.180.81428.300.4240.420.0542.040.12Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.1Also referred to as “wet weight” in the test guideline.2Sample size (n) represents the number independent replicates

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DER Template Version: 22 September 2011 No thyroid gland hypertrophy or atrophy was detected in any tadpoles from the controls or treatment

groups. Mild follicular cell hypertrophy was detected in both controls and all treatment groups, though

did not appear to be treatment-related and was interpreted to be within normal limits. No follicular cell

hyperplasia was detected. Further, there was no reported evidence of any inflammatory or degenerative

changes in the thyroid glands examined from all control and treatment tadpoles. All other

histopathological criteria, such as the overall size of the gland, the follicular lumen area, amount and

type of colloid, and the follicular cell type and arrangement, of all tadpoles in the treatment groups

were comparable to those of the controls.

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DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis

.

Treatment

(mg a.i./L)

[measured] Diagnostic 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 8 20 20

1 20 0 20 0 20 12 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

Solvent Control 0 20 20 20 20 20 11 20 20

1 20 0 20 0 20 9 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0027 0 20 20 20 20 20 9 20 20

1 20 0 20 0 20 11 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0141 0 20 20 20 20 20 10 20 20

1 20 0 20 0 20 10 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0744 0 20 20 20 20 20 7 20 20

1 20 0 20 0 20 13 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

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

2=Moderate, 3=Severe. See OECD No. 82 for reference.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page34of67 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. Not AssessedTreatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANASolvent Control0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA0.00270NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANAData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page35of67 DER Template Version: 22 September 2011Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence0.01410NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA0.07440NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANAAbbreviations:NANot applicable.1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.

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DER Template Version: 22 September 2011 No other clinical signs or sublethal effects were noted in the controls or treatment groups.

Table 15: Clinical Signs in Xenopus laevis

.

Treatment

(mg a.i./L)

[measured] Clinical Signs

Type n Incidence

Negative Control None 80 0

Solvent Control None 80 0

0.0027 None 80 0

0.0141 None 80 0

0.0744 None 80 0

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DER Template Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

Median values were inspected for each measured endpoint by treatment level to determine if the

response was monotonic with increasing concentration. Analyses of the continuous data set, including

normalized HLL, wet weight, and SVL, were analyzed with the Jonckheere-Terpstra test in a stepdown manner if the data were consistent with a monotonic concentration -response. If these endpoints

were monotonic, data were assessed for normality (Shapiro-Wilk’s test) and homogeneity of variance

(Levene’s test). If data met the assumptions of ANOVA, then a significant treatment effect was

determined using Dunnett’s test. A significant treatment effect for development stage was determined

from the step-down Jonckheere-Terpstra test from the 20 th 

### to the 80 th 

### percentile. All analyses were

conducted at Į = 0.05, with the exception of the Shapiro-Wilk test, which was conducted at Į = 0.01.

Throughout the duration of the exposure period, no mortality or sublethal effects were noted. On Days

7 and 21, there were no signs of advanced or delayed development, treatment-related effects on

growth, or asynchronous development in any of the treatment groups relative to the pooled control.

Further, no treatment-related effects were noted for histopathological observations.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: No mortalities were observed, therefore, no statistical analysis of mortality data

was required. No data appeared to exhibit a monotonic trend. Among the endpoints, only the median

values for developmental stage (Days 7 and 21) did not satisfy parametric assumptions and were

analyzed using the Mann-Whitney U test. Wet weight (Days 7 and 21), SVL, HLL and normalized

HLL data satisfied the assumptions of normality (determined using Shapiro-Wilk’s test) and variance

homogeneity (determined using Levene’s test) and were therefore analyzed using Dunnett’s test. All

analyses were conducted using SAS ver. 8.1. Unless otherwise noted, all comparisons were conducted

using the negative control.

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DER Template Version: 22 September 2011 The reviewer compared replicate negative and solvent control data for wet weight, SVL, HLL, and

normalized HLL (Days 7 and 21) using a two-sided Student’s t-Test assuming unequal variance via

Microsoft Excel. No differences (p>0.05) were detected between the two controls for any endpoint,

on either day.

No asynchronous development was detected. Histopathological observations are provided in Table 13.

Conclusions:

Growth parameters ( i.e. , wet weight, SVL, HLL) were unaffected in all treatment groups relative to the

negative control on Day 7 (Dunnett’s test; p > 0.05). On Day 21, however, wet weight was significantly

greater in the lowest concentration relative to the negative control (10% increase, Dunnett’s test; p =

0.018). SVL and HLL on Day 21 were comparable to the negative control (Dunnett’s test; p > 0.05).

Median NF stage on Days 7 and 21 were comparable in all treatment groups compared to the negative

control (Mann-Whitney; p  0.48). Day 7 and Day 21 normalized HLL were also comparable between

treatment groups and the negative control response (Dunnett’s test; p > 0.05). No clear concentration

-related trends were observed for histopathological observations or sublethal effects.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page39of67 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Benfluralin.Treatment(mg a.i./L)[TWA-measured]NF Developmental StageHind Limb Length3Thyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 21Median pMedian p% Diff. p% Diff. pTreatment-Related Effects?(Yes/No)Negative Control54NA57NA0NA0NANoSolvent Control54NA57NA-2.100.393.200.44No0.002754>0.9957>0.99-2.080.85-3.210.75No0.014154>0.99570.480.00>0.991.600.95No0.0744540.62571.06.250.1711.600.95NoStatistical testMann-Whitney UMann-Whitney UDunnett’sDunnett’sNAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Hind-limb length is normalized to snout-vent length (SVL).

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DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 

### in the AMA with Benfluralin.

Treatment

(mg a.i./L)

[TWA-measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

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

Negative Control 0 NA 0 NA 0 NA 0 NA



### Solvent Control 4.40 0.17 3.50 0.08 2.50 0.82 6.90 0.12



### 0.0027 -0.90 0.97 2.92 0.11 -5.71 0.72 10.0 0.018



### 0.0141 6.03 0.10 1.33 0.62 11.52 0.23 3.67 0.51



### 0.0744 -2.41 0.70 0.27 0.99 -0.01 >0.99 3.94 0.50



### Statistical test Dunnett’s Dunnett’s Dunnett’s Dunnett’s

Abbreviations: Diff. 

### Difference. NA 

### Not applicable.

1 

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

2 

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

E. Study Deficiencies

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER.

This study met all of the validity and performance criteria with the exception that the CV for measured

concentrations ranged from 27.3 to 31.1%, exceeding the guideline performance criterion of  20%.

This departure from a guideline performance criterion and deviations from guideline recommendations

did not impact the interpretation of the study.

F. Reviewer’s Comments

The study authors reported no significant differences between treatment groups and the pooled control

for any growth or development endpoints on Days 7 and 21. Conversely, the reviewer’s analysis

detected a significant increase of wet weight at the lowest concentration on Day 21 (10%, p = 0.018),

relative to the negative control. No other treatment-related effects, including those examined for

histopathology were noted. There were no differences indicated between the results interpretation for

the OCSPP 890.1100 and historical EFED methodologies.

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DER Template Version: 22 September 2011 The study authors found high variability in the measured test concentrations over the duration of the

exposure period (CV 24.3-35.6%) with the greatest variability at the lowest concentration . The study

authors suggest that the decrease in measured concentrations can be explained both by the absorptive

(K OC 

### = 9840 -22,660 L/kg) and lipophilic (log P OW 

### = 5.29) nature of the test substance and the

dynamic (e.g., flow-through) test system. Specifically, when tadpoles were initially added to the test

system and also as the tadpoles grew (increasing both their biomass and their waste production),

water concentrations of benfluralin likely decreased due to a greater availability of lipophilic “sinks” in

the test system.

For the histopathology, the study author stated that “Five juveniles that corresponded to the control

median developmental stage were randomly selected from each replicate tank if there were a sufficient

numbers of tadpoles in each replicate tank. If there were an insufficient number of juveniles at the

median developmental stage of the controls, then randomly selected individuals from the next lower

and upper developmental stages were alternatively selected to reach a total sample size of five

tadpoles/ replicate.”

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DER Template Version: 22 September 2011 III. REFERENCES

Coady, K., Marino, T., Thomas, J., Currie, R., Hancock, G, Crofoot, J., McNalley, L., McFadden, L., Geter,

D., and Klecka, G. (2010). Evaluation of the amphibian metamorphosis assay: Exposure to the goitrogen

methimazole and the endogenous thyroid hormone L-thyroxine. Environmental Toxicology and Chemistry

29 (4), 869-880.

Day, E., Saunders, D., and Loh, A. (1983). Octanol/Water Partition Coefficient of Benefin: Lab Project

Number:I0EWD-93-92. Unpublished study prepared by Lilly Research Labs. 9p.

Filary, M. (2010). General Quantitative (Sera Micron Tadpole Food, Lab Dilution Water). Analytical Report,

The Dow Chemical Company, Midland, Michigan.

Hollander, M. and Wolfe, D.A. (1973). Nonparametric Statistical Methods . John Wiley & Sons, New York.

Koors, B. (2009). Recertification of purity and identity of TSN100037, Benefin technical grade active

ingredient. FA&PC Number 09-227482.

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

New York.

Nishiuchi, Y. (1980). Toxicity of formulated pesticides to fresh water organisms. LXXIV Suisan Zoshoku

28 (2), 107-122.Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page43of67 DER Template Version: 22 September 2011APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:TABLE OF STATISTICAL INTERPRETATION: EDSP VS EFED METHODSEndpointMonotonic?Parametric?EDSPEFEDComments7-day wet weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect7-stage medianNoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05EDSP and EFED same conclusions, no effect7-day SN-ventlengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect7-day hind-limblengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect7-day normalizedHLLNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect21-day stagemedianNoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05EDSP and EFED same conclusions, no effect21-day wet weightNoYesDunnett’s:Dose 1 p=0.018Dunnett’s:Dose 1 p=0.018EDSP and EFED same conclusions. The lowest dosewas significantly (p<0.05) increased 10% relative tocontrol.21-day SN-ventlengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectData Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis AssayEPA MRID Number 48614901Page44of67 DER Template Version: 22 September 2011EndpointMonotonic?Parametric?EDSPEFEDComments21-day HLLNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect21-day normalizedHLLNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effect

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DER Template Version: 22 September 2011 Benfluralin 084301 48614901 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=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.962 0.689 2.232 0.137 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 0.43 0.03 0.02 7.85 0.38, 0.48
Dose1 4 0.41 0.02 0.01 3.93 0.38, 0.43
Dose2 4 0.48 0.05 0.02 9.97 0.40, 0.56
Dose3 4 0.43 0.05 0.03 11.74 0.35, 0.51

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

Ctrl 0.43 0.39 0.48 . .

Dose1 0.40 0.39 0.43 94.29 5.71

Dose2 0.48 0.42 0.54 111.52 -11.52

Dose3 0.42 0.38 0.49 99.99 0.01

******************************************************************************** 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 2.49 0.110

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 0.43 . 0.44 . . . . . .

Dose1 0.41 0.715 0.44 0.703 . . . . .

Dose2 0.48 0.230 0.44 0.737 0.085 . . . .

Dose3 0.43 1.000 0.43 0.636 0.815 0.330 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

3 4.96 0.175

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.43 . .

Dose1 0.40 0.346 0.124

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DER Template Version: 22 September 2011 Dose2 0.48 0.235 0.879

Dose3 0.42 1.000 0.646

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 2.49 0.110

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 -0.43 . -0.42 . . . . . .

Dose1 -0.41 0.715 -0.42 0.753 . . . . .

Dose2 -0.48 0.230 -0.45 0.250 0.085 . . . .

Dose3 -0.43 1.000 -0.45 0.259 0.815 0.330 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

3 4.96 0.175

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 -0.43 . .

Dose1 -0.40 0.346 0.876

Dose2 -0.48 0.235 0.121

Dose3 -0.42 1.000 0.354

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

Benfluralin 084301 48614901 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=0.01 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

0.768 0.001 1.000 0.426 USE NON-PARAMETRIC TESTS

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DER Template Version: 22 September 2011 ********************************************************************************

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 53.50 0.58 0.29 1.08 52.58, 54.42
Dose1 4 53.50 0.58 0.29 1.08 52.58, 54.42
Dose2 4 53.50 0.58 0.29 1.08 52.58, 54.42
Dose3 4 53.75 0.50 0.25 0.93 52.95, 54.55

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

Ctrl 53.50 53.00 54.00 . .

Dose1 53.50 53.00 54.00 100.00 0.00

Dose2 53.50 53.00 54.00 100.00 0.00

Dose3 54.00 53.00 54.00 100.47 -0.47

******************************************************************************** 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 0.20 0.894

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 53.50 . 53.56 . . . . . .

Dose1 53.50 1.000 53.56 0.648 . . . . .

Dose2 53.50 1.000 53.56 0.683 1.000 . . . .

Dose3 53.75 0.862 53.56 0.702 0.920 0.920 . . .

******************************************************************************** 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 0.71 0.870

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 53.50 . .

Dose1 53.50 1.000 0.500

Dose2 53.50 1.000 0.500

Dose3 54.00 0.624 0.744

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

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DER Template Version: 22 September 2011 3 12 0.20 0.894

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 -53.50 . -53.50 . . . . . .

Dose1 -53.50 1.000 -53.50 0.583 . . . . .

Dose2 -53.50 1.000 -53.50 0.618 1.000 . . . .

Dose3 -53.75 0.862 -53.75 0.356 0.920 0.920 . . .

******************************************************************************** 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 0.71 0.870

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 -53.50 . .

Dose1 -53.50 1.000 0.500

Dose2 -53.50 1.000 0.500

Dose3 -54.00 0.624 0.256

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

Benfluralin 084301 48614901 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=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.966 0.776 1.964 0.173 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 16.58 0.59 0.29 3.53 15.64, 17.51
Dose1 4 16.43 0.13 0.06 0.77 16.22, 16.63
Dose2 4 17.58 0.74 0.37 4.19 16.40, 18.75
Dose3 4 16.18 0.81 0.41 5.03 14.88, 17.47

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

Ctrl 16.60 15.90 17.20 . .

Dose1 16.40 16.30 16.60 99.10 0.90

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

EPA MRID Number 48614901

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DER Template Version: 22 September 2011 Dose2 17.55 16.70 18.50 106.03 -6.03

Dose3 16.35 15.10 16.90 97.59 2.41

******************************************************************************** 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 3.86 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 16.58 . 16.86 . . . . . .

Dose1 16.43 0.973 16.86 0.815 . . . . .

Dose2 17.58 0.104 16.86 0.845 0.093 . . . .

Dose3 16.17 0.700 16.17 0.253 0.940 0.035 . . .

******************************************************************************** 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 6.46 0.091

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 16.60 . .

Dose1 16.40 1.000 0.442

Dose2 17.55 0.156 0.976

Dose3 16.35 0.580 0.663

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 3.86 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 -16.58 . -16.50 . . . . . .

Dose1 -16.43 0.973 -16.50 0.653 . . . . .

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

EPA MRID Number 48614901

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DER Template Version: 22 September 2011 Dose2 -17.58 0.104 -16.88 0.326 0.093 . . . .

Dose3 -16.17 0.700 -16.88 0.337 0.940 0.035 . . .

******************************************************************************** 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 6.46 0.091

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 -16.60 . .

Dose1 -16.40 1.000 0.558

Dose2 -17.55 0.156 0.024

Dose3 -16.35 0.580 0.337

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

Benfluralin 084301 48614901 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=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.959 0.642 1.929 0.179 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 2.03 0.10 0.05 4.73 1.87, 2.18
Dose1 4 1.95 0.06 0.03 2.96 1.86, 2.04
Dose2 4 2.13 0.17 0.09 8.04 1.85, 2.40
Dose3 4 2.08 0.15 0.08 7.23 1.84, 2.31

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

Ctrl 2.05 1.90 2.10 . .

Dose1 1.95 1.90 2.00 96.30 3.70

Dose2 2.15 1.90 2.30 104.94 -4.94

Dose3 2.10 1.90 2.20 102.47 -2.47

******************************************************************************** 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 1.39 0.293

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

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

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DER Template Version: 22 September 2011 Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

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

Ctrl 2.03 . 2.04 . . . . . .

Dose1 1.95 0.743 2.04 0.669 . . . . .

Dose2 2.13 0.563 2.04 0.704 0.258 . . . .

Dose3 2.08 0.899 2.04 0.723 0.525 0.942 . . .

******************************************************************************** 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 3.41 0.333

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 2.05 . .

Dose1 1.95 0.320 0.111

Dose2 2.15 0.402 0.752

Dose3 2.10 0.671 0.807

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 1.39 0.293

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 -2.03 . -1.99 . . . . . .

Dose1 -1.95 0.743 -1.99 0.746 . . . . .

Dose2 -2.13 0.563 -2.10 0.267 0.258 . . . .

Dose3 -2.08 0.899 -2.10 0.277 0.525 0.942 . . .

******************************************************************************** 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 3.41 0.333

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

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

EPA MRID Number 48614901

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DER Template Version: 22 September 2011 Level Median MannWhit p-value Jonckheere p-value

Ctrl -2.05 . .

Dose1 -1.95 0.320 0.889

Dose2 -2.15 0.402 0.248

Dose3 -2.10 0.671 0.193

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

Benfluralin 084301 48614901 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=0.01 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

0.842 0.010 1.636 0.233 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 0.12 0.00 0.00 0.00 . , .
Dose1 4 0.12 0.00 0.00 4.26 0.11, 0.13
Dose2 4 0.12 0.01 0.00 6.80 0.11, 0.13
Dose3 4 0.13 0.01 0.00 3.92 0.12, 0.14

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

Ctrl 0.12 0.12 0.12 . .

Dose1 0.12 0.11 0.12 97.92 2.08

Dose2 0.12 0.11 0.13 100.00 0.00

Dose3 0.13 0.12 0.13 106.25 -6.25

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

Numerator df Denominator df F-stat P-value

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 0.12 . 0.12 . . . . . .

Dose1 0.12 0.851 0.12 0.713 . . . . .

Dose2 0.12 1.000 0.12 0.748 0.912 . . . .

Dose3 0.13 0.171 0.12 0.766 0.091 0.254 . . .

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

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

EPA MRID Number 48614901

Page 53 of 67

DER Template Version: 22 September 2011 NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

3 6.16 0.104

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 0.12 . .

Dose1 0.12 0.478 0.159

Dose2 0.12 1.000 0.461

Dose3 0.13 0.102 0.964

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 2.57 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 -0.12 . -0.12 . . . . . .

Dose1 -0.12 0.851 -0.12 0.713 . . . . .

Dose2 -0.12 1.000 -0.12 0.618 0.912 . . . .

Dose3 -0.13 0.171 -0.13 0.046 0.091 0.254 . . .

******************************************************************************** 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 6.16 0.104

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 -0.12 . .

Dose1 -0.12 0.478 0.841

Dose2 -0.12 1.000 0.539

Dose3 -0.13 0.102 0.036

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL

Williams Dose3

Jonckheere Dose3

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

EPA MRID Number 48614901

Page 54 of 67

DER Template Version: 22 September 2011 Benfluralin 084301 48614901 890.1100 ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.565 <.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 57.00 0.00 0.00 0.00 . , .
Dose1 4 57.00 0.00 0.00 0.00 . , .
Dose2 4 57.25 0.50 0.25 0.87 56.45, 58.05
Dose3 4 57.00 0.00 0.00 0.00 . , .

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

Ctrl 57.00 57.00 57.00 . .

Dose1 57.00 57.00 57.00 100.00 0.00

Dose2 57.00 57.00 58.00 100.44 -0.44

Dose3 57.00 57.00 57.00 100.00 0.00

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

Numerator df Denominator df F-stat P-value

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 57.00 . 57.08 . . . . . .

Dose1 57.00 1.000 57.08 0.763 . . . . .

Dose2 57.25 0.389 57.08 0.796 0.515 . . . .

Dose3 57.00 1.000 57.00 0.636 1.000 0.515 . . .

******************************************************************************** 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 57.00 . .

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

EPA MRID Number 48614901

Page 55 of 67

DER Template Version: 22 September 2011 Dose1 57.00 1.000 .

Dose2 57.00 0.478 0.890

Dose3 57.00 1.000 0.673

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 -57.00 . -57.00 . . . . . .

Dose1 -57.00 1.000 -57.00 0.583 . . . . .

Dose2 -57.25 0.389 -57.13 0.315 0.515 . . . .

Dose3 -57.00 1.000 -57.13 0.326 1.000 0.515 . . .

******************************************************************************** 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 -57.00 . .

Dose1 -57.00 1.000 .

Dose2 -57.00 0.478 0.110

Dose3 -57.00 1.000 0.327

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

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

EPA MRID Number 48614901

Page 56 of 67

DER Template Version: 22 September 2011 Benfluralin 084301 48614901 890.1100 ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.930 0.245 1.519 0.260 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 1.96 0.03 0.02 1.72 1.91, 2.02
Dose1 4 2.16 0.04 0.02 1.85 2.09, 2.22
Dose2 4 2.03 0.11 0.05 5.33 1.86, 2.21
Dose3 4 2.04 0.12 0.06 5.88 1.85, 2.23

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

Ctrl 1.96 1.93 2.00 . .

Dose1 2.16 2.12 2.19 109.99 -9.99

Dose2 2.05 1.89 2.14 103.66 -3.66

Dose3 2.06 1.88 2.16 103.94 -3.94

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

Numerator df Denominator df F-stat P-value

3 12 3.66 0.044

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 1.96 . 2.06 . . . . . .

Dose1 2.16 0.018 2.06 0.969 . . . . .

Dose2 2.03 0.514 2.04 0.950 0.217 . . . .

Dose3 2.04 0.459 2.04 0.958 0.249 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 7.32 0.062

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 1.96 . .

Dose1 2.16 0.067 0.990

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

EPA MRID Number 48614901

Page 57 of 67

DER Template Version: 22 September 2011 Dose2 2.05 0.346 0.810

Dose3 2.06 0.346 0.646

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 3.66 0.044

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 -1.96 . -1.96 . . . . . .

Dose1 -2.16 0.018 -2.08 0.046 . . . . .

Dose2 -2.03 0.514 -2.08 0.049 0.217 . . . .

Dose3 -2.04 0.459 -2.08 0.050 0.249 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 7.32 0.062

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 -1.96 . .

Dose1 -2.16 0.067 0.010

Dose2 -2.05 0.346 0.190

Dose3 -2.06 0.346 0.354

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

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

EPA MRID Number 48614901

Page 58 of 67

DER Template Version: 22 September 2011 Benfluralin 084301 48614901 890.1100 ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.962 0.698 0.879 0.479 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 28.23 0.30 0.15 1.06 27.75, 28.70
Dose1 4 29.05 0.37 0.18 1.27 28.46, 29.64
Dose2 4 28.60 0.82 0.41 2.88 27.29, 29.91
Dose3 4 28.30 0.42 0.21 1.47 27.64, 28.96

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

Ctrl 28.20 27.90 28.60 . .

Dose1 29.05 28.60 29.50 102.92 -2.92

Dose2 28.70 27.50 29.50 101.33 -1.33

Dose3 28.30 27.80 28.80 100.27 -0.27

******************************************************************************** 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 2.08 0.157

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 28.23 . 28.64 . . . . . .

Dose1 29.05 0.107 28.64 0.918 . . . . .

Dose2 28.60 0.624 28.60 0.922 0.624 . . . .

Dose3 28.30 0.994 28.30 0.721 0.227 0.845 . . .

******************************************************************************** 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 5.78 0.123

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 28.20 . .

Dose1 29.05 0.082 0.985

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

EPA MRID Number 48614901

Page 59 of 67

DER Template Version: 22 September 2011 Dose2 28.70 0.343 0.907

Dose3 28.30 0.889 0.537

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 2.08 0.157

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 -28.23 . -28.23 . . . . . .

Dose1 -29.05 0.107 -28.65 0.161 . . . . .

Dose2 -28.60 0.624 -28.65 0.171 0.624 . . . .

Dose3 -28.30 0.994 -28.65 0.177 0.227 0.845 . . .

******************************************************************************** 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 5.78 0.123

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 -28.20 . .

Dose1 -29.05 0.082 0.015

Dose2 -28.70 0.343 0.093

Dose3 -28.30 0.889 0.463

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

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

EPA MRID Number 48614901

Page 60 of 67

DER Template Version: 22 September 2011 Benfluralin 084301 48614901 890.1100 ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.949 0.477 2.105 0.153 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 13.20 0.65 0.32 4.91 12.17, 14.23
Dose1 4 13.15 0.45 0.23 3.43 12.43, 13.87
Dose2 4 13.73 1.30 0.65 9.49 11.65, 15.80
Dose3 4 13.50 0.60 0.30 4.44 12.55, 14.45

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

Ctrl 13.25 12.40 13.90 . .

Dose1 13.15 12.60 13.70 99.62 0.38

Dose2 13.35 12.70 15.50 103.98 -3.98

Dose3 13.40 13.00 14.20 102.27 -2.27

******************************************************************************** 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 0.43 0.733

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

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

Ctrl 13.20 . 13.39 . . . . . .

Dose1 13.15 1.000 13.39 0.716 . . . . .

Dose2 13.73 0.698 13.39 0.750 0.756 . . . .

Dose3 13.50 0.916 13.39 0.769 0.929 0.979 . . .

******************************************************************************** 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 0.66 0.882

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 13.25 . .

Dose1 13.15 1.000 0.500

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

EPA MRID Number 48614901

Page 61 of 67

DER Template Version: 22 September 2011 Dose2 13.35 0.780 0.696

Dose3 13.40 0.671 0.787

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 0.43 0.733

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

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

Ctrl -13.20 . -13.18 . . . . . .

Dose1 -13.15 1.000 -13.18 0.601 . . . . .

Dose2 -13.73 0.698 -13.61 0.313 0.756 . . . .

Dose3 -13.50 0.916 -13.61 0.324 0.929 0.979 . . .

******************************************************************************** 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 0.66 0.882

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 -13.25 . .

Dose1 -13.15 1.000 0.500

Dose2 -13.35 0.780 0.304

Dose3 -13.40 0.671 0.213

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

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

EPA MRID Number 48614901

Page 62 of 67

DER Template Version: 22 September 2011 Benfluralin 084301 48614901 890.1100
ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.974 0.898 0.650 0.598 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 0.47 0.02 0.01 5.35 0.43, 0.51
Dose1 4 0.45 0.02 0.01 3.77 0.43, 0.48
Dose2 4 0.48 0.03 0.02 7.19 0.42, 0.53
Dose3 4 0.48 0.02 0.01 5.01 0.44, 0.51

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

Ctrl 0.47 0.44 0.50 . .

Dose1 0.46 0.43 0.47 96.79 3.21

Dose2 0.47 0.44 0.52 101.60 -1.60

Dose3 0.48 0.45 0.50 101.60 -1.60

******************************************************************************** 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 0.68 0.581

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

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

Ctrl 0.47 . 0.47 . . . . . .

Dose1 0.45 0.751 0.47 0.583 . . . . .

Dose2 0.48 0.954 0.47 0.618 0.617 . . . .

Dose3 0.48 0.954 0.47 0.636 0.617 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 1.77 0.621

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 0.47 . .

Dose1 0.46 0.489 0.190

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

EPA MRID Number 48614901

Page 63 of 67

DER Template Version: 22 September 2011 Dose2 0.47 0.888 0.588

Dose3 0.48 0.888 0.746

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 0.68 0.581

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

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

Ctrl -0.47 . -0.46 . . . . . .

Dose1 -0.45 0.751 -0.46 0.743 . . . . .

Dose2 -0.48 0.954 -0.48 0.436 0.617 . . . .

Dose3 -0.48 0.954 -0.48 0.451 0.617 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 1.77 0.621

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 -0.47 . .

Dose1 -0.46 0.489 0.810

Dose2 -0.47 0.888 0.412

Dose3 -0.48 0.888 0.254

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

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

EPA MRID Number 48614901

Page 64 of 67

DER Template Version: 22 September 2011 REVIEWER’S COMPARISON OF NEGATIVE AND SOLVENT CONTROL DATA

t- Test: Two-Sample Assuming Unequal

Variances

Day 7 Wet Weight Negative
Control Solvent
Control

Mean 0.430175 0.44075

Variance 0.001140556 0.006833017

Observations 4 4 Hypothesized Mean Difference 0

df 4

t Stat -0.236855731 P(T<=t) one-tail 0.412202146

t Critical one-tail 2.131846782 P(T<=t) two-tail 0.824404291

t Critical two-tail 2.776445105

t-Test: Two-Sample Assuming Unequal

Variances

Day 7 SVL Negative
Control Solvent
Control

Mean 16.575 17.3

Variance 0.3425 0.5

Observations 4 4 Hypothesized Mean Difference 0

df 6

t Stat -1.579730661 P(T<=t) one-tail 0.082624573

t Critical one-tail 1.943180274 P(T<=t) two-tail 0.165249146

t Critical two-tail 2.446911846

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

EPA MRID Number 48614901

Page 65 of 67

DER Template Version: 22 September 2011 t-Test: Two-Sample Assuming Unequal

Variances

Day 7 HLL Negative
Control Solvent
Control

Mean 2.025 2.025

Variance 0.009166667 0.0225

Observations 4 4 Hypothesized Mean Difference 0

df 5

t Stat 0 P(T<=t) one-tail 0.5

t Critical one-tail 2.015048372 P(T<=t) two-tail 1

t Critical two-tail 2.570581835

t-Test: Two-Sample Assuming Unequal

Variances

Day 7 Normalized HLL Negative
Control Solvent
Control

Mean 0.12 0.1175

Variance 0 0.000025

Observations 4 4 Hypothesized Mean Difference 0

df 3

t Stat 1 P(T<=t) one-tail 0.195501109

t Critical one-tail 2.353363435 P(T<=t) two-tail 0.391002218

t Critical two-tail 3.182446305

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

EPA MRID Number 48614901

Page 66 of 67

DER Template Version: 22 September 2011 t-Test: Two-Sample Assuming Unequal

Variances

Day 21 Wet Weight Negative
Control Solvent
Control

Mean 1.962025 2.0983

Variance 0.001141816 0.014486633

Observations 4 4 Hypothesized Mean Difference 0

df 3

t Stat -2.180159382 P(T<=t) one-tail 0.058665751

t Critical one-tail 2.353363435 P(T<=t) two-tail 0.117331502

t Critical two-tail 3.182446305

t-Test: Two-Sample Assuming Unequal

Variances

Day 21 SVL Negative
Control Solvent
Control

Mean 28.225 29.225

Variance 0.089166667 0.6425

Observations 4 4 Hypothesized Mean Difference 0

df 4

t Stat -2.338155339 P(T<=t) one-tail 0.03976936

t Critical one-tail 2.131846782 P(T<=t) two-tail 0.079538721

t Critical two-tail 2.776445105

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

EPA MRID Number 48614901

Page 67 of 67

DER Template Version: 22 September 2011 t-Test: Two-Sample Assuming Unequal

Variances

Day 21 HLL Negative
Control Solvent
Control

Mean 13.2 14.125

Variance 0.42 1.275833333

Observations 4 4 Hypothesized Mean Difference 0

df 5

t Stat -1.420627262 P(T<=t) one-tail 0.107334782

t Critical one-tail 2.015048372 P(T<=t) two-tail 0.214669564

t Critical two-tail 2.570581835

t-Test: Two-Sample Assuming Unequal

Variances

Day 21 Normalized HLL Negative
Control Solvent
Control

Mean 0.4675 0.4825

Variance 0.000625 0.000691667

Observations 4 4 Hypothesized Mean Difference 0

df 6

t Stat -0.826767382 P(T<=t) one-tail 0.22000113

t Critical one-tail 1.943180274 P(T<=t) two-tail 0.44000226

t Critical two-tail 2.446911846

# DATA EVALUATION RECORD

BENFLURALIN

Study Type: O CS PP 890.1150, Androgen Receptor Binding (Rat Prostate Cytosol)

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614902)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature:
David A. McEwen, B.S. Date: 11 / 13 /2011

Secondary Reviewer Signature: Michelle J. Sharpe

- Kass, M.S

. Date: 11 / 15 /2011

Program Manager: Signature: Jack D. Early, M.S. Date: 11 / 16 /2011

Quality Assurance: Signature: Jack D. Early, M.S. Date: 11/16/ 2011

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel

.

## 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 i s 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).

### BENFLURALIN BENEFIN /084301 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I 

### Pagel of l3

OCSPP 890.1150/ OECD None



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

. T.



## Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D.



## Health Effects Division

# :::::~:~~~ Date: 

# "'/lr:/ t-?

- Template version 08/201 l



## DATA EVALUATION RECORD

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



## PC CODE: 084301 

## DP BARCODE: D399309



## TXR#: 0054123 

## CAS No.: 1861-40-1



## TEST MATERIAL (PURITY): Benfluralin (97.5% a.i.)



## SYNONYMS: Benefin; N-Butyl-N-ethyl-2

, 6-dinitro-4-(trifluoromethyl)benzenamine



## CITATION: LeBaron, M

. J.

, Schisler

, M.R.

, and Visconti

, N

. R. (2011). Evaluation ofBenefin

in an in vitro androgen receptor binding assay

. Toxicology 

## & Environmental

Research and Consulting

, The Dow Chemical Company, Midland

, MI. Laboratory Project ID : 111097

, October 21, 2011. MRID 48614902. Unpublished.



## SPONSOR: Dow AgroSciences LLC

, 9330 Zionsville Rd, Indianapolis, 

## IN 46268



## TEST ORDER#: EDSP-084301-15



## EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48614902),

v entral prostate cytosol from Sprague Dawley rats was used as the source of AR. Prior to conducting the AR competitive binding experiments, saturation binding experiments were
conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable

numbers and was functioning with appropriate affinity for the radiolabeled ligand. The saturation binding experiment was conducted in duplicate and summarized in a submitted study profile
( MRID 48614902)

.

The competitive binding assay was conducted to measure the binding of a single concentration of [3H]-Rl 881 (10 nM) in the presence of increasing concentrations of benfluralin (logarithmic

increase from 10- 1 0 

## to 10- 3 

## M). Ethanol was used as a vehicle at a final assay concentration of

< 3 %. The assa y included dexamethasone as a weak positive control

, and Rl881 as the ligand

reference standard. Three independent runs were conducted with 3 replicates per concentration

per run.

In the saturation binding experiment, the maximum binding capacity (Bm ax ) was 3 .245 fmol / 100

μg protein and the dissociation constant (Kd) 

## was 0.4641 nM. These values were below the

range of values from the validation studies (7 to 16 fmol/100 μg protein and 0.685-1.57 nM, respectively). The Scatchard plot indicated a linear response across the concentrations of ligand

added. Nonspecific binding as a percent of total binding was less than 20% across the entireAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 2 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## concentration range in the saturation binding assays (range 6.2-19.8% ), with the exception of the high concentration (10 nM) in one assay, which was 24.6%.

The estimated log IC 50 

## s for R -1881 and the weak positive control (dexamethasone) were -8.975

and -4.509 M , respectively . The mean RBA for the positive control was 0.0034%. The solvent control responses indicated no drift in the study assay. All performance criteria were met, with the exception of the bottom (% binding) of R1881. In Run 2, the bottom was 2.1, just outside the

recommended range of -2.0 to 2.0%. The range for the other two runs was -1.0 to -1.7, with a mean value for the three assays of -1.6%. The reviewers consider this a minor deviation and find that the performance criteria were met.

The lowest percent total [ 3 

## H] -R1881 binding at all concentrations of benfluralin was 77 %, 7 8 %,

and 75 % resulting in classification for the runs as non-binder, non-binder and equivocal in runs 1, 2, and 3, respectively. An estimated mean log IC 50 

## for benfluralin could not be determined since benfluralin did not displace more that 50% of the radioligand at any concentration.

Based on the results from the three runs , benfluralin is classified as a N on-Binder in the Androgen Receptor Binding Assay.

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

a ssay (OCSPP 890.1150).

COMPLIANCE : Signed and dated GLP Compliance and Quality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 3 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test f acility : Toxicology & Environmental Research and Consulting, The Dow Chemical Company

Location: Midland, MI Study Director: M.J. LeBaron

Other Personnel: M.R. Schlisler and N.R. Visconti Study Period: June 27, 2011 to July 5, 2011



## 2

. Test substance : Ben fluralin

Description: Yellow solid

Source: Dow AgroSciences LLC, Indianapolis, IN (Ref. No. TSN100037)

Lot #: ACD13683 Purity: 97.5 % Solubility : Soluble in ethanol up to 30 mM; less than 0.001 g/L in water Volatility : Vapor pressure 2.8 mmHg Stability: 4 yrs shelf life Storage conditions: Ambient

CAS #: 1861

- 40

- 1 Molecular weight: 335.3

Structure:



## 3

. Non

- labeled ligand : R1881

Supplier : Perkin

- Elmer, Boston, MA Catalog # : Not reported

Batch #: 614156 Purity: >97%

CAS #: 965

- 93

- 5



## 4

. Radioactive ligand : [ 3 

## H]

- R1881

Supplier : Perkin

- Elmer , Boston, MA Catalog and Batch #: NET590250UC; Lot # 614814 Date of production: July 1, 2010

Date of use: June 27 , 2011 to July 5, 2011 Radiochemical purity: >97 % Specific activity : 85.1 Ci/mmol *Information on adjusted specific activity was not available.

Concentration in stock : 1.0 m Ci/m L



## 5

. Positive control : Dexamethasone

Supplier : Sigma, St. Louis, MO Catalog # : Not reported

Batch #: BCBC9269 Purity: 98.9%

CAS # : 50

- 02

- 2



## 6

. Solvent/vehicle control : Ethanol

Justification for choice of solvent : Test substance has v ery low solubility in water (>0.001 g/L)

Final c oncentration : < 3% F F

F N O 2 N

O 2 N CH

3 CH

3Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 4 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol : The rat ventral prostate tissue was purchased from Charles River (Wilmington, MA). M ale Sprague Dawley rats (number not reported) were castrated at approximately 90 days of age and were euthanized 24 hours

later . The ventral prostate tissues were collected and s tored at approximately -80°C until use, and were processed as a batch and used for multiple studies.

The cytosol was prepared by adding low- salt TEDG buffer [0.01 M TRIS buffer, 10% glycerol, 1 mM sodium molybdate, 1.5 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM phenylmethylsulfonyl fluoride (PMSF) with fresh dithiothreitol (DTT)] at pH 7.4, to the ventral prostate tissues at 10 mL/g of tissue. The tissues were minced, homogenized on

ice , and centrifuged for 30 min at 30,000 × g at 4 °C. The s uper natant was pooled, aliquoted, and stored at approximately -80°C for up to 6 months. Protein concentration of the cytosol prepared (on June 2, 2011) for this study was determined to be 6.566 mg/mL using the Pierce BCA method (Thermo Scientific Pierce Resea rch Lab, Rockford, IL).

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

## H] -R1881 was performed to demonstrate that the AR was present in reasonable concentrations and had the appropriate affinity for the R1881 ligand. The conditions for the saturation binding experiment are summarized in

Table 1.

TABLE 1. Summary of Condition s for Saturation Binding Experiment a


### Source of receptor Rat prostate cytosol
Concentration of radioligand (as serial dilutions) 0.25

- 10 nM

Concentration of non

- labeled ligand (100X [radioligand]) 25

- 1000 nM
Optimization of receptor concentration Sufficient to bind 8.6

- 9.0%b of radioligand at 0.25 nM
Temperature ~2

- 8 oC

Incubation time ~16 hours
Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10%
Phenylmethylsulfonyl fluoride 1.0 mM

DTT 1.0 mM

a Data were not included in the study report, but are reported in the study profile (MRID 48675402).

b As indicated in the guideline for assay performance the receptor concentration bound less than 25 to 35% of the

radiolabeled R1881.



## On the day of the assay, the specif ic activity of the stock solution [ 3 

## H] -R1881 was not adjusted for decay over time, and serial dilutions in TEDG buffer were prepared to achieve

the final concentrations in cytosol of 0.25, 0.5, 0.7, 1.0, 1.5, 2.5, 5.0, and 10.0 nM to

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

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

## H] -R1881 was measured. For each batch of cytosol, the optimal protein concentration was dete r mine dAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 5 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## by calculating specific binding to differing amounts of protein per tube, using 0.25 nM radiolabeled R1881. The optimal protein concentration was determined to be 1.97 mg protein/assay tube, which resulted in the binding of 8.6-9.0% of the total radioactivity added. As indicated in the guideline for assay performance the receptor concentration bound

less than 25 to 35% of the radiolabeled R1881

. 

## Cytosolic protein used in this assay was thawed fresh for this experiment at ~4°C and maintained at ~4°C during the binding assay. Each run contained three concurrent replicates at each concentration, resulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment 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) [3H]

- 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 not included in the study report, but are reported in the study profile (MRID 48675402).

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

c T ubes 1

- 24 contained 50 μL of triamcinolone acetonide and 7.5

- 45 μL [ 3 

### H]

- R1881 . Samples were dried, and 300 μl of prostate cytosol were added.

d T ubes 25

- 48 contained 50 μL of triamcinolone acetonide and 7.5

- 45 μL [ 3 

### H]

- R1881. R1881 was added in a 100

- fold molar excess of [ 3 

### H]

- R1881 in a volume of 7.5

- 45 μL. Samples were dried, and 300 μl of prostate cytosol were added.

e Tubes 49

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

### H]

- R1881 or 7.5, 15, 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 for approximately 16

hours at 2-8°C. Samples were maintained at temperatures of ~4°C except during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, a nd the supernatant was aspirated and discarded. The samples were washed 3 times in 50 mM TRIS buffer. Following the last wash and decanting of the Tris buffer, pellets were then extracted by addition of 2 ml ethanol. The samples were vortexed 3 times

at 5 minute intervals. Samples were maintained on ice at all times between vortexing. Each ethanol supernatant was then decanted into a scintillation vial, and the radiation was quantified by liquid scintillation counting. A total of 4 runs were performed on 2 batches of cytosol with similar results. For the batch of cytosol used for the competitive assay, 2 runs were performed rather than runs as recommended in the test guideline; however, both runs

had highly similar binding profiles.

3. Competitive Binding Experiment : A summary of the assay conditions for the competitive binding experiment is presented in Table 3.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 6 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



### TABLE 3 . Summary of Conditions for Competitive Binding Experiment a


### Source of receptor Rat ventral prostate cytosol

Concentration of radioligand 10 nM
Optimization of receptor c oncentration Sufficient to bind 4.3

- 5.2% b 

### of 1.0 nM radioligand.
Concentration of test substance (as serial dilutions) 10

- 10 

### to 10

- 3 

### mM

Incubation Temperature 4

- 8° C

Incubation time 16 hours
Composition of assay buffer Tris 0.01 M (pH 7.4 )

EDTA 1.5 mM
Glycerol 10 %
Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM

a Data were obtained from page s 16 and 19 of the study report. b As indicated in the test guideline, the receptor concentration bound less than 10

- 15% of the radiolabeled

R1181.



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

the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures the binding of a single concentration of [ 3 

## H] -R1881 (adjusted specific activity of 85.1

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

Ethanol was used as a vehicle, and no precipitation was observed in the 3 x 10 -2 

## M stock or the 1 mM final concentration by visual assessment. Results from the saturation binding experiment demonstrated that 6.566 mg/ml or 1.97 mg/assay tube of cytosolic protein

contain enough receptor to bind no more than 10-15% of the [ 3 

## H] -R1881, and that nonspecific binding met the guideline recommended value of <20%.

Dilutions of the test substance, reference standard (R1881 ), weak positive control ( dexamethasone ), and solvent control (ethanol) were prepared to achieve the concentrations

shown in Table 4. Each assay consisted of three independent runs on three different days, and each run contained three replicates at each concentration, resulting in a total of 85 samples per run

.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 7 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



### TABLE 4

. Competitor F inal Molar (M) C oncentrations in Competitive Binding Assay a b



### Solvent Control Reference standard Weak positive control Test Chemical None Ethanol R1881 Dexamethasone Benfluralin

Tubes 1 1

- 13 and 8 0

- 8 2 Tubes 1 4

- 3 1 and 8 3

- 8 5 c 

### Tubes 32

- 55 Tubes 56

- 79 Tubes 3

- 8

1×10

- 6 

### 1×10

- 3 

### 1×10

- 3



### 1×10

- 7 

### 1×10

- 4 

### 1×10

- 4



### 1×10

- 8 

### 1×10

- 5 

### 1×10

- 5



### 1×10

- 9 

### 1×10

- 6 

### 1×10

- 6



### 1×10

- 10 

### 1×10

- 7 

### 1×10

- 7



### 1×10

- 11 

### 1×10

- 8 

### 1×10

- 8



### -- 1×10

- 9 

### 1×10

- 9



### -- 1×10

- 10 

### 1×10

- 10



### a Data were obtained from page s 18

- 20 and 38

- 43 of the study report.

b Each concentration of each chemical was run in triplicate for a total of 8 5 tubes per run. Tubes 11-8 5 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

### H]

- R1881. Samples were dried, and 300 μl of prostate cytosol

were added. Tubes 11-8 5 also contained 10 μL of the solvent control, reference standard (non

- radiolabeled R

1881), weak positive control, or test substance, with the exception of Tubes 14- 16 and 83 -8 5 that contained 30 μL of non

- radiolabeled R1881 (used to evaluate non

- specific binding). Tubes 3-8 contained only 30 μl of
[ 3 

### H]

- R1881.

c Tubes 14-1 6 and 83-8 5 were used to evaluate non

- specific binding by adding 100X of cold (non

- radiolabeled)

R1881

.



## 4. Data Analysis : The maximal binding capacity (B
max 

## ) and dissociation constant (K d 

## ) were not reported, and as the results of the saturation binding experiment were not provided in the current study they could not be determined. The inhibition concentration (IC 50 

## ) w as calculated using nonlinear regression analysis using Graph Pad Prism v. 5 ( GraphPad Software, Inc., La Jolla, CA ).

5. Definitions

a. Classification of test material

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

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

bound.

Equivocal : The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at least 25% but less than 50%), or the curve falls outside the range for the weak positive control (-0.6 to -1.4).

Non

- Binder : The average lowest portion of curves across runs is greater than 75% activity ( i.e. less than 25% displacement of radioligand), or the data do not fit the model.

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

- 6 

## M and the binding curve does not cross 50%, the chemical is judged to be untestable.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 8 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



### TABLE 5. Data Interpretation Criteria Specified in the Test Guideline (890.1150).

Criteria Classification Values

Data fit 4

- parameter nonlinear regression

model Average curve across runs crosses 50%* Binder 2 Average lowest portion of curves across runs is between 50% and 75% activity** Equivocal 1

Average lowest portion of curves across runs is greater than 75% activity** Non

- Binder 0

Data do not fit the

model ----

* If the curve fell outside the range for the weak positive control (see test performance criteria), the run as classified as equivocal.

** If the test compound was not soluble above 10 -6 

### M and the binding curve did not cross 50%, the chemical was judged to be untestable.



## Chemical classification is based on the average value of the three valid runs performed for a

test chemical:

• binder: average ≥ 1.5

• equivocal: 0.5 ≤average <1.5

• non-binder: average < 0.5

b. Descriptors for receptor binding

B max 

## : maximal binding capacity (Not provided)

K d 

## : dissociation constants (Not provided)

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 e xperiment parameters are presented in Table 6. The dissociation constant (Kd) for [3H]-R1881 was 0.4641, and the estimated Bmax (nM) was 0.06392 for the batch of prostate cytosol that was used for this study. The Kd was below the range reported in the EPA validation program (0.685-1.57 nM). Both runs provided similar results.

TABLE 6. Saturation Binding Experiment of R

- 1881 with Androgen Receptor from Rat Prostate Cytosol a



### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean Runs 1

- 2 c



### R 2 

### (unweighted) 0.9937 0.9871 ND 0.9871

- 0.9937

B max 

### (nM) 0.06194 0.06590 ND 0.06392

B max 

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

K d 

### (nM) 0.4359 0.4922 ND 0.4641

a Data were not included in the study report, but are reported as a separate validation report.

b Two saturation runs were performed for this batch of cytosol.

c The range of R 2 

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

R 2 

### Goodness of fit for curve calculated for specific binding,

ND not determinedAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 9 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## Non -specific, specific, and total binding curves for [ 3 

## H] -R1881 to the androgen receptor are shown in Figure 1. The specific binding reached a plateau and the non-specific binding was generally less than 20% of total binding at all concentrations (range 6.2%-19.8%) except at the highest concentration (10 nM) in Run 1 (24.6%).

FIGURE 1. Binding of [ 3 

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

Figure 2 is a Scatchard plot that illustrates the binding of [ 3 

## H] -R1881 to the androgen receptor.

The data fit results in a line ar plot.

FIGURE 2. Scatchard Plot of the Binding of [ 3 

## H] -R1881 to the Androgen Receptor.

COMPETITIVE BINDING EXPERIMENT : The results from the 3 c ompetitive binding

e xperiment s are summarized in Table 7 and shown graphically in Figures 3-6. The estimatedAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 10 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## log IC 50 

## s for R-1881 and the weak positive control (dexamethasone) were -8.975 and -4.509

M , respectively. The mean RBA for the positive control was 0.0034%. Confidence in these numbers is high due to the small variation. The solvent control responses indicated no drift in the study assay. The lowest percent total [ 3 

## H] -R1881 binding at all concentrations of

benfluralin was 77 %, 7 8%, and 75 % resulting in run classifications of non-binder, nonbinder and equivocal in runs 1, 2, and 3, respe ctively. An estimated mean log IC 50 

## for benfluralin could not be determined since benfluralin did not displace more that 50% of the radioligand at any concentration. Based on the results of the three runs, benfluralin is

classified as a non -binder in the AR binding assay.

TABLE 7 . Competitive Binding Assay of Benfluralin with A R from Rat Prostate Cytosol a



### Parameter Assay 1 Assay 2 Assay 3 Mean

r 2 

### (unweighted) R1881 0.9999 1.0000 0.9999 0.9999 to 1.0000

Positive control 0.9992 0.9997 0.9985 0.9985 to 0.999 7

Test substance 0.9912 0.9888 0.9861 NA Log IC 50 

### (M) R1881

- 8.965

- 8.980

- 8.981

- 8.975

Positive control

- 4.522

- 4.431

- 4.550

- 4.509

Test substance NA NA NA NA

IC 50 

### (M) R1881 1.084×10

- 9 

### 1.048×10

- 9 

### 1.044×10

- 9 

### 1.060×10

- 9



### Positive control 3.006×10

- 5 

### 3.707×10

- 5 

### 2.819×10

- 5 

### 3.094×10

- 5



### Test substance NA NA NA NA

RBA (as % IC 50 

### ) c 

### P ositive control 0.0036 0.0028 0.0037 0.0034

Test substance NA NA NA NA

a Data were obtained from page s 31 and 32 of the study report.

NA N ot applicable.

r 2 

### Goodness of fit (r 2 

### is more appropriately expressed as a range, as opposed to a mean).

c Calculated by reviewer: [ IC 50 

### (in M) positive control or benfluralin / IC 50 

### (in M) R1881] x 100% RBA (%) relative binding affinity



## FIGURE 3. Relative binding activity in Assay 1.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 11 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## FIGURE 4. Relative binding activity Assay 2.

FIGURE 5. Relative binding activity in Assay 3.

FIGURE 6. Mean relative AR binding activity for Benfluralin

.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 12 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## C. PERFORMANCE CRITERIA : To ensure that the competitive binding assay was functioning properly, each run was evaluate d using the following criteria (Table 8) :

T ABLE 8

. Criterion a 

### Tolerance Limit(s) b 

### Value Yes No

Ligand depletion is minimal. The recommended r atio of total binding in the absence of competitor to total amount of [ 3 

### H]

- R1881 added per assay tube. ≤15% 4.8 to 5.5% X

Test chemical Top (% binding) 80 to 115 97.9 to 106.5 X

R1881 fitted curve parameters Top (% binding) 82 to 114 99.8 to 102.5 X Bottom (% binding)

- 2.0 to 2.0

- 1.0 to 2.1 X Hill Slope

- 1.2 to

- 0.8

- 0.9 to

- 1.0 X Weak positive control (dexam e thasone ) fitted curve parameters Top (% binding) 87 to 106 99.9 to 102.7 X Bottom (% binding)

- 12 to 1 2

- 1.5 to 0.3 X Hill Slope

- 1. 4 to

- 0. 6

- 1.0 X Saturation Binding Experiment K d 

### (nM) 0.685

- 1.57 0.4641 X

Non

- specific binding (%) c 

### <20% 6.5

- 7.4% X

a Data were obtained from page s 31 and 32 of the study report.

b These values represent ran ges from the validation study.

c Calculated by reviewer from page 38

- 42. Avg ethanol (minus background) / Avg NSB (minus background) x

100%.



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

## H] -R1881 in a manner consistent with one-site binding, as indicated by a descent from 90% to 10% binding over approximately an 81-fold i ncrease in concentration of R1881 (i.e., covering approximately 2 log units). Examination across the runs indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus.

The percentage of the total specific binding in the solvent controls was approximately 5%.
This was within the less than the ≤ 10 -15% recommended in the guideline. Ligand depletion was also minimal. Sufficient optimization of the number of specific bind ing sites is supported curves for controls of acceptable steepness, low non-specific binding, and low variability.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : The Investigators concluded that the collective responses of three independent assays for Benfluralin in the AR binding assay indicated a reproducible slight decrease (75.0-78.1% of control) in radiolabeled R1881 binding at the assay limit concentration (1 mM, 10 -3 

## M). Two out of three assays did not reach the equivocal criterion described in the guideline (50-75% radioligand binding ). There were no apparent alterations in radiolabeled R1881 binding at any of the lower concentrations tested. The means of the three independent assays are shown in Figure 4 above. Benflur alin was classified as non -binding based on the average of the three valid assays with a mean value of “0.33” ( i.e., (0+0+1)/3 runs).

B. AGENCY COMMENTS : In the competitive binding experiment, t he estimated mean log IC 50 

## for the test material could not b e determined as it did not cross 50% binding. TheAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 13 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0.1150 / OECD None



## estimated log IC 50 

## s for R1881 and the weak positive control (dexamethasone) were -8.975

and -4.509 M , respectively . The mean RBA for the positive control was 0.0034%. The solvent control responses indicated no drift in the study assay. All performance criteria were met, with the exception of the bottom (% binding) of R1881. In Assay #2, the bottom was 2.1%, just outside the recommended range of -2.0 to 2.0%. The range for the other two

assays was -1.0 to -1.7% , with a mean value for the three assays of -1.6%. The reviewers consider this a minor deviation and find that the performance criteria were met.

The lowest percent total [ 3 

## H] -R1881 binding at all concentrations of benfluralin was 77 %, 78%, and 75 % resulting in run classifications of non-binder (0), non-binder (0) and equivocal (1) in runs 1, 2, and 3, respectively. The chemical classification is based on the average value for the three valid runs. The mean value for the three runs is 0.33 (<0.5=Non-Binder), therefore the classification for benfluralin is non-binder in the Androgen Receptor Binding Assay.

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

• The Kd for saturation binding (0.4641 nM) was less than the recommended range of

0.685- 1.57 nM.

• The Bottom (% binding) for the R1881 was slightly outside of tolerance limits (an upper end of 2.1 instead of 2.0). Th is minor deviation did not impact the study results as evidenced by expected IC50 and Hill Slope values for R1881.

# DATA EVALUATION RECORD

B ENFLURALIN

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614903)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Scott D. Studenberg, Ph.D., D .A.B.T. Signature:

Date: 11/17 /2011

Secondary Reviewer : Signature:
David A. McEwen, B.S. Date: 11/18 /2011

Program Manager: Signature: Jack D. Early, M.S. Date: 11/18/2011

Quality Assurance: Signature: Jack D. Early, M.S. Date: 11/18/2011

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel

.

## 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 stre ngths 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).BENFLURALIN (BENEFIN) I 084301



## Primary Reviewer: Vincent Chen, M.S.



## Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D

. 

## Health Effects Division Aromatase (Human Recombinant) Assay (2011) I Page 1 of 13

OCSPP 890.1200 I OECD None

# Signature:~ Date: zy ""3" vf\J z..o..l-5 

## Signature:**~** 

# A-:-:

Date: <;, 

## k Y- 

### It r Template version 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



## PC CODE: 084301 

## DP BARCODE: D399309



## TXR: 0054123 

## CAS: 1861-40-1



## TEST MATERIAL (PURITY): Benfluralin TGAI (97.5% a.i

. )



## SYNONYMS: Benefin



## IUPAC: N-butyl-N-ethyl-a,a,a-trifluoro-2,6-dinitro-p-toluidine



## CAS: N-butyl-N-ethyl-2,6-dinitro-4-(trifluoromethyl)benzenamine



## CITATION: Coady 

## K.K., Sosinski 

## L.K. (2011)

. Benefin: Evaluation ofBenefin in the Human

Recombinant Aromatase Assay. Toxicology & 

## Environmental Research and Consulting (The Dow Chemical Company [Midland, MI 48674, USA]). Laboratory Report Number: 101143, 04 October 2011. MRID 48614903. Unpublished.



## SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Rd, Indianapolis, 

## IN 46268



## TEST ORDER: EDSP-084301-15



## EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48614903),

Benfluralin (97.5% a.i., Lot No. ACD13683, TSN100037) was incubated with human

recombinant aromatase and tritiated androstenedione (1-P [3H(N)]-androst-4-ene-3, 17-dione;
[3H]ASDN) in dimethyl sulfoxide (DMSO) at concentrations of 0, 10- 10

, 

## 10- 9

, 

## 10- 8

, 

## 10

- 1

, 

## 10- 6

, 

## 1



# o- 5 5

, 

## 10 4

, 

## or 10- 4 5 

## M for 15 minutes to assess the potential of benfluralin to inhibit aromatase activity. Evidence of test material insolubility was observed at 10- 3 

## and 10- 4 

## M therefore the highest test concentration analyzed was 10- 4 5 

## M.

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- 10 

## to 10- 5 ) 

## with a known inhibitor ( 4-hydroxyandrostenedione; 4-0H ASDN), and the test chemical series (1 o-io 

## to

10- 4 5 ) 

## with three repetitions per concentration. The proficiency evaluation data for the investigating laboratory regarding the aromatase assay were not included in the study.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 2 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## The response of the full activity controls and background controls were acceptable for each run, although the mean background count from assay Run 3 was approximately 100- fold greater compared to the previous two assay run s.

The standard curves for 4- OH A SD N met three out of four performance criteria in Run 1, all four

criteria in Run 2, and only two out of four performance criteria in Run 3. These deviations from the guideline recommendations for performance criteria were considered minor by the investigators because acceptable Hill slope values and goodness of fit values were obtained for all three assay runs. In general, values in Run 3 were more variable compared to the previous two assay runs, including a higher background count and higher %CVs for the replicate tubes.

Aromatase activity was similar across the tested concentration range for benfluralin (10

- 10 

## M to

10

- 4.5 

## M). The a romatase activity curves after exposure to benfluralin either did not fit the model (Run 1) or were not lower than 75% of full acti vity (Run s 2 and 3). The log IC 50 

## of the test

material was -8.0 M.

Based on the data from the average response curve, and despite the large variability introduced by the data from assay Run 3, benfluralin is classified as a Non- inhibitor of aromatase activity in this assay.

The assay satisf ies the EDSP Tier 1 Test Order requirement s for an Aromatase a ssay (OCSPP 890.1200).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality Assurance statements were provided.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 3 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : Benefin TGAI Description: Yellow S olid

Source: Dow AgroSciences LLC [Indianapolis, Indiana, USA]

Lot/Batch #: Lot No. ACD13683, TSN100037 Purity: 97.5 % Storage conditions: 5 o C

- Ambient Stability: Not reported

Solvent : DMSO Solubility (in Test S olvent): 0.1 M Highest Concentration Tested: 1x10

- 4.5 M

Stock Solution Preparation : Serial D ilution

Molecular W eight: 335.3 g/mol

CAS #: 1861

- 40

- 1

Structure:



## 2. Non

- L abeled S ubstrate : Androstenedione (ASDN)

CAS # : 63

- 05

- 8

Source: Steraloids Inc. [ Newport, RI ]; Catalog No. A6030

- 100

Lot/Batch #: Batch No. L1627 Purity: 98 .36 %



## 3

. Radiolabeled S ubstrate : 1

- β [ 3 H(N)]

- Androst

- 4

- ene

- 3,17

- dione ; ( [ 3 H] ASDN)

Source: Perkin Elmer Life and Analytical Sciences [ Boston, MA ]

Lot/Batch #: Lot s: 3615304 and 619344 Radiochemical Purity (Supplier): Lot 619344: > 97 %

Lot 3615304: R adiochemical purity was not reported Specific activity: Lot 619344: 26.3 Ci/mmol

Lot 3615304: 20.7 Ci/mmol Radiochemical Purity (In

- lab determination): > 95%



## 4. Positive C ontrol : 4

- hydroxyandrostenedione (4

- OH ASDN)

CAS # 566

- 48

- 3

Source: Sigma Aldrich [ St. Louis, MO ]; Catalog No. F2552

Lot/Batch #: Lot 081K2133 Purity: 99.6 %



## 5. Solvent (V ehicle C ontrol ) : DMSO

Lot/Batch #: Sigma Aldrich [ St. Louis, MO ]; Catalog No. D5879

Lot 037K0766

Justification for Choice of S olvent Greater solubility compared to ethanol Concentration (% of Total Volume in A ssays) : 1% N

O 2 N NO 2

CF 3 H 3 CH 2 C (CH 2 ) 3 CH 3Aromatase (Human Recombinant) Assay ( 2011 ) / Page 4 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## 6. Test M icrosomes : Human R ecombinant A romatase ( CYP19 ) M icrosomes

Source : Gentest [ Woburn, MA ]; C atalog No. 456260

Lot/Batch #: Lot 74101

Protein C oncentration : 0.004 mg protein per mL
Cytochrome C R eductase A ctivity: 560 nmole cytoch rome c reduced/mg protein/min

Aromatase A ctivity: 4.4 p mol product/ pmol P450 /min



## B. METHODS

1. Assay C omponent s and Preparations : A mixture of non- labeled and radiolabeled
[ 3 

## H]ASDN was prepared such that the final concentration of ASDN in the assay was approximately 2 μ M, and the amount of tritium added to each incubation tube was approximately 0.2 μCi in a volume of 100 μL (approximately twice the radioactive content suggested in the guideline

, OCSPP 890.1200). No reason was given for this deviation.

Test chemical(s) stock solutions were prepared such that the total volume of each test chemical formulation used per assay was no more than 1% of the total assay volume to minimize the potential for the solvent to inhibit the enzyme. Selection of the solv ent(s),

DMSO , was based upon the physical properties of the test chemical ( greater solubility of

benfluralin in DMSO relative to ethanol ).

A stock solution of the positive control substance, 4- OH ASDN, was formulat ed in DMSO

. Fresh dilutions of the stock solution w ere prepared in the same solvent as the stock solution on the day of use. Dilutions were prepared such that the target concent rations of the positive control substance (0.1–10,000 nM; Table 4 ) wer e achieved by the addition of 20 μ L of the dilution for a final assay volume of 2 mL.

Human recombinant microsomes were purchased from Gentest, and stored at approximately -80oC. The length of the storage period was not reported . Microsomes were portioned into individual vials based on the protein concentration of the batch (i.e., approximately 0.004 mg/mL microsomal protein per tube) and stored at approximately

- 80oC.

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

TABLE 1

. Assay Components and Conditions

Assay Factor Values

S odium phosphate buffer (pH 7.4) 0.1M

Microsomal Protein 0.004 mg/mL a

NADPH 0.3 mM
[ 3 H]ASDN 100 nM Propylene Glycol 5% Temperature 37°C

Incubation Time 15 min

a The concentration of microsomal protein was optimized for microsomes that produce d approxi mately 4.4 pmol product/pmol P450/min.



## 2. Suitability Ass es s ments : The protein concentration was reported to have been determined on each day the aromatase assay was run. Protein concentration was determined inAromatase (Human Recombinant) Assay ( 2011 ) / Page 5 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## accordance with SOP No. TOX

- BMT -192.05 (Quantitation of Proteins), but protein concentration results were not reported ; the SOP No. TOX

- BMT -192.05 was not included. The microsomes were diluted with phosphate buffer to yield approximate protein concentrations in the final reaction mixture of 0.004 mg/mL.

Aromatase activity in each lot of human recombinant microsomes was determined to demonstrate the presence of sufficient activity for analysis of benfluralin. The aromatase activity was determined to range from 0.1222 to 0.1802 nmol/mg

- protein/min for the three

aromatase assay runs , which wa s greater than the minimum acceptable aromatase activity of 0.1 nmol/mg- protein/min.

3. Aromatase A ssay : Each assay run contained 4 tubes for the full enzyme activity and background activity controls, respectively, and a full concentration curve in duplicate for the positive control and in triplicate for the test substance. The aromatase assay was conducted 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 liquid

scintillation counting (LS C ), and aromatase a ctivity was reported in units of

nmol

∙ mg -protein -1



## ∙ min -1



## .

4. Demonstration of P roficiency : There was n o mention of proficiency testing prior to the conduct of the assay (i.e., at least one single run of the positive control experiment and three full scale runs of the proficiency chemicals were conducted to demonstrate assay proficiency of the laboratory).

a. Positive Control

(1) Initial Demonstration of Laboratory P roficiency : The positive control data (new/historical data for laboratory) were not reported in regards to the following criteria:

• Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol/mg

protein/min.

• Mean background control activity was ≤ 15% of the full activity control.

• Coefficient of variation (% CV) for replicates within each sample type and

concentration of 4

- OH ASDN was <15%.

Performance criteria were reported for the test runs only (Table 2), and served as guidance in identifying runs that provided parameters in the preferred ranges.

(2) Demonstration of Proficiency of New Technician for Conducting As say (when applicable) : Demonstration of proficiency by a new technician, if applicable, was not reported.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 6 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



### TABLE 2. Performance Criteria for the Positive Control

Parameter Lower Limit Criteria Upper Limit Criteria Actual Lower Limit Actual Upper Limit
Slope

− 1.2

− 0.8

– 0.94

– 0.91
Top (%) 90 110 98 117
Bottom (%)

− 5 +6

– 5.2 0.33
Log IC 50 (M)

− 7.3

− 7.0

– 7.4

– 7.3

a Data were obtained from page 27 of the study report.



## b. Proficiency C hemicals : The proficiency of the laboratory was not described or reported. Historical proficiency data with the aromatase assay from the laboratory/technicians was not included in lieu of current proficiency data.

T ABLE 3. Proficiency Chemicals

Compound CAS# Class Concentrations

Econazole 24169

- 02

- 6 Inhibitor Not reported

Fenarimol 60168

- 88

- 9 Inhibitor Not reported

Nitrofen 1836

- 75

- 5 Inhibitor Not reported

Atrazine 1912

- 24

- 9 Non

- inhibitor Not reported



## 5. Determination of Aromatase A ctivity with T est C hemical(s) : T he response of aromatase activity to the presence of eight concentrations of a t est chemical per run, in triplicate, was tested during three ind ependent runs.

T ABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repe titions (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

Benfluralin Conc 1 3 All test components plus test chemical 1×10

- 4.5

Benfluralin Conc 2 3 All test components plus test chemical 1×10

- 5

Benfluralin Conc 3 3 All test components plus test chemical 1×10

- 5.5

Benfluralin Conc 4 3 All test components plus test chemical 1×10

- 6

Benfluralin Conc 5 3 All test components plus test chemical 1×10

- 7

Benfluralin Conc 6 3 All test components plus test chemical 1×10

- 8

Benfluralin Conc 7 3 All test components plus test chemical 1×10

- 9

Benfluralin Conc 8 3 All test components plus test chemical 1×10

- 10

a The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH

.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 7 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## C. D ATA ANALYSIS

1. Raw D ata : Raw data were converted to aromatase activity (nmol/mg protein/min) and percent control for the positive control, proficiency chemicals and benfluralin. The following raw data and calculated endpoints for each run were included in the report (Table 5 ).

TABLE 5

. Raw and Calculated Data

Raw/Calculated Data Included (X)

DPM/ 0.5 mL for each portion of extracted aqueous incubation mixture X Average DPM/ 0.5 mL for each aqueous portion (after extraction) X

Total DPM for each aqueous portion (after extraction) 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/ mi n X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations

DPM Disintegrations per minute



## 2. S tatistical M ethods : Statistical analyses and displays such as summary tables, graphical displays, analysis of variance, and multiple comp arisons were conducted

.

The response curve was fitted by weighted least squares nonlinear regression analysis with Graph Pad Prism version 4.0. For each run, the individual percent of control values were plotted versus logarithm of the test chemical concentration. The fitted concentration

response curve was superimposed on the plot, with individual plots prepared for each run. The average percent of control values versus logarithm of test chemical concentration for

the individual runs for each test chemical (with different s ymbols for each run) were included on the same graph with their respective fitted response curves. In a ddition, t he average percent of control values for each run versus the logarithm of test chemical concentration were plotted on a separate graph along with t he avera ge concentration response curve across runs were superimposed on the same plot.

In order t o determine the consistency among runs, the s lope and log IC 50 

## for the positive

control and test chemical were compared across runs based on one- way random effects analysis of variance (ANOVA) , with the runs treated as random effects. The parameters

were graphed within each run with associated 95 percent confidence intervals based on the

within -run standard error, and the average across-run standard error, with the associated 95 percent confidence interval incorporating run

- to -run variation.

3. Interpretation of Results : Interpretation of the assay results was based on the average of three runs, with the categories presented in Table 6.

TABLE 6 : Interpretation of Results

Criteria Interpretation

Data fit 4

- parameter nonlinear regression model Averag e curve across runs crossed 50% Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity Equivocal

Average lowest portion of curves across ru ns is greater than 75% activity Non

- inhibitor

Data do not fit model

---Aromatase (Human Recombinant) Assay ( 2011 ) / Page 8 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## II

. RESULTS

A. CONTROL ACTIVITY : Aromatase activity in the full activity controls ranged from

0.1148 to 0.1922 nmol

∙ mg

- protein -1



## ∙ min -1 

## for the three test runs, with a mean and standard

deviation of 0.1553 ± 0.0298 nmol

∙ mg -protein -1



## ∙ min -1 

## . Activity in the background controls averaged 2.29 % of the full control activity. The response of the full activity controls and background controls were acceptable for each run, although the background counts from

assay Run 3 were approximately 100- fold greater comp ared to the previous two assay runs.

A different (an d older) lot of [ 3 

## H]ASDN was used for the third assay run.

B. POSITIVE CONTROL : For the positive control substance (4

- OH A SD N) , a romatase activity averaged 0.1651 ± 0.0276 nmol

∙ mg -protein -1



## ∙ min -1 

## at the lowest tested concentration (10

- 10 

## M; two out of three runs) and -0.0013 ± 0.0040 nmol

∙ mg -protein -1



## ∙ min -1


## at the highest tested concentration (10 -5 

## M)

. The standard curves for 4- OH A SD N met three out of four performance criteria in assay Run 1, all four criteria in assay Run 2, and only two

out o f four performance criteria in assay Run 3. These deviations from the guideline recommendations for performance criteria were considered minor by the investigators because acceptable Hill slope values and goodness of fits were obtained for all three assay

runs. The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across all three runs is presented in Table 7, along

with the o verall s tandard deviation and % CV. T he inhibition response curves for the positive control are shown in Figure 1.

TABLE 7

. Effect of Benfluralin on Aromatase Activity (as percent of control) from Independent Runs

Chemical Concentration ( Log M ) # Runs Overall Mean Overall SD Overall SEM a Overall %CV a

4

- OH A SD N (P ositive C ontrol) - 5 3

- 1.28 a 3.10 a 1.26

- 242

- 6 3 4.68 a 2.36 a 0.96 50.4

- 6.5 3 13.3 a 2.69 a 1.10 20.2

- 7 3 32.3 a 4.41 a 1.80 13.6

- 7.5 3 62.2 a 4.92 a 2.01 7.92

- 8 3 84.3 a 5.19 a 2.12 6.16

- 9 3 105 a 8.71 a 3.55 8.26

- 10 3 110 a 7.31 a 3.66 6.63

Benfluralin

- 4.5 3 102 b 4.15 b 2.45 7.18

- 5 3 106 b 3.34 b 2.40 6.77

- 5.5 3 102 b 12.9 b 5.11 15.0

- 6 3 102 b 3.85 b 2.28 6.71

- 7 3 104 b 6.88 b 3.04 8.77

- 8 3 101 b 11.1 b 4.60 13.6

- 9 3 99.8 b 4.24 b 1.93 5.81

- 10 3 100 b 3.71 b 1.93 5.79

a Statistics were calculated by the reviewer.

b Statistics were reported on page 29 (Table 3) of the study report.

SD Standard Deviation

SEM Standard Error of the Mean

CV Coefficient of VarianceAromatase (Human Recombinant) Assay ( 2011 ) / Page 9 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## FIGURE 1. Inhibition R esponse C urve s for 4

- OH A SD N.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 10 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## C. TEST SUBSTANCE : For benfluralin , aromatase activity averaged 0.1543 ± 0.0209 nmol

∙ mg -protein -1



## ∙ min -1 

## at the lowest tested concentration (10

- 10 

## M) and 0.1576 ± 0.0201 nmol

∙ mg -protein -1



## ∙ min -1 

## at the highest tested concentration (10

- 4.5 

## M) . The mean aromatase activity of benfluralin (expressed as % full control activity) for each

concentration tested across all three runs is presented in Table 7, along with the overall

standard deviation and % CV. Inhibition response curves for benfluralin from each run are shown in Figure 2, and the average inhibition response curve across all runs is shown in Figure 3.

FIGURE 2. Inhibition R esponse C urves for Benfluralin F rom E ach T est R un.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 11 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## FIGURE 3. Mean I nhibition R esponse C urve for Benfluralin

.

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

estimated log IC 50 

## ranged from -9.0 to -5.9 M for the test material (average not determined) compared to the positive control 4- OH A SD N, which averaged -7.3 M . The average slope of the concentration response curve could not be determined for benfluralin , but was -0.88

for 4

- OH A SD N. Confidence in the 4- OH A SD N data is high due to the small variation , but confidence is low for benfluralin due to the high variation in the IC 50 

## and slope data. The best fit Hillslope and values for the 4-OH ASDN standard curves were not significantly different based on a one-way random effects ANOVA (p = 0.9475 and p = 0.0699, respectively). The best fit Hillslope and values for the benfluralin standard curves could not be compared by a one- way ANOVA because the data from assay Run 1 did not converge on the sigmoidal dose response variable slope curve.

TABLE 8

. Effect of Be nfluralin on Aromatase Activity (as P ercent of C ontrol) F rom Independent Runs

Chemical Run 1 Run 2 Run 3 Mean SD %CV Log IC 50 (M)

Benfluralin ND

- 9.0

- 5.9

- 8.0 b ND ND

4

- OH A SD N

- 7.4 a

- 7.3

- 7.3

- 7.33 b NR NR Slope

Benfluralin ND 7.5 0.54 18 b ND ND

4

- OH A SD N

- 0.93

- 0.91

- 0.94

- 0.9 3 b NR NR

a The log IC 50 was outside the guideline recommended performance criteria range of

- 7.3 to

- 7. 0

b Mean log IC 50 and slope calculated from the benfluralin average response curve.

SD Standard Deviation

ND Not determined, did not converge

NR Not reported



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS ’ CONCLUSIONS : The investigators concluded that there was no inhibition of aromatase at any concentration of benfluralin (up to 10

- 4.5 

## M, the limit of solubility) based on the average results from the three aromatase assay runs. Therefore,Aromatase (Human Recombinant) Assay ( 2011 ) / Page 12 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## under the conditions of the study, benfluralin was classified as a “non-inhibitor” of aromatase activity.

B. AGENCY COMMENTS : Aromatase activity in the full activity controls varied no more

than two-fold between the three test runs with a mean and stand ard deviation of 0.1553±0.0298 nmol∙mg -protein -1 

## ∙min -1 

## . The response of the full activity controls and background controls were acceptable for each run, although the mean background count from assay Run 3 was approximately 100-fold greater compared to the previous two assay runs. The use of a different (and older) lot of [ 3 

## H]ASDN may have contributed to this

difference.

The investigators stated that i f the concentration response curve generated for 4

- OH ASDN met 3 of the 4 listed proficiency parameters and it fit the sigmoidal dose response variable slope model (R 2 

## > 95%) the test would be considered valid despite one parameter being out of range . This qualification is not stated in the guideline ( OCSPP 890.1200). The standard

curves for 4

- OH A SD N met three out of four performance criteria in assay Run 1, all four criteria in assay Run 2, and only two out of four performance criteria in assay Ru n 3. These deviations from the guideline recommendations for performance criteria were considered minor by the investigators because acceptable Hillslope values and goodness of fit value s were obtained for all three assay runs. The log IC 50 

## value in assa y Run 1 was -7.4 M (recommended range -7.3 to -7.0 M) and the bottom and top values were -5.2% and 117% in

assay Run 3 (recommended ranges of -5 to 6 and 90 to 110, respectively). In general, assay Run 3 was more variable compared to the previous two assay runs, with a higher background count. A lack of uniformity in the pipetting rate and the timing of termination

w ere reported as potential reasons for the issues with the background count. As described previously, a different (and older) lot of [ 3 

## H]ASDN was used for assay Run 3 only. The failure to meet these performance criteria is not considered by the Reviewer to invalidate the benfluralin aromatase inhibitory activity data determined in the present study.

A romatase activity was similar across the tested concentration range for benfluralin (10

- 10 

## M

to 10

- 4.5 

## M). The aromatase activity curves after exposure to benfluralin either did not fit the model (assay Run 1) or were not lower than 75% of full activity (assay run Nos. 2 and 3 ).

The estimated log IC 50 

## from the mean benfluralin response curve was -8.0 M compared to

the log IC 50 

## from the mean positive control 4- OH A SD N curve, which averaged -7.3 M ; the

Hill slope of the mean benfluralin response curve was 18 compared to -0.88 for the 4- OH

A SD N Hillslope . Confidence in the 4- OH A SD N data is high due to the small variation, but

low er for benfluralin due to the considerably high er variation reported for the IC 50 

## and

Hill slope data (95% confidence intervals of 10 -7 

## to 10 7 

## and 10

- 10 

## to 10 10 

## , respe ctively)

.

Based on the data from the average response curve , and despite the large variability introduced by the data from assay Run 3, the reviewers agree that the results support the

conclusion that benfluralin is classified as a n on-inhibitor of aromatase activity in this assay.

The proficiency evaluation data for the investigating laboratory regarding the aromatase assay were not reported in the study.

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:Aromatase (Human Recombinant) Assay ( 2011 ) / Page 13 of 13

BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1200 / OECD None



## • Expiration dates were not reported for test materials (reagents, positive control, and test chemical)

.

• Two different lots of test substrate [ 3 

## H]A SD N were used separately for different assay runs; initial radiochemical purity of the older lot (No. 36153054, 770 days) was not reported

.

• The s tabilities of the 4

- OH A SD N positive control and benfluralin stock solutions were not verified analytically

.

• The maximum stor age period for the human recombinant microsomes used in the present study was not reported.

• Overall mean and SD values were not reported for the 4- OH A SD N percent aromatase activity from the individual assay runs (or the average curve), and SEM and %CV val ues were not reported for 4- OH A SD N or benfluralin percent aromatase activity. These values were calculated by the Reviewer and reported in Table 7 of the DER.

# DATA EVALUATION RECORD

BENFLURALIN

Study Type: O CSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614904)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Dynamac Corporations

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Michelle Sharpe

- Kass, M.S. Date: 11/14 /201 1

Secondary Reviewer Signature:
David A. McEwen, B.S. Date: 11/15 /201 1

Program Manager: Signature: Jack D. Early, M.S. Date: 11/16 /201 1

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 11/16 /201 1

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel

.

## 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).Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) I Page 1 of 14

BENFLURALIN (BENEFIN)/084301



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

Health Effects Division Secondary Reviewer: Greg Akerman, Ph

. D.

Health Effects Division

DAT A EVALUATION RECORD OCSPP 890.1250/ OECD None

Template version 08/2011



## STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC);

OCSPP 890.1250

PC CODE: 084301 DP BARCODE: 0399309

TXR#: 0054123 CAS No.: 1861-40-1

TEST MATERIAL (PURITY): Benefin (97.5%)

SYNONYMS: Benfluralin; N-Butyl-N-ethyl-2,6-dinitro-4-(trifluoromethyl)benzenamine

CITATION: LeBaron, M.J., Schisler, M

. R., Visconti, N.R. (2011). Evaluation ofBenefin in

an in vitro Estrogen Receptor Binding Assay. Toxicology & 

## Environmental

Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory Project ID: 111123, October 25, 2011. MRID 48614904. Unpublished.

SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Rd, Indianapolis, IN 46268

TEST ORDER#: EDSP-084301-15

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48614904),

uterine cytosol from Sprague-Dawley rats was used as the source of estrogen receptors (ER) to

evaluate the potential for benfluralin to displace the bound reference estrogen, radiolabeled [3H]-**17~-estradiol** 

## from the ER. A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the ER in the uterine cytosol was present in adequate numbers and functioning with the appropriate affinity for the radiolabeled ligand.

Saturation binding data were not presented in the study report; however, saturation binding data

was presented in the study profile submitted separately by the test order recipient (study profile,

MRID 48675404).

The competitive binding experiment was conducted to measure the binding of a single

concentration of [3H]-l**7~-estradiol** 

## ( 1 nM) in the presence of increasing concentrations of

benfluralin (logarithmic increase from 10- 10 

## to 10- 3 

## M). Ethanol was used as a vehicle at a final

concentration of < 3%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and**17-~-estradiol** 

## as the natural ligand reference

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

In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100

μg protein and the dissociation constant (Kct) was 0.1032 nM. The Bmax fell within the expectedEstrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 2 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## 0.03 to 1.5 nM. Nonspecific binding as a percent of total binding was 1.7%-8.6% across the entire concentration range in the saturation binding assay. Only a single run of the saturation binding e xperiment was conducted on the batch of cytosol preparation used for the c ompetitive binding e xperiment.

For the c ompetitive binding experiment, all performance criteria were met for appropriate responses by 17 β -estradiol and 19-norethindrone. T he l og IC 50 

## and RBA could not be

determined for benfluralin as it did not displace ≥ 50% for the radiolabeled estradiol from the ER at any concentration . The bottom of the curve for percent of total binding at the 95% confidence interval for benfluralin was 87.7%, 70.2%, and 75.9% in Runs 1, 2, and 3, respectively.

Based on the results f rom the three runs, benfluralin is classified as Not I nteractive in th e Estrogen R eceptor Binding A ssay

.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE : Signed and dated GLP Compliance and Quality Assurance statements were provided.Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 3 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Toxicology & Environmental Research and Consulting

Location: Dow Chemical Company, Midland MI Study Director: M. R. Schisler, B.S

Other Personnel: M. J. LeBarons, Ph.D., Lead Scientist; M.R. Visconti, M.S, Research Biologist. Study Period: September 6, 2011

– October 25

, 2011



## 2

. Test substance : Benfluralin

Description: Technical Grade, Yellow Solid

Source: Dow AgroSciences, LLC, Indianapolis, IN

Lot/Batch #: Lot# ACD13683, TSN100037 Purity: 97.5 % Solubility : Ethanol up to 30 mM; less than 0.001 g/L in water Volatility : Vapor Pressure 2. 8 mmHg Stability: 4 years shelf life Storage conditions: Ambient

CAS #: 1861

- 40

- 1 Molecular weight: 335.3

Structure:



## 3

. Non

- labeled ligand : 17 β

- estradiol

Supplier : Sigma, St. Louis, MO Catalog # Not Reported

Batch #: 098K1372 Purity: 100%

CAS #: 50

- 28

- 2



## 4

. Radioactive ligand : [ 3 

## H]

- 17 β

- estradiol

Supplier : Perkin

- Elmer, Boston, MA Catalog #: NET517001MC

Batch #: 639068 Radiochemical p urity: >97 % Specific activity : Not Reported

Concentration of stock : 162.9 Ci/mmol *Information on adjusted specific activity was not available.



## 5

. Positive control : 19

- Noreth indrone

Supplier: Sigma, St. Louis, MO Catalog # Not Reported

Batch #: 030M1359 Purity: 99%

CAS # : 68

- 22

- 4



## 6

. Negative control : Octyltriethoxysilane

Supplier: Sigma, St. Louis, MO Catalog # Not Reported

Batch #: 72596AMV Purity: 98.58%

CAS # : 2943

- 75

- 1Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 4 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## 7

. Solvent/vehicle control : Ethanol

Justification for choice of

solvent : Not reported; The reviewer assumes it is due to low solubility in water (<0.001 g/L)

Final Concentration : < 3 %



## B. METHODS

1. Preparation of R at U terine Cytosol (RUC) : Trimmed uterine tissue from an unreported number of female Sprague-Dawley rats was purchased from Charles River (Wilmington, MA). Animals were 85 -100 days old at the time of euthanasia, and rats had been ovariectomized approximately 7 -10 days prior to tissue harvest. The tissues were stored at -80°C and used within 6 months of collection. The study authors did not report weighing the uteri, but tissues were homogenized in ice

- cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer and centrifuged for 10 min at 2500 × g at 4oC. Supernatant was transferred and centrifuged for 60 minutes at 105,000 × g , discarding the resulting pellets. Protein c oncentration of the cytosol was determined to be 3.573 mg/mL using the Pierce BCA method (Thermo Scientific Pierce Research Lab, Rockford, IL)

. Cytosol was divided into aliquots (volume not reported ) and stored at -80 ° C for up to 90 days

until use.

2. Sa turation (radioligand) B inding E xperiment : A saturation binding e xperiment was conducted to measure total and non-specific binding of [ 3 

## H]

- 17β

- estradiol to demonstrate that the ER in the cytosolic preparation was present in reasonable numbers and had the appropriate affinity for the native ligand. A summary of the conditions for the saturation binding e xperiment are presented in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a


### Source of receptor Rat uterine cytosol
Concentration of radioligand (as serial dilutions) 0.03

- 3.0 nM

Concentration of non

- labeled ligand (100X [radioligand]) 3.0

- 300 nM

Concentration of receptor Sufficient to bind 40.77% of radioligand
at 0.03 nM b



### Temperature ~2

- 8 oC

Incubation time ~16 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 D ata were not included in the study report, but are reported in the study profile, submitted separately (MRID 48675404).

b T his value was slightly higher than the suggested range in the guideline; however, all other values, including minimal ligand depletion, indica ted acceptable performance in the assay.



## The specific activity of the stock solution [3H]- 17β -estradiol was not adjusted for decay over time at the time of the experiment. Serial dilutions in TEDG + PMSF buffer were prepared to

achieve the final concentra tions of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of nonlabeled 17β

- estradiol were prepared in a similar manner to achieve concentrations that were 100

-Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 5 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## fold greater than each respective radiolabeled concentration to result in final concentr ations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. The optimal protein concentration was determined to be 0.1191 mg protein/assay tube, which resulted in the binding of 40.77% of the total radioactivity added. This value was slightly higher than the recommended range in the guideline. Cyto solic protein used in this assay was thawed fresh for this experiment at ~4°C and maintained at ~4°C during the binding assay. Each run contained three concurrent replicates at each concentration, resulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment 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 [3H]

- 17β

- estradiol (8 serial dilutions) e



### --- 50 μL

--- Non

- labeled 17β

- estradiol (8 serial dilutions, 100x each respective labeled concentration) f



### 100 μL 100 μL

--- Uterine cytosol (diluted to appropriate conc.)

500 μL 500 μL 50 μL Total volume in each assay tube

a D ata were not included in the study report, but are reported in the study profile, submitted separately (MRID 48675404).

b T otal binding = [3H]

- 17β

- estradiol bound to ER.

c N on

- specific binding = [3H]

- 17β

- estradiol and 100

- fold greater non

- labeled bound to ER.

d T otal [3H]

- 17β

- estradiol alone for dpm determination at each concentration.

e F inal concentrations of [3H]

- 17β

- estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f F inal concentrations of non

- labeled 17β

- estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



## 3. Competitive B inding E xperiment : A summary of the experiment conditions for the Competitive Bind ing Experiment is presented in Table 3.

TABLE 3 . Summary of Conditions for Competitive Binding Experiment a



### Source of receptor Rat U terine C ytosol

Concentration of radioligand 1 nM

Concentration of receptor Sufficient to bind 5.66

- 7.15 % of radioligand

Concentration of test substance (as serial dilutions) 10

- 10 

### to 10

- 3 

### M

Temperature 4

- 8 ° C

Incubation time 16

- 20 hours
Composition of assay buffer Tris 10 mM (pH not reported )

EDTA 1.5 mM

Glycerol 10%

Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM

a Data were obtained from page s 17

, 22

- 23 , 41, 43 and 45 of the study report.



## The solubility of benfluralin and each positive and negative control chemical in ethanol was reported. On the day of the assay, the specific activity of the stock solution [ 3 

## H] -17 β

estradiol was not reported, but the study authors stated that it was ad justed for decay overEstrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 6 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## time. [ 3 

## H] -17 β

- estradiol was diluted in TEDG + PMSF buffer to achieve a final

concentration of 50 nM.

Serial dilutions of the test substance, positive control (19-norethindrone), negative control (octyltriethoxysilane), and reference material ( non- labeled 17β

- estradiol) were prepared to

ach ieve the concentrations shown in Table 4. E ach assay co nsisted of three runs, and each

run contained three replicates at each co ncentration, resulting in a total of 306 samples.

TABLE 4

. Molar (M) concentrations in Co mpetitive Binding Assay Run a , b



### Benfluralin Positive control Negative control Reference Chemical

19

- Norethindrone Octyltriethoxysilane Non

- labeled 17β

- estradiol

Tubes 4

- 24 and 100

- 102 c 

### Tubes 25

- 48 c 

### Tubes 49

- 72 c 

### Tubes 1

- 3, 7 3

- 96 , and

97

- 99 c



### 10

- 10 

### 3.16 x 10

- 9 

### 10

- 10 

### Solvent control d



### 10

- 9 

### 3.16x10

- 8 

### 10

- 9 

### 10

- 11



### 10

- 8 

### 10

- 7 

### 10

- 8 

### 10

- 10



### 10

- 7 

### 3.16 x 10

- 7 

### 10

- 7 

### 3.16 x 10

- 10



### 10

- 6 

### 10

- 6 

### 10

- 6 

### 10

- 9



### 10

- 5 

### 3.16 x 10

- 6 

### 10

- 5 

### 3.16 x 10

- 9



### 10

- 4 

### 3.16 x 10

- 5 

### 10

- 4 

### 10

- 8



### 10

- 3 

### 10

- 4 

### 10

- 3 

### 10

- 7



### a Data were obtained from page s 19

- 21 and 41

- 46 of the study report.

b Each tube contains: 10μL of the test substance, positive control, negative control, solvent control, or non

labeled 17β

- estradiol; 390 μL of TEDG + PMSF buffer with [ 3 

### H]

- 17β

- estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL.

c Each concentration of each chemical was run in triplicate, for a total of 102 tubes per run.

d Solvent was ethanol (<3%) in Tubes 1

- 3 and 97

- 99.



## Tubes were incubated with gentle vortexing for 16-20 hours at 4-8 ° C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed 4

times at five -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 discarded. 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 temperature was maintained at 4 -8oC throughout the assay prior

to extraction with ethanol.

C. D ATA ANALYSIS : For the c ompetitive binding a ssay , total binding and non- specific binding data were modeled via non-linear regression using Graph Pad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA). This software was used to fit a curve (for 17β -estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated log

IC 50 

## as a parameter to be estimated. For the c ompetitive 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 [software not reported].Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 7 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## 1. Definitions

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

three runs. Each run wa s first individually classified a s follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estradiol has been displaced from the ER).

Not interactive = there are usable data points at or above 10 -6 

## M and either the lowest point on the fitted response curve within the range of the data is above 75 % (i.e.,

<25 % of the radiolabeled estradiol has been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

Equivocal up to the limit of concentrations tested = there are no data points at or above a

test chemical concentration of 10 -6 

## M and either a binding curve can be fit but ≤50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be fit and the lowest average percent binding among concentration groups in the data is >50%.

Equivocal = A run is classified as equivocal if it does n ot fall into any of the categories

above.

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

Run Classification Numerical Value

Interactive 2 Equivocal 1

Not interactive 0 Equivocal up to the limit of concentrations tested “missing”

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

Test Material Classification Numerical Range

Interactive average ≥1.5 Equivocal 0.5 ≥ average <1.5

Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”

b. Descriptors for receptor binding :

B max 

## : maximum specific binding number (not provided)

K d 

## : dissociation constant (not provided)

IC 50 

## : concentration of the test substance at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity ( RBA % ): IC 50 

## of 17 β

- estradiol ÷ IC 50 

## of test substance × 100Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 8 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## II. RESULTS

A. SATURATION BINDING EXPERIMENT : Saturation binding e xperiment parameters are presented in Table 5. The K d 

## for [ 3 

## H]

- 17β -estradiol was 0.1032 nM and the B
max 

## (nM) was 0.07097 for the prepared rat uterine cytosol used in these experiments. The K d 

## for the run was within the expected range of 0.03 to 1.5 nM. The B
max 

## was also within the expected range of 10

- 150 fmol/100 μg protein. The data produced a linear Scatchard plot.

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) 0.9617 N/A N/A N/A

B
max 

### (nM) 0.07097 N/A N/A N/A

B
max 

### (fmol/100 μg protein) 59.28 N/A N/A N/A

K d 

### (nM) 0.1032 N/A N/A N/A

a 

### Data were not includ ed in the study re por t, but are report ed in the study profile, submitted separately (MRID 48675404).

b 

### Only a sing le ru n of the satur ati on binding experiment was condu cted on the b atch of cytosol us ed for this

comp etit ive binding experiment.

r 2 

### G ood ness of f it

for cu rv e calc u lated for sp eci f ic binding



## Specific, no n-specific, and total bindi ng curves for [3H]- 17β -e st ra diol to t he estr og en re ce pto r are presented in Figure 1 below

. The sp ec ific binding re ac hed a pla te au, a nd non-sp ec if ic binding w a s le ss than 20% of to ta l bindi ng at all concent ra tions (ra nge 1.7%-8.6 %) . Figure 2 is

a S ca tch ar d plot th at illus t ra tes the bindi ng of [ 3 

## H]- 17β -est ra diol to the estrogen re ceptor. T he

da ta f

it re sults in a lin ea r plot.

F IGURE 1. Bi nd i ng of [3H]- 17β -est ra diol to t he ER during t he Satu ra tion Bi nd i ng Exp er im ent.Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 9 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## F IGURE 2. Scatchard Plot of the Binding of [3H]- 17β -est ra diol to t he ER during the Saturation Binding Experiment.

B. COMPETITIVE BINDING EXPERIMENT : The non-specific binding was less than 4.1% in all assays, and was calculated by reviewers for each assay by dividing the average ethanol concentration (minus background) by the average non-specific binding (on pages 41-45 of the study report) and multiplying by 100%. Non-specific binding was considered

well below the recommended maximum limit of 50%. Ligand depletion wa s stated in the study report data tables on pages 41, 43, and 45 and was ≤7.15%. This was within the

recommended tolerance limit of 15%.

The results from the 3 c ompetitive binding experiment s are summarized in Table 6 and shown graphically in Figures 3-6 . The estimated mean log IC 50 

## for the test material could not be determined as it did result in 50% displacement of radiolabeled estradiol a t any
concentration. The mean log IC 50 

## for 17 β

- estradiol was

− 8.934 M and the positive control

was

− 5.442 M. The mean RBA was 100 % for the positive control, but could not be calculated for benfluralin. Confidence in these numbers is high due to the small variation. Repeat runs were unnecessary.

The bottom of the curve for percent of total binding at the 95% confidence interval for benfluralin was 87.7%, 70.2%, and 75.9% in runs 1, 2, and 3, respectively. Using the guideline classification criteria, the runs were classified as not interactive (0), equivocal (1) and not interactive (0), respectively. Based on an average of all three runs, benfluralin is classified as not interactive in this assay (Table 7).Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 10 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



### TABLE 6 . Competitive Binding Assay of Benfluralin with Estrogen Receptor from Rat Uterine Cytosol a



### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean c



### r 2 

### (unweighted), 17 β

- estradiol 0.9989 0.9990 0.9997 0.9989

- 0.9997

Positive control 0.9983 0.9999 0.9997 0.9983

- 0.9997

Benfluralin 0.9433 0.9835 0.9708 NA Log IC 50 

### (M), 17 β

- estradiol

- 8.913

- 8.948

- 8.934

- 8.934

Positive control

- 5.431

- 5.462

- 5.435

- 5.442

Benfluralin NA NA NA NA

IC 50 

### (M), 17 β

- estradiol 1.223 x 10

- 9 

### 1.127 x 10

- 9 

### 1.142 x 10

- 9 

### 1.165 x 10

- 9



### Positive control 3.710 x 10

- 6 

### 3.448 x 10

- 6 

### 3.673 x 10

- 6 

### 3.612 x 10

- 6



### Benfluralin NA NA NA NA RBA ( as % IC 50 

### ) d 

### , Positive

control 100 100 100 100

Benfluralin NA NA NA NA

a Data were obtained from pages 34 and 35 of the study report.

r 2 

### Goodness of fit

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

c The range of R 2 

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

d Calculated by reviewer: [ IC 50 

### (in M) positive control or benfluralin / IC 50 

### (in M) R1881] x 100% RBA (%) R elative binding affinity

NA Not applicable

TABLE 7

. Binding Classification of Benfluralin with Estrogen Receptor a



### Run 1 2 3 Mean Binding Classification c 

### Classification category value b 

### 0 1 0 0 .3 Not interactive

a Data were obtained from page 35 of the study report.

b Classification category value: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean).

c Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 11 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## F IGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test Compound. Assay #1.

F IGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test Compound. Assay #2.

F IGURE 5. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test Compound. Assay #3.Estrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 12 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



## F IGURE 6. Mean and Standard Error of the Mean for 3 Independent Assays - R elative ER Binding Activity for B enfluralin

.

C. PERFORMANCE CRITERIA : To ensure that the competitive binding assay function ed properly, each run was evaluated using the following criteria in Table 8 :

T ABLE 8. Criterion a 

### Tolerance Limit(s) Value Yes No

17 β

- estradiol fitted curve parameters Log
e 

### residual SD ≤2.35

- 0.1812 X Top (% binding) b 

### 94 to 111 99 X Bottom (% binding)

- 4 to 1

- 0.8 X Hill Slope (log 10 

### (M)

- 1 

### )

- 1.1 to

- 0.7

- 0.99 X

Weak Positive control (norethynodrel) fitted curve parameters Log
e 

### residual SD ` ≤2.60

- 0.2331 X Top (% binding) b 

### 90 to 110 100 X Bottom (% binding)

- 5 to 1

- 0.7 X Hill Slope (log 10 

### (M)

- 1 

### )

- 1.1 to

- 0.7

- 1.013 X

Solvent concentration

Ethanol ≤3% <3% 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% 13.5 c 

### X

a Data were obtained from page 34 of the study report.

b Calculated by reviewer by subtracting the least total binding from 100%.



## Additionally, the curve for the reference material showed that increasing concentrations of unlabeled 17β

- estradiol displaced [ 3 

## H]

- 17β -estradiol in a manner consistent with one- site binding, as indicated by a descent from 91.4% to 10.6% binding over approximately a 100- fold increase in concentration of the test chemical (i.e., covering approximately 2 log units).

Benfluralin was tested over a concentration range that fully defined the top of t he curve. T h e percent binding at th is top plateau 100.4% was within 25 percentage points of the value for

the lowest concentration of the estradiol standard 97.9%. Examination across the runsEstrogen Receptor Binding Assay Using Rat Uterine Cytosol ( 2011 ) / Page 13 of 13 BENFLURALIN (BENEFIN) / 084301 O CSPP 89 0. 1250/ OECD None



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

The percentage of the total specific binding in the solvent controls was approximately 7%.
This was within the less than the ≤ 10 -15% recommended in the guideli ne. Ligand depletion was also minimal. Sufficient optimization of the number of specific binding sites is supported by curves for controls of acceptable steepness, low non-specific binding, and low variability.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Based on the combined responses in each of the three independent estrogen receptor binding assays, t he i nvestigators concluded that benfluralin had no appreciable effect on the binding of 17 β

- estradio l at any concentration up

to 10 -3 

## M. The results of the in vitro estrogen receptor binding assay using rat uterine cytosol indicate that, under the conditions of this s tudy benfluralin was negative for estrogen receptor binding up to 10 -4 

## M.

B. AGENCY COMMENTS: The results of the saturation binding experiment were not reported in the study report; however, summary saturation data including graphs showing specific, no n-specific, and total bindi ng curves for [ 3 

## H]- 17β -e st ra diol to the estrogen re ce pto r were reported in a study profile (MRID 48675401). The maximum binding capacity (B
max 

## ) was 59.28 fmol/100 μg protein and the dissociation constant (K d 

## ) was 0.1032 nM. The B
max 

## fell within the expected range of 10 to 150 fmol/100 μg protein and the K d 

## for the run was within the exp ected range of 0.03 to 1.5 nM. Only one saturation binding run was performed instead of the recommended three runs. For the c ompetitive binding experiment,

the l og IC 50 

## and RBA could not be calculated for benfluralin as it did result in 50% displacement of radiolabeled estradiol from the ER at any concentration. All performance criteria were met for appropriate responses by unlabeled 17β -estradiol and 19-norethindrone. The bottom of the curve for percent of total binding at the 95% confidence interval for benfluralin was 87.7%, 70.2%, and 75.9% in assays 1, 2, and 3, respectively. Based on

results of all three runs, benfluralin is classified as not interactive in this assay

.

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

• Summary saturation binding data were provided in the submitted study profile (MRID 48675404); however, individual saturation data were not provided for independent

verification.

• Only a single run of the saturation binding experiment was performed on the cytosol preparation used for the competitive binding experiment instead of the recommended three runs/cytosol prepar ation s.

# DATA EVALUATION RECORD

BENFLURALIN (BENEFIN)

Study Type: OPPTS 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614905)

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,

Buildin g 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Michelle Sharpe

- Kass , M.S. Date: 11/ 09 /11

Secondary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 11/ 1 1 /11

Program Manager: Signature: Jack D. Early, M.S. Date: 11/ 1 7 /11

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 11/ 1 7 /11

Disclaimer

This Data Evaluation Record may have been altered by the Endocrine Disruptor Review Team 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 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 a cross 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 det ermine 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).Estrogen Receptor Transcriptional Activation (2009) I Page I of9

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ OECD 455



## Primary Reviewer: Sheila Healy, Ph.D. 

## Sign't ·

# ~L.1-+-1~6!!11i:f...-~- Health Effects Division Date:

_**__.:.~~~L:=..-...,,..-** 

## Secondary Reviewer: Minerva Mercado-Feliciano, Ph

. D. DABT**Signatur t:** **'-:?~:Z:: "'="'¥=** **=-** 

## Health Effects Division Date: 

## h-M- IS

... Template 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: 084301 

## DP BARCODE: D399309



## TXR#: 0054123 

## CAS No.: 1861-40-1



## TEST MATERIAL (PURITY): Benefin (97.5% a.i.)



## SYNONYMS: Benfluralin, N-Butyl-N-ethyl-2,6-dinitro-4-(trifluoromethyl)benzenamine



## CITATION: LeBaron, M.J

. ; Kan, H.L. (2011) Evaluation ofBenefin in an in vitro Estrogen Receptor Transcriptional Activation Assay in Human Cell Line HeLa-9903. Toxicology & 

## Environmental Research and Consulting

, Dow Chemical Company,
Midland, MI. Laboratory Project ID: 101184, October 20

, 2011. MRID

48614905. Unpublished.



## SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, 

## IN

TEST ORDER#: EDSP-084301-15



## EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID
48614905) conducted by Toxicology & 

## Environmental Research and Consulting, hERa-HeLa9903 cells cultured in vitro were exposed to benfluralin (97

. 5% purity Lot# ACD13683,

TSN100037 at concentrations of 10

- 4

, 

## 10

- s
, 10

- 6

, 

## 10

- 7

, 

## 10

- 8

, 

## 10

- 9 

## and 10

- 10 

## Min DMSO (final

concentration 0.1 %) for 24 hours. The experiments were performed using 96-well plates and

each benfluralin concentration was tested in triplicate (3 wells / plate) in each experiment. Cells

were exposed to the test substance for approximately 24 hours to induce reporter (luciferase)

gene products. Luciferase expression in response to activation of the estrogen receptor was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity.

Benfluralin was tested up to the limits of both cytotoxicity and solubility ; 10

- 3 5 

## M was both cytotoxic to the cells (79.1 % viability compared to controls) and precipitated in the cell culture

media at the end of the 24-hour treatment duration. The positive control and reference chemicals

induced the appropriate responses
, although the assay's responsiveness to 17a-methyltestosterone

indicated increased sensitivity to weak agonists. The RPCMax (maximum level of response induced by chemical expressed a percentage of the response induced by 1 nM estradiol (E2) on the same plate) was 13.3-19

. 1%. The PCM ax was the limit of solubility and cytotoxicity, 10

- 4 

## M.Estrogen Receptor Transcriptional Activation ( 2009 ) / Page 2 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## Because the RPC Max 

## > PC 10 

## in all three assay runs , benfluralin was considered positive for estrogen receptor transcriptional activation in this test system

.

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

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

statements were provided.Estrogen Receptor Transcriptional Activation ( 2009 ) / Page 3 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance : Benfluralin (Benefin)

Description: Yellow Solid

Source: Dow AgroSciences

Lot/Batch #: Lot# ACD13683, TSN100037 Purity: 97.5 % Solubility : DMSO 0.1M. Water 0.001 g/L Volatility : Not provided Stability: 3.5 years shelf life Storage conditions: Ambient
Vapor pressure : 2.8 mmHg

CAS #: 1861

- 40

- 1

Structure :



## 2. Reference substances 17β

- estradiol (strong estrogen; positive control)

Supplier : Sigma, St. Louis, Missouri Catalogue and Batch #: Cat# E

- 8875, Lot # 079 KI 0131 Purity: 100%

CAS # : 50

- 28

- 2



## 17α

- estradiol (weak estrogen)

Supplier : Sigma, St. Louis, Missouri Catalogue and Batch #: Cat# E

- 8750 Lot# 029 K 4116 Purity: ≥ 99.5%

CAS # : 57

- 91

- 0



## Corticosterone (negative compound)

Supplier : Sigma, St. Louis, Missouri Catalogue and Batch #: Cat# C

- 2505 , Lot# 010M2010 Purity: 99%

CAS # : 50

- 22

- 6



## 17α

- methyltestosterone (very weak agonist)

Supplier : Sigma, St. Louis, Missouri Catalogue and Batch #: Cat# M

- 7252, Lot# 060M1543V Purity: 99%

CAS # : 58

- 18

- 4



## 3. Vehicle(s)

Solvent: DMSO; Sigma

- Aldrich, Cat# 276855, Lot# 68996LMV and 3696DM, Purity ≥99.9%

Solvent control (final conc en tration ): 0.1%Estrogen Receptor Transcriptional Activation ( 2009 ) / Page 4 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## B. METHODS

1. C ell C ulture : Stably

- transfected hERα

- HeLa -9903 cell s were obtained from the Japanes e Collection of Research Bioresources (JCRB) Cell Bank and were verified to be free of mycoplasma infection using DNA Stain or Direct Culturing by ATCC Laboratory . Cells

were maintained in Eagles Minimum Essential Medium (EMEM) without phenol red, supplemented with kanamycin (60 mg/L) and 10% dextran- coated charcoal

- treated fetal bovine serum (DCC- FBS ; Hyclone Laboratories, Inc., Logan, Utah; Lot # DCC

- FBS 062410) , in an incubator under 5% CO 2 

## at 37oC. C ells were subcultured at least twice (% confluency at passage not reported) prior to exposure to the test material , and were used for up to 40 passages. DCC

- FBS was prepared in -house following a protocol identical to that found in Appendix 3 of Guideline 890.1300. No analysis of the presence or absence of estrogenic compounds in the serum was performed.

2. Transcriptional Activation Assay s : For each test, cells were plated in 96- well microplates (Estrogenic activity of the plates was not reported) at a density of 1.0-1.5 × 10 4 

## cells per

100 μL medium/well in a 96

- well plate and allowed to attach for 3 hours. The Growth media was replaced with media containing serial log dilutions of benfluralin in DMSO . Cells were

incubated for 24 hours at approximately 37°C. The final concentration of DMSO was 0.1%

in the medium. Cytotoxicity was determined by a colorimetric modified MTT cell viability

assay

.

Transcriptional activation of the estrogen receptor was determined using a standard Luciferase Assay System (Promega, Madison, WI, Cat # E1501 ). C ells containing luciferase were lysed and luciferase substrate was added. The resulting chemiluminescence was measured immediately after adding luciferas e reagents in a Packard TopCount NXT

luminescence counter . I n a typical in vitro cell culture assay, TopCount has a det ection limit

of ~5 cells in 1000- μL portion of medium.

a. P reliminary T est : A preliminary test evaluating concentrations of benfluralin ranging from

10

− 3.5 

## to 10

− 10 

## M was conducted to determine the appropriate concentration range and to determine concentrations resulting in ins olubility and/or cytotoxicity.

b. Proficiency Chemicals : The proficiency chemicals test w as not provided.

c. Reference Chemicals : To ensure th e stability of the response from the cell line, six concentrations of each of the following reference chemicals were included in each plate in the current assay, along with the test chemical :

Reference Chemical CAS No. Concentration R ange Class 17β

- estradiol 50

- 28

- 2 10

− 14 to 10

− 8 Strong estrogen

17α

- estradiol 57

- 91

- 0 10

− 12 to 10

− 6 Weak estrogen

Corticosterone 50

- 22

- 6 10

− 10 to 10

− 4 Negative compound

17α

- methyltestosterone 58

- 18

- 4 10

− 11 to 10

− 5 Very weak agonist



## 3. Data analysis : To obtain t he relative transcriptional activity to the 1 nM E2 positive control (PC) , the luminescence signals from the concurrent plate were analyzed by subtracting the

mean value of the vehicle control from each well value to norm alize t he data ; eachEstrogen Receptor Transcriptional Activation ( 2009 ) / Page 5 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## normalized value was then divid ed by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to express relative transcriptional activity as a percentage of the PC. Graph Pad Prism v. 5 ( GraphPad Software, In c., La Jolla, CA) was

used to calculate t he EC 50 

## , PC 10 

## , PC 50 

## , RPC Max 

## , and PC Max 

## for b en fluralin when applicable.

T he test material was defined as positive for inducing estrogen receptor transcriptional

activation if the RPC Max 

## ≥ PC 10 

## in at least 2 of 2 (or 2 of 3) runs. LogEC 50 

## and Hill slop e values are calculated only if a positive response is observed. Coefficients of variation (CV)

w ere calculated for the luminescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of insolubili ty were excluded from analyses.

4. Definitions

EC 50 

## = concentration of agonist that induces a response halfway 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 : Initial testing of benfluralin was conducted at 10

− 9 

## to 10

− 3.5 

## M. Cytotoxicity and compound precipitation were observed at 10

− 3.5 

## M. Based on these results,

concentrations of 10

− 4 

## , 10

− 5 

## , 10

− 6 

## , 10

− 7 

## , 10

− 8 

## , 10

− 9 

## and 10

− 10 

## M were selected for the main

ass ay (Table 1)

.

TABLE 1

. Preliminary T est for Solubility , C ytotoxicity , and Concentration

- Selection for Benfluralin a

Concentration (M) % Viability b Comments c

10

− 3 .5 79.1 10

− 3.5 is a cytotoxic dose, cell viability < 80% and cell wells had some precipitation after 24

- hr incubation

10

− 4 112.7

10

− 5 133.2

10

− 6 111.2

10

− 7 114.3

10

− 8 138.0

10

− 9 116.5

10

− 10 NR e

E2 1nM 137.7

VC d 100.0

a Data were obtained from page 48 of the study report.

b If viability is <80%, the concentration is considered cytotoxic.

c Include comments related to solubility issues, if applicable.

d VC = Vehicle control

e NR = Not ReportedEstrogen Receptor Transcriptional Activation ( 2009 ) / Page 6 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## B. Positive and Negative Reference Chemicals

1. Proficiency Chemicals : The responsiveness of cells to the required proficiency chemicals

was not included in this report, although the registrant stated that these non- GLP, unpublished experimental results demonstrated laboratory proficiency and assay validation.

2. Reference C hemicals : Values derived from the concentration response curve ( e.g ., l og PC 50, 

## log PC 10, 

## log EC 50, 

## and H ill slope) for the four concurrently run reference materials are

included in Table 2. The reference compounds were within the acceptable range with the exception of 17 α

- methyltestosterone which had a l og PC 10 

## value lower than the acceptable range for all three runs. This indicates that the assay's responsiveness is more sensitive to weak agonists. Additionally, the Hill slope was lower than the accep table value for 17 α

estradiol in one run.

TABLE 2

. Performance Criteria for Reference Chemicals a

Reference Chemical

Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 4 Yes No
17β

- estradiol Log PC 50 − 11.4 to

− 10.1

− 10.6

− 10.6

- 11.0 X Log PC 10 <

− 11

− 11.9

− 12.4

- 12.9 X Log EC 50 − 11.3 to

− 10.1

− 10.5

− 10.7

- 11.2 X Hill Slope 0.7 to 1.5 1.0 1.4 0.7 X Test range 10

− 14 to 10

− 8 M 10

− 14 to 10

− 8

M 10

− 14 to 10

− 8

M 10

− 14 to 10

− 8

M

17α

- estradiol Log PC 50 − 9.6 to

− 8.1

− 8.7

− 9.0

− 9.4 X Log PC 10 − 10.7 to

− 9.3

− 10.0

− 10.0

− 10.6 X Log EC 50 − 9.6 to

− 8.4

− 8.7

− 8.9

− 9.0 X Hill Slope 0.9 to 2.0 1.0 0.7 1.0 X Test range 10

− 12 to 10

− 6 M 10

− 12 to 10

− 6

M 10

− 12 to 10

− 6

M 10

− 12 to 10

− 6

M

Corticosterone Test range 10

− 10 to 10

− 4 M 10

− 10 to 10

− 4

M 10

− 10 to 10

− 4

M 10

− 10 to 10

− 4

M

17α

- methyltestosterone Log PC 50 −6.0 to −5 .1

− 5.4

− 6.1

− 8.0 X Log PC 10 −8.0 to − 6.2

− 8.2

− 8.8

− 8.9 X Test range 10

− 11 to 10

− 5 M 10

− 11 to 10

− 5

M 10

− 11 to 10

− 5

M 10

− 11 to 10

− 5

M

a Data were obtained from page 49 of the study report

.



## C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls : Data for the vehicle and positive controls are included in

Table 3.Estrogen Receptor Transcriptional Activation ( 2009 ) / Page 7 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



### TABLE 3

. Transcriptional Activation (TA) Response of Vehicle and Positive Control a



### Sample Vehicle Control Positive Control b



### Runs Mean SD Mean SD Fold Induction c



### 1 1539.6 244.4 11729.0 1251.0 7.6

2 3395.4 598.3 19724.6 3250.5 5.8

4 d 

### 2323.4 261.9 14 4 43.2 1361.8 6.2

a Data were obtained from page 50 of the study report.

b Positive control was 17β

- estradiol (E2) at 1 nM.

c Fold

- induction = (mean TA of PC)/(mean TA of VC)

d Data from run #3 was not analyzed due to insufficient positive control induction, therefor e the assay was repeated.



## 2. Test Material : Relative (to the PC) transcriptional activation at each concentration of the test chemical during the 3 assay runs is presented in Table 4. C oncentration

- response curve s depicting fold induction of relative transcriptional acti vation are presented in Figure 1 below.

The RPC M ax 

## was 13.3-19.1 %, and the associated PC M ax 

## was the limit of solubility and cytotoxicity, 10

− 4 

## M for all three runs . Because the RPC Max 

## ≥ PC 10 

## in all three runs, b enfluralin was considered positive for estrogen receptor transcriptional activation.

The PC 50 

## could not be determined.

TABLE 4 . Relative Transcriptional Activation (RTA) of Benfluralin a



### Parameter RTA (mean ± SD); % of Positive Control (PC)

Run 1 Run 2 Run 4 c


### Conc. (M) Mean SD Mean SD Mean SD

10

− 3.5 

### 23.0 2.8 NR NR NR NR

10

− 4 

### 19.1 0.3 18.8 12.2 13.3 2.1

10

− 5 

### 7.2 1.4 14.3 2.9 11.7 7.3

10

− 6 

### 0.1 3.9 5.4 2.6 5.0 5.4

10

− 7



### − 4.8 1.5

− 1.3 4.4

− 2.5 4.3

10

− 8



### − 2.8 3.2

− 2.9 3.8

− 2.3 6.8

10

− 9 

### 1.3 3.3 0.4 4.1

− 5.8 3.6

10

− 10 

### NR NR 1.3 1.4 0.3 1.3

Log EC 50 b



### − 4.7

− 5.6

− 6.0

Hill Slope b 

### 1.1 1.1 1.3

RPC M ax 

### 19.1% 18.8% 13.3%

PC M ax 

### 10

− 4 

### 10

− 4 

### 10

− 4



### PC 50 

### NA NA NA

PC 10 

### 10

− 4.8 

### 10

− 5.5 

### 10

− 5.3

a Data were obtained from page 51 of the study report.

b Report if appropriate

c Data from run #3 was not analyzed due to insufficient positive control induction, therefor e the assay was repeated. NR = Not Reported NA= Not ApplicableEstrogen Receptor Transcriptional Activation ( 2009 ) / Page 8 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of Benfluralin compared to the Positive Control.

VC= Vehicle Control PC= Positive Control (1 nM E2)



## 3. Performance C riteria : The laboratory proficiency assay data with the required reference compounds were not included in the study report and were not available to the reviewer. The

concurrent reference chemicals 17β

- estradiol and 17α

- estradiol included in each experimentEstrogen Receptor Transcriptional Activation ( 2009 ) / Page 9 of 9

BENFLU RALIN/PC Code 084301 O CSPP 89 0.1300 / OECD 455



## fell within the acceptable range (Table 3). However

, the PC 50 

## and PC 10 

## for

17α

- methyltestosterone were consistently lower than the acceptable range. This indicates an increase in the test sensitivity which may result in a false positive reaction for the test

chemical. Triplicate samples of the positive control (PC, 1 nM E2) and the vehicle control (VC, 0. 1% DMSO) were included in each plate, and the mean lucifera s e activity of the positive control was greater than 4-fold that of the mean vehicle control on each plate. PC 10 

## values of concurrent PC w ere greater than 1+2 standard deviations of the concurrent VC

. Variability was minimal and the results were reproducible among runs.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS ’ CONCLUSIONS : At the two highest concentrations of benf luralin (10

− 5 

## and 10

− 4 

## M), there was a reproducible increase in estrogen receptor

- mediated transcriptional activity, as determined by the guideline -criterion of at least 10% of the response of the positive control. The RPC M ax 

## induced by benfluralin was 13.3-19.1% of the response induced by the positive control. The results of the in vitro estrogen receptor transcriptional activation study indicate that, under the conditions of the study benfluralin had a weak effect in increasing estrogen receptor

- mediated transactivation.

B. AGENCY COMMENTS : Benfluralin was tested up to the limit of solubility and cytotoxicity, both of which occurred in the highest dose of 10

− 3.5 

## M in the first run of the assay. Precipitation occurred at the end of the 24-hour exposure, which may be due to the change in pH of the media due to respiration of the cells. In each of the three reported runs,

the RPC M ax 

## for benfluralin was greater than 10% of the response of the positive control (13.3-19.1%) indicating a positive result. The PC 10 

## was 10

− 5.3 

## to 10

− 4.8 

## M, but the activity of benfluralin was not strong enough to allow the calculation of a PC 50



## . It is pertinent to note

that increased sensitivity to a known weak agonist of the estrogen receptor, 17 α

methyltestosterone , may result in a false positive response.

C. STUDY DEFICIENCIES : The following deficiencies were noted:

• The proficiency test results were not provided.

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

EPA MRID Number 48614906

Page 1 of 91 

### Data Requirement: EPA DP Barcode D399308

OECD Data Point

EPA MRID 48614906

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Benefin TGAI Purity: 97.5%

Common name: Benfluralin

Chemical name: IUPAC: N-butyl-N-ethyl- Į

, Į

, Į -trifluoro-2,6-dinitro-p-toluidine.

CAS name: N-Butyl-N-ethyl-2,6-dinitro-4-(trifluoromethyl)benzenamine.

CAS No.: 1861-40-1

Synonyms:

EPA PC Code: 084301

Primary Reviewer: Joan Gaidos Signature:

Senior Scientist, Cambridge Environmental, Inc. Date: 04/23/2012

Secondary Reviewer: Teri S. Myers Signature:

Senior Scientist, Cambridge Environmental, Inc Date: 05/30/2012

Primary Reviewer: Patience Browne Signature:

USEPA/OCSPP/OSCP Date: 06/12/2012

Additional Reviewer: Amy Blankinship Signature:

USEPA/OCSPP/OPP/EFED/ERB3 Date:

Final Additional Reviewer: Robin Sternberg Signature:

USEPA/OCSPP/OPP/EFED/ERB1 Date: 05/28/2015

Date Evaluation Completed: 05/28/2015 Digitally signed by ROBIN STERNBERG
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN
STERNBERG, dnQualifier=0000039126

Date: 2015.06.03 15:25:44 -04'00' Digitally signed by PATIENCE BROWNE DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff,
cn=PATIENCE BROWNE, dnQualifier=0000048202

Date: 2015.06.03 16:19:26 -04'00'



### AMY

BLANKINSHIP Digitally signed by AMY BLANKINSHIP
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY
BLANKINSHIP, dnQualifier=0000040917

Date: 2015.06.05 10:07:03 -04'00'

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

EPA MRID Number 48614906

Page 2 of 91 

### CITATION: Marino, T.A., K.K. Coady, R.R. Hukkanen, D.W. Louch, N.A. Malowinski, L.K. Sosinski. 2011.

Benefin: A Fish Short-Term Reproduction Assay with the Fathead Minnow, Pimephales promelas

.
Unpublished study performed by Toxicology and Environmental Research and Consulting, Midland,

Michigan. Lab Study No.: 101165. Study sponsored by Dow AgroSciences LLC, Indianapolis, Indiana.

Study completed September 22, 2011; revision completed October 17, 2011.

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

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

the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within

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

should not be considered in isolation but rather should be considered in the context of other assays in the

battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has

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

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

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

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

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

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

EPA MRID Number 48614906

Page 3 of 91 

### EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of benfluralin with fathead minnows ( Pimephales promelas ) was

conducted under flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each

group; 1 group/replicate and 4 replicates/control or treatment ; ca . 6 months old) were exposed to negative

and solvent (0.05 mL/L dimethylformamide; DMF) controls and benfluralin (97.5% purity) at nominal

concentrations of 0.00033, 0.0033, and 0.033 mg a.i./L; time-weighted average (TWA), measured

concentrations were <0.0000505 (<LOQ; controls), 0.000302, 0.00314, and 0.0365 mg a.i./L. The test

system was maintained at 24.3 to 25.5 o 

### C and a pH of 7.5 to 7.8.

Unless otherwise indicated in this DER, all effects are reported based on comparison to the negative control.

Overt toxicity ( e.g. , mortality, lethargy, loss of equilibrium, bloating) was observed among fish exposed to the

high treatment level with  38% of the fish affected; therefore, this treatment group was removed from the study

and excluded from any subsequent statistical analyses. In the low and mid treatment levels, no effect on

mortality was observed; overall mortality was 4.2% in the negative and solvent controls and low treatment

group and 0% in the mid treatment group. No treatment-related effects on secondary sex characteristics or

clinical signs ( i.e. , behavioral and other sublethal effects) were observed; however, one female in the mid

treatment group was observed to have slight vertical banding present. Benfluralin exposure was not associated

with any significant changes (p>0.05) in body weight or length in males or females relative to the negative

control.

With the exception of one negative control replicate and two solvent control replicates, spawning occurred every

4 days. Fecundity in the negative and solvent controls was 30.7 and 18.8 eggs/female/day/replicate,

respectively (range for negative control: 13.2-49.0; range for solvent control: 11.0-26.9). Mean fertilization

success in the negative and solvent controls was >99%. No significant differences (p>0.05) in fecundity or

fertilization success were observed in the benfluralin treatment groups relative to the negative control. Plasma

vitellogenin (VTG) was not significantly different (p>0.05) in males or females compared to the negative

control. Gonado-somatic index (GSI) was significantly increased (p<0.05) by 27% in males at the mid

treatment level compared to the negative control; there were no significant differences (p>0.05) in GSI in

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

EPA MRID Number 48614906

Page 4 of 91 

### females compared to the negative control. Male nuptial tubercle score was not significantly different (p>0.05)

relative to the negative control; no tubercles were observed in females.

No treatment-related effects on gonadal histopathology were observed in males. The incidences of grade 4

(severe) oocyte atresia (9 or more atretic oocytes in a section) were slightly higher in the solvent control

group (n=6) and the low treatment group (n=7) relative to the negative control and the mid treatment group

(n=0 for both). However, the reason for this increase was not clear and was deemed not to be treatment

related. Five of the six reported incidences of severe atresia in the solvent control group occurred in the two

replicates in which a decrease in fecundity was observed, and all four females in the low treatment replicate

that produced no eggs also had severe oocyte atresia.

While not statistically significant (p>0.05), mean fecundity in the solvent control group was reduced by 39%

compared to the negative control. Therefore, there is uncertainty as to whether there were solvent effects in

the study. Two of the four replicates in the solvent control had fecundity rates of 11 eggs/female/day, and

these replicates did not spawn every four days; the fecundity rates for these two replicates seven days prior

to the exposure period were 27.3 and 20.6 eggs/female/day, suggesting adequate spawning prior to test

initiation. Fecundity in one replicate of the negative control was also reduced compared to the pre-exposure

period, and the replicate did not spawn every four days (13.2 eggs/female/day in the 21-day exposure period

versus 31.1 eggs/female/day in the 7 days prior to the exposure period). Although the observed decline in

these replicates may be due to biological variation, the possibility of a solvent interference cannot be

conclusively ruled out. One replicate in the low treatment group did not produce any eggs during the exposure

period. While not statistically different (p>0.05) from the negative control, decreases of 29 and 21% in fecundity

in the low and mid concentration groups, respectively, were of a similar magnitude as that for the solvent

control. Therefore, the results of the study do not suggest a significant treatment-related effect on fecundity.

The study met all of the validity and performance criteria with the exception that: 1) the mean coefficients of

variation (CV) of measured concentrations for the low and mid treatment levels were 43.4 and 27.1%,

respectively, exceeding the guideline criterion of <20%; and 2) not all replicates in the control groups (one in

negative control and two in the solvent control) produced  15 eggs/female/reproductive day or spawned at

least every four days. These deviations did not impact the interpretation of the study.

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

EPA MRID Number 48614906

Page 5 of 91 

### This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay

(OCSPP Guideline 890.1350).

Results Synopsis

Test organism age at test initiation: ~6 months

Mean body weight at test initiation: 3.8 g for males, 1.8 g for females

Mean length at test initiation: Not measured

Test type: Flow-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page6of91Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Benfluralin.Treatment(mg a.i./L)[TWAmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.000302NoNoNoNoNoNoNoNoNoNoNANANANA0.00314NoNoNoNoYesNoNoNoNoNoNANANANA0.03653NRNRNRNRNRNRNRNRNRNRNRNRNRNRAbbreviations:E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.NRNot reported.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’sAnalysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.3The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequent analysis due to overt signs oftoxicity.

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

EPA MRID Number 48614906

Page 7 of 91 

### I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350:

“Fish Short-Term Reproductive Assay”; and OECD 229 (2009). The

following deviations were noted:

1. In one negative control and two solvent control replicates, fecundity (13.2, 11.1, and 11.0

eggs/female/reproductive day, respectively) and frequency of spawning did not meet performance

criteria ( ca . 15 eggs/female/reproductive day and at least every 4 days, respectively).

2. The mean CV of the measured concentrations for the two lowest treatments were 43.4 and 27.1%,

respectively, exceeding the guideline criterion of <20%. This variability reflects the expected loss of

test substance in the water column primarily during the first week of the study.

3. The total organic carbon in the test water was not reported.

4. Unionized ammonia and residual chlorine levels were reportedly not detected, but the LODs for these

parameters were higher than the OCSPP 890.1350 guideline-recommended maximum levels, so it is

unclear if these performance criteria were met.

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

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims

statements were provided. This study was conducted in accordance with GLP

Standards as published by the U.S. EPA (40 CFR Parts 160), and OECD

Principles of GLP [ENV/MC/CHEM(98)17].

A. TEST MATERIAL: Benefin (benfluralin) TGAI

Description: Yellowish-orange crystalline solid; Water solubility 0.1 mg/L (at 25°C); Log

Octanol/water of 5.29; Stable under normal storage conditions; Expiration

date not reported; recertification date February 27, 2013.

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

EPA MRID Number 48614906

Page 8 of 91 

### OECD recommends describing water so lubility, melting/boili ng point stability in water and light, pKa,

Pow or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : ACD13683, TSN100037 (Lot No.)

Purity: 97.5%

Impurities: Not reported

Stability of Compound: Analytical verification performed every 2-4 days during the study on test

solutions yielded average recoveries ranging from 83.7 to 123% of TWA

concentrations. At 21 days (test termination), recoveries ranged from 86.1 to

135% of TWA (0.033 mg/L not sampled). DMF-based stock solutions

yielded average recoveries of 85.6 to 91.1% of TWA concentrations. The

relative standard deviation (coefficient of variation) of the measured

concentrations for all of the replicate tanks in the study (specifically in the

two lowest treatments, since the high treatment was terminated on Day 11)

exceeded 20% ranging from 21.9 to 52.5%, reflecting the expected loss of

test substance in the water column primarily during the first week of the

study. Due to the low solubility and adsorptive properties of benfluralin and

anticipated loss of material in the test system, the test solutions were dosed

at 2.5X the target (nominal) concentration. Therefore, the test concentrations

were substantially higher at test initiation and for the first part of the test but

had stabilized near nominal concentrations by Day 11. Due to this decrease

in measured concentrations over time, the CV’s were >20% and timeweighted averages were used to reported measured concentrations during

the study.

Storage Conditions of

Test Chemicals: The test material was stored at 5°C to ambient conditions.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page9of91B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?YesNew England Bioassay, Inc. (Manchester,Connecticut) 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:20 days EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page10of91ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities 0.7% during 20 day acclimationperiod; 0.5% in7 days prior to pre-exposureperiod.Fish did not receive any treatment fordisease during acclimation, pre-exposure orexposureperiods.Moribund fish or fish with clinical signs ofdisease were not used inthe study. EPA recommends that mortality during the 7 days prior to the pre -exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5- 10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the

fish not be used.Type of food: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:Brine Shrimp Direct(Ogden, Utah)Frequency of feeding:2 times/dayDetails on feeding:Equal measured portions of shrimp were fedto each tank.Food was withheld for atleast 12 hours prior to treatment terminationto aid in histological processing.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page11of91Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca. 6 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):3.8 gBased on all male fish selected for the test(pre-exposure). 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 testinitiation (ifdetermined):Not reportedIndividual weights within ±20% of theestimated mean.Average male weight was3.8 ± 0.6 g (CV=15.0%; n=60)Mean weight of females at testinitiation (if determined):1.8 gBased on all female fish selected for the test(pre-exposure).Range of individual weights(females) at test initiation (ifdetermined):Not reportedIndividual weights within ±20% of theestimated mean. Average female weightwas 1.8 ± 0.3 g (CV=17.1%; n=120)Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:16 days EPA recommends a minimum of 14 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page12of91ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitivetest?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:30 tanks10 extra replicates were established andincluded in the totalnumber of pre-exposuretanks (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.Number of males per tank:2 males/tankNumber of females per tank:4 females/tankPre-exposure fecundity:>15 eggs/female/reproductive day/replicate EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:>2 times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page13of91ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on pre-exposure:2 pre-exposure replicates were excludeddue to mortality and 4 pre-exposurereplicates were excluded due to poorreproductive performance.C. 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.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelinesfor the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page14of91ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Continuous flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:90 mL/minThe flow rate is greater than the USEPAguideline (45 ml/min) and was chosen to helpmaintain test concentrations during the exposuredue to the adsorption properties of the testsubstance. Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day .2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page15of91ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:A continuous-flow syringe pump delivery usinggas-tight syringes continuously delivered stocksolution at designated rate to designatedstainless steel mixing chamber and mixed withdilution water; no test substance or solventstock were added to thenegative control.Concentrations of the solvent in the treatmentand solvent control were identical. Testsolution was split equally among four replicatetest vessels (maintained in temperaturecontrolled water trough) via Teflon manifold andstopcock. Diluter operation was monitored atleast two times daily during the test and flowrates assessed. The flow rates of the dosestock solutions were measured 6 times/weekand the diluent measured daily during exposureand targeted not to vary more than 20% duringexposure.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 s ystem is recommended; 4) The recommended flow splitting accuracy is within

10%.Aeration?Not reportedDissolved oxygen levels were maintained at>60% saturation. EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page16of91ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Natural waterLaboratory dilution water was Lake Huron watersupplied by the City of Midland WaterTreatment Plant and limed and flocculated withferric chloride. The water was pumped to thelab prior to municipal treatment for humanconsumption. Before use, water was sandfiltered, pH-adjusted with gaseous CO2, carbonfiltered, and UV-irradiated. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and 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?YesPeriodically, the water is analyzed for totalorganic carbon (TOC), total suspended solids(TSS), pesticides, organics, metals and otherinorganics.Test vessel type/materials:Glass aquaria sealed withclear silicone adhesive.EPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:39 x 20 x 25 (L x W x H)cm deep. Water depth ca.13 cm by a screen coveredoverflow drain. EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page17of91ParameterValue(s)Details or RemarksGuideline RecommendationsFill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material:Aged PVCSpawning tiles were constructed of ca. 9 cmlong x 10 cmdiameter PVC pipe cut andsectioned lengthwise and inverted to form asemicircle arch. Each tile positioned on alipped tray of stainless steel ( ca. 13 cm long)to contain eggs that do not adhere to tile and astainless steel screen to prevent fish fromaccess to any fallen eggs. 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: Ca. 9 cm long and 10 cmdiameter PVC pipeData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page18of91ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:The test substance was absorptive (Koc =9840-11,660 L/kg) and lipophilic (log Pow =5.29). Therefore, the exposure system wasequilibrated at least 8 days prior to initiatingexposure to achieve equilibrium. Nominal‘dosing’ concentrations were increased to 2.5Xof target exposure concentrations (i.e.0.083,0.0083, and 0.00083 mg/L) in an effort toachieve and maintain the target exposureconcentrations (0.033, 0.0033, and 0.00033mg/L) in the water column of the test vessels.Water samples from all replicate vessels andconcentrated stock solutions were sampled onDays 0, 1, 2, 4, 7, 11, 14, 18 and 21 to bettercharacterize benfluralin exposure.Finally, testsolution flow rate was doubled from 45 mL/minto 90 mL/min (6 to 12 turnovers/day).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page19of91Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.3 25.5 24.9Weekly in all replicatetanks and continuouslyin one replicate tank. EPA recommends temperature 25±1 oC; interreplicate and inter -treatment differentials should not exceed 1 o C.pH7.57.87.6Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)6.4 8.2 7.5 Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3)32 66 47Weekly from thenegative controlandhighest treatment levelwith surviving fish. EPA recommends total alkalinity >20 mg/L

as CaCO 3.Hardness(mg/L as CaCO3) 60 92 74Weekly from thenegative controlandhighest treatment levelwith surviving fish.Total organic carbon (mg/L)NotreportedMeasured periodicallyfrom source water. EPA recommends that total organic carbon in dilution water be <2 mg/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page20of91ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsUnionized ammonia (Pg/L) Ammonia (as Nitrogen); <0.1 mg/L (<LOD)Measured periodicallyfrom source water;results from mostrecent analysis. EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L) <0.02 mg/L (<LOD)Measured periodicallyfromsource water;results from mostrecent analysis. EPA recommends that residual chlorine in dilution water be <10 P g/L.Other(pesticides, organics,inorganics, metals)Measured concentrations of pesticides, organics,metals, and other inorganics were belowlevels ofconcern.Measured periodicallyfrom source water;results from mostrecent analysis. 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page21of91D. 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?Not applicable 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:Not applicableLife stage:Not applicable 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:Not applicable EPA recommends a 96-hour exposure.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page22of91ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:Not applicable 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 concentrati on-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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page23of91Table 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:Other reference study(1/3rdthe 96-hour LC50)The acute toxicity 96-hour LC50 value forbenfluralinwith fathead minnows wasreported to be0.100 mg/L (Mayer andEllersieck, 1986). From the acute toxicitystudy, the high test concentration for thepresent study was selected as 1/3 of0.100mg/L, equal to 0.033 mg/L. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg /L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page24of91ParameterValue(s)Details or RemarksGuideline RecommendationsReference study citation (ifapplicable):Mayer, F. M. andEllersieck, M. R. (1986).Manual of Acute Toxicity:Interpretation and DataBasefor 410 Chemicalsand 66 Species ofFreshwater Animals.Resource Publication 160.U. S. Department of theInterior, Fish and WildlifeService.Washington, D.C.Separation of test concentrations:0.1 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 EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?YesData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page25of91ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Time-weighted averageLimit of quantification (LOQ):0.0000505 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:Test concentrationsmeasured on 0, 2, 4, 7,11, 14, 18 and 21 from allreplicate tanks.Additional analysis of test concentrationswas necessary to characterize benfluralinexposure concentrations due to theadsorptive and lipophilic nature of the testchemical. It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two

replicates per treatment level.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre - exposure. Tanks are then ranked according to pre -exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page26of91ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (ifapplicable):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 EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page27of91ParameterValue(s)Details or RemarksGuideline RecommendationsWas a solvent used?YesDose selection determined from the limitconcentration (100 ug/L) reported inpublished literature and the solubility limit ofthe test chemical. The study reportrecognizes use of a solvent (DMF) toprepare test chemical solutions is a lastresort. However, test chemicalconcentrations in an aqueous solution werelikely to be unstable and the solubility limitsof test chemical made saturator columnsimpractical for the selected testconcentrations.Solvent type (if applicable):DMFN’, N-dimethylformamide.Maximum solvent concentration (ifapplicable):0.05 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 other3Hutchinson 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page28of91ParameterValue(s)Details or RemarksGuideline Recommendations adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):Not applicablePositive control concentration(s) (ifapplicable):Not applicablePhotoperiod:16 hrs light :8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:635-999 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Description of test solution appearance wasnot located in study report.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page29of91Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Benfluralin.Treatment IDNominalConcentration(mg a.i./L)TWA MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative control0<LOQNAThe coefficient of variation for measuredconcentrations of 0.000302 and 0.00314mg a.i./L exceeded 20%, ranging from 21.9to 52.5%, reflecting the loss of testsubstance in the water column during thefirst week of the study(the 0.033 mg a.i./Lwas terminated at 11 days). Severalmeasures were taken to compensate for theuncontrollable loss of test substance in thewater column to provide exposureconcentrations near the target (seeadditional details on exposure system). EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent control0<LOQNATreatment 10.000330.00030243.4Treatment 20.00330.0031427.1Treatment 30.0330.036512.5Abbreviations:CVCoefficient of variation.NANot applicable.LOQ=0.0000505 mg a.i./LData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page30of91E. ObservationsBiological Endpoints:Daily, observations were conducted for mortality, external abnormalities (e.g. hemorrhage, discoloration) and any abnormalbehavior relative to the controls (e.g. hyperventilation, loss of equilibrium, uncoordinated swimming, atypical quiescence andfeeding abstinence), fecundity and fertilization. At test termination (Day 21) fish were observed for behavioral and secondarysex characteristics (SSC), body weight and length, nuptial tubercle score, gonado-somatic index (GSI), plasma vitellogenin,gonadal staging and histopathology.Were raw (individual) data provided?Yes, with the exception of daily external abnormalities and behavior, and general SSC; data provided onsurviving fish at test termination. 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 st aging 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page31of91II. RESULTS AND DISCUSSIONA. ResultsDuring the exposure, signs of overt toxicity (e.g. mortality, lethargy, loss of equilibrium, bloating) were observed among fishexposed to 0.0365 mgbenfluralin/L with38% of the fish affected. Therefore, this treatment group was terminated and removed from the study on exposure Day 11 andexcluded from any subsequent statistical analysis. The toxicity in the high treatment group may have been due to the necessityof dosing the testsolution at 2.5X the target exposure concentrations to achieve target water concentrations during the exposure. The nominal dosing concentration forthe high treatment group was 0.083 mg a.i./L, which was near the reported 96-hour LC50value for benfluralin with fathead minnows of 0.100 mg/L(Mayer and Ellersieck, 1986). The mortality rate in the negative control and solvent control were both 4.2% (4.2% pooled controls); therefore, thesurvival rate in the control groups satisfied the minimum acceptable control value criteria of90% according to the USEPA OCSPP 890.1350guideline. Excluding the highest treatment level, minimal signs of abnormal behavior and appearance were observed in the othertreatment groupsduring the exposure and were not treatment related.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page32of91Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]MalesFemalesn# Surviving% Survivaln# Surviving% SurvivalNegative control (<LOQ)88100161594Solvent control (<LOQ)881001615940.000302881001615940.003148810016161000.03651NANANANANANAAbbreviations:NANot applicable.LOQ=0.0000505 mg a.i./L1The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequent analysis due to overt signs oftoxicity.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page33of91Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Body WeightLengthMalesFemalesMalesFemalesn1Mean(g)±SDn1Mean(g)±SDn1Mean(mm)±SDn1Mean(mm)±SDNegative control(<LOQ)44.730.7424 1.910.200457.63.35444.41.49Solvent control(<LOQ)44.030.3864 1.960.208453.51.07444.42.010.00030244.260.26941.820.119455.21.32443.90.760.0031444.640.40541.890.182456.11.53443.71.660.03652NANANANANANANANANANANANAAbbreviations:NANot applicable.SDStandard deviation.LOQ=0.0000505 mg a.i./L1n indicates the number of replicates, not the total number of fish.2The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequent analysis due to overt signs oftoxicity.

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### All control replicate vessels did not meet the specified test validity (performance) criteria for the control group,

i.e. , spawning at least every 4 days or approximately 15 eggs/female/reproductive day and fertility greater

than 95%. Replicates failing to meet these criteria were one negative control and two solvent control vessels

which had reported fecundity of 13.2, 11.1, and 11.0 eggs/female/reproductive day; spawning did not occur at

least every four days in these replicates. Mean fecundity for the negative control, solvent control, were 30.7,

and 18.8 eggs/female/reproductive day/replicate, respectively.

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA-measured] Fecundity 1 Fertilization Success (%) 2

Mean ± SD Mean ± SD

Negative control (<LOQ) 30.7 15.5 99.2 0.13

Solvent control (<LOQ) 18.8 8.90 99.1 0.58

0.000302 21.7 17.7 98.8 4 0.52

0.00314 24.2 8.39 98.7 0.83

0.0365 3 NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

4 The study author reported a 0% fertilization rate for the one replicate that did not produce any eggs and a mean

fertilization rate of 74.1% with a SD of 49.4, the reviewer concluded that fertility could not be assessed for that replicate

because no eggs were produced and therefore excluded it from the mean calculation and statistical analyses.

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### There were no tubercles noted among female fathead minnows at the end of the exposure period. Median

male tubercle scores were 30.3, 26.3, 25.3, and 29.5 for the negative control, solvent control, 0.000302

and 0.0365 mg a.i./L treatment groups, respectively.

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

.

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 n indicates the number of replicates, not the total number of fish.

2 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

3 Mean tubercle scores: 28, 26, 26, and 29 for the negative control, solvent control, and mean-measured 0.000302 and

0.00314 mg a.i./L treatment levels, respectively. Treatment

(mg a.i./L)

[TWA-measured] Males Females

n 1 Median Tubercle

Score n 1 Median Tubercle

Score

Negative control (<LOQ) 4 30 4 0

Solvent control (<LOQ) 4 26 4 0

0.000302 4 25 4 0

0.00314 4 30 4 0

0.0365 2 NA NA NA NA

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### Mean male GSI was 1.15, 1.20, 1.44 and 1.46 and mean female GSI was 14.1, 13.1, 12.8 and 14.3 in the

negative control, solvent control, and TWA-measured 0.000302 and 0.00314 mg a.i./L treatment levels,

respectively.

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA-measured] Males Females

n 2 Mean GSI 1 (%) ±SD n 2 Mean GSI 1 (%) ±SD

Negative control (<LOQ) 4 1.15 0.141 4 14.1 1.93

Solvent control (<LOQ) 4 1.20 0.134 4 13.1 0.622

0.000302 4 1.44 0.427 4 12.8 2.41

0.00314 4 1.46 0.125 4 14.3 1.39

0.0365 3 NA NA NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.

2 n indicates the number of replicates, not the total number of fish.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

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### Gonad staging was performed in general accordance with the procedures described in Appendix E of the

USEPA test guideline. The pattern of predominant germ cell distribution (staging) in ovaries or testes was

comparable between the controls and all treated groups.

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

.

Treatment

(mg a.i./L)

[TWA-measured] Males Females

n Median Stage 1 n Median Stage 2

Negative control (<LOQ) 8 2 15 3

Solvent control (<LOQ) 8 2 15 3

0.000302 8 3 15 2

0.00314 8 3 16 3

0.0365 3 NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early

spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early

development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

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### There were no treatment-related histopathologic changes either in the testes or ovaries of fathead minnows

exposed to benfluralin at all concentrations evaluated. No histopathological findings were observed in the testis

of male fish; all diagnostic observations were “within normal limits” (severity grade 0). In females, the incidences

of grade 4 oocyte atresia (9 or more atretic oocytes in a section) were slightly higher in the solvent control

group and the 0.000302 mg a.i./L group as compared to the negative control and the 0.00314 mg a.i./L

group. However, according to the study authors, the reason for this increase was not clear and was deemed

to not be treatment-related; the study authors stated no treatment-related effects on female ovaries. According

to the raw data, 5 of the 6 reported incidences of severe atresia in the solvent control occurred in the two

replicates in which a decrease in fecundity (compared to output during pre-exposure) was observed (<15

eggs/female/day or not spawning every 4 days; 2 of the 4 females in replicate C and all 3 females in

replicate D) and all four female fish in the 0.000302 mg a.i./L replicate that produced no eggs also reported

severe oocyte atresia.

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

.

Treatment

(mg a.i./L)

[TWA

- measured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of TestisOva Increased

Testicular

Degeneration Interstitial Cell

Hypertrophy/

Hyperplasia

n Incidence 2 n Incidence 2 n Incidence 2 n Incidence 2

Negative control

(<LOQ) 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

Solvent control

(<LOQ) 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

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(mg a.i./L)

[TWA

- measured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of TestisOva Increased

Testicular

Degeneration Interstitial Cell

Hypertrophy/

Hyperplasia

n Incidence 2 n Incidence 2 n Incidence 2 n Incidence 2

0.000302 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.00314 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.0365 3 0 NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA

4 NA NA NA NA NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 Diagnostic observation data for the specific criteria were not provided in this present study unless “findings” (severity

grading 1-4) were observed for a specific criterion. Thus, di agnostic observations in this table are reported as severity

grade 0 if a diagnostic criterion was not mention in the report. Note: This present study reported that no

histopathological findings were observed in the testis of male fish; all diagnostic observations were “within normal

limits”.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page40of91Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronous GonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidence2nIncidence2nIncidence2nIncidence2nIncidence2Negative control (<LOQ)0888888888818080808080280808080803808080808048080808080Solvent control (<LOQ)08888888888180808080802808080808038080808080480808080800.0003020888888888818080808080280808080803808080808048080808080Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page41of91Treatment(mg a.i./L)[TWA-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronous GonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidence2nIncidence2nIncidence2nIncidence2nIncidence20.0031408888888888180808080802808080808038080808080480808080800.036530NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAAbbreviations:NANot applicable.LOQ=0.0000505 mg a.i./L1Gonadal 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.2Diagnostic observation data for the specific criteria were notprovided in this present study unless “findings” (severity grading 1-4) were observed for a specific criterion.Thus, diagnostic observations in this table are reported as severity grade 0 if a diagnostic criterion was not mention in the report. Note: This present study reported that noData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page42of91histopathological findings were observed in the testis of male fish; all diagnostic observations were “within normal limits”.3The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequent analysis due to overt signs oftoxicity.

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

.

Treatment

(mg a.i./L)

[TWA-measured] Diagnostic Observations 1

Severity Increased Oocyte Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation

n Incidence 2 n Incidence 2 n Incidence 2

Negative control (<LOQ) 0 15 4 15 15 15 15

1 15 7 15 0 15 0

2 15 1 15 0 15 0

3 15 2 15 0 15 0

4 15 1 15 0 15 0

Solvent control (<LOQ) 0 15 5 15 15 15 15

1 15 3 15 0 15 0

2 15 0 15 0 15 0

3 15 1 15 0 15 0

4 15 6 15 0 15 0

0.000302 0 15 4 15 15 15 15

1 15 1 15 0 15 0

2 15 0 15 0 15 0

3 15 3 15 0 15 0

4 15 7 15 0 15 0

0.00314 0 16 2 16 16 16 16

1 16 10 16 0 16 0

2 16 2 16 0 16 0

3 16 1 16 0 16 0

4 16 1 16 0 16 0

0.0365 3 0 NA NA NA NA NA NA

1 NA NA NA NA NA NA

2 NA NA NA NA NA NA

3 NA NA NA NA NA NA

4 NA NA NA NA NA NA

Abbreviations: NA Not applicable.

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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.

2 Diagnostic observation data for the specific criteria were not provided in this present study unless “findings” (severity

grading 1-4) were observed for a specific criterion. Thus, di agnostic observations in this table are reported as severity

grade 0 if a diagnostic criterion was not mention in the report. Note: This present study reported that no

histopathological findings were observed in the testis of male fish; all diagnostic observations were “within normal

limits”.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.



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

promelas)

.

Treatment

(mg a.i./L)

[TWA

- measured] Additional Diagnostic Observations 1

Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence 2 n Incidence 2 n Incidence 2 n Incidence 2

Negative control

(<LOQ) 0 15 15 15 15 15 14 15 15

1 15 0 15 0 15 1 15 0

2 15 0 15 0 15 0 15 0

3 15 0 15 0 15 0 15 0

4 15 0 15 0 15 0 15 0

Solvent control

(<LOQ) 0 15 15 15 15 15 15 15 15

1 15 0 15 0 15 0 15 0

2 15 0 15 0 15 0 15 0

3 15 0 15 0 15 0 15 0

4 15 0 15 0 15 0 15 0

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(mg a.i./L)

[TWA

- measured] Additional Diagnostic Observations 1

Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence 2 n Incidence 2 n Incidence 2 n Incidence 2

0.000302 0 15 0 15 15 15 15 15 15

1 15 0 15 0 15 0 15 0

2 15 0 15 0 15 0 15 0

3 15 0 15 0 15 0 15 0

4 15 0 15 0 15 0 15 0

0.00314 0 16 16 16 16 16 16 16 16

1 16 0 16 0 16 0 16 0

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

0.0365 3 0 NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA

4 NA NA NA NA NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 Diagnostic observation data for the specific criteria were not provided in this present study unless “findings” (severity

grading 1-4) were observed for a specific criterion. Thus, di agnostic observations in this table are reported as severity

grade 0 if a diagnostic criterion was not mention in the report. Note: This present study reported that no

histopathological findings were observed in the testis of male fish; all diagnostic observations were “within normal

limits.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

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### Table 18b: Additional Gonadal Histopathology Observations in Female Fathead Minnows ( Pimephales

promelas ).

Treatment

(mg a.i./L)

[TWA

- measured] Diagnostic Observation 1

Severity Inflammation;

Subacute to Chronic;

multifocal Edema;

Interstitium;

Bilateral

n Incidence 2 n Incidence 2

Negative control

(<LOQ) 0 15 8 15 14

1 15 6 15 1 4

2 15 1 15 0

3 15 0 15 0

4 15 0 15 0

Solvent control

(<LOQ) 0 15 11 15 15

1 15 2 15 0

2 15 2 15 0

3 15 0 15 0

4 15 0 15 0

0.000302 0 15 12 15 15

1 15 3 15 0

2 15 0 15 0

3 15 0 15 0

4 15 0 15 0

0.00314 0 16 13 16 16

1 16 3 16 0

2 16 0 16 0

3 16 0 16 0

4 16 0 16 0

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(mg a.i./L)

[TWA

- measured] Diagnostic Observation 1

Severity Inflammation;

Subacute to Chronic;

multifocal Edema;

Interstitium;

Bilateral

n Incidence 2 n Incidence 2

0.0365 3 0 NA NA NA NA

1 NA NA NA NA

2 NA NA NA NA

3 NA NA NA NA

4 NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 Diagnostic observation data for the specific criteria were not provided in this present study unless “findings” (severity

grading 1-4) were observed for a specific criterion. Thus, di agnostic observations in this table are reported as severity

grade 0 if a diagnostic criterion was not mention in the report. Note: This present study reported that no

histopathological findings were observed in the testis of male fish; all diagnostic observations were “within normal

limits.

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

4 Graded as “slight”; not 1, 2, 3, or 4.

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### Male mean plasma vitellogenin ranged from 1.45x10 -3 mg/mL plasma in the solvent control to 8.49x10 -4 mg/mL

plasma in the negative control. 

### Female meanplasma vitellogenin ranged from 82.67 mg/mL plasma in the TWA

0.000302 mg a.i./L treatment group to 106.12 mg/mL plasma in the negative control.



### Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[TWA-measured] Plasma Vitellogenin (VTG)

Males Females

n 1 Mean

(mg/mL plasma) ±SD n 1 Mean

(mg/mL plasma) ±SD

Negative control (<LOQ) 4 8.49x10 -4 2.34x10 -4 4 106.53 37.97

Solvent control (<LOQ) 4 1.45x10 -3 1.20x10 -3 4 96.35 14.50

0.000302 4 5.95x10 -4 2.88x10 -4 4 82.67 33.24

0.00314 4 6.39x10 -4 2.53x10 -4 4 106.12 43.38

0.0365 2 NA NA NA NA NA NA

Abbreviations: NA Not applicable. SD Standard deviation.

1 n indicates the number of replicates, not the total number of fish.

2 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.

LOQ=0.0000505 mg a.i./LData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page49of91Plasma testosterone (T) and 17ȕ-estradiol were not measured.Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas)). Not measured.Treatment(mg a.i./L)[TWA-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative control (<LOQ)NANANANANANANANANANANANASolvent control (<LOQ)NANANANANANANANANANANANA0.000302NANANANANANANANANANANANA0.00314NANANANANANANANANANANANA0.0365NANANANANANANANANANANANAAbbreviations:NANot applicable.SDStandard deviation.LOQ=0.0000505 mg a.i./LData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page50of91During the exposure, signs of overt toxicity ( e.g., mortality, lethargy, loss of equilibrium, bloating) were observed among fish exposed to 0.0365 mgbenfluralin/L with38% of the fish affected. Therefore, this treatment group was terminated and removed from the study on exposure Day 11 and excludedfrom any subsequent statistical analysis. Excluding the highest treatment level, minimal signs of abnormal behavior and appea rance were observed during theexposure and were not treatment related. Overall, no concentration-dependent overt signs of toxicity were observed during the study except for the hightreatment level. One female in the 0.00314 mg a.i./L group was reported as having slight vertical bands present. Dorsal fat pads were observed, but notableeffects were not mentioned, except for the one fish in the negative control which was reported as not having a very pronouncedfatpad.Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Secondary Sex Characteristics and Clinical Signs in Surviving Fish at Test TerminationMalesFemalesTypen2IncidenceTypen2IncidenceNegative control(<LOQ)Fatpad not very pronounced81Injured pectoral fin151Very thin81No notable effects1514No notable effects86Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page51of91Treatment(mg a.i./L)[TWA-measured]Secondary Sex Characteristics and Clinical Signs in Surviving Fish at Test TerminationMalesFemalesTypen2IncidenceTypen2IncidenceSolvent control (<LOQ)No notable effects88Slight darkening of body151Bloated151No notable effects15130.000302No notable effects88No notable effects15150.00314Loss of territorial aggressiveness81Slightly bloated161No notable effects87Slight vertical banding present161Aggressive behavior161No notable effects16130.03651NANANANANANALOQ=0.0000505 mg a.i./L1The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequentanalysis due to overt signs oftoxicity.2n is the number of fish present at test termination, not replicates

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### B. Study Author’s Analysis and Conclusions

The appropriate units of statistical analyses were the measures of central tendency from the replicate test

vessels. The statistical significance of all tests was judged at the 0.05 significance level, with the exception

of the Shapiro-Wilk test, which was judged at the 0.01 significance level. All biological response data, apart

from mortality, fecundity and fertility, were analyzed and reported separately by sex. Endpoints were

statistically evaluated with the Jonckheere-Terpstra test (Hollander and Wolfe, 1973) in a step down manner

if the measures of central tendency for that endpoint were consistent with a monotone concentrationresponse. If the endpoint response was not consistent with a monotone concentration-response, the data

were assessed for normality using the Shapiro-Wilk test and variance homogeneity using Levene’s test.

Where non-normality or variance heterogeneity was observed, normalizing and/or variance stabilizing

transformations were applied. If the data were non-monotonic and normally distributed with homogeneous

variances, then a significant treatment effect was determined using the one way ANOVA followed by

Dunnett’s test. If the data were normally distributed with heterogeneous variance, the Mann-WhitneyWilcoxon U test was used. If no normalizing transformation was found, the Mann-Whitney-Wilcoxon U test

using a Bonferroni-Holm adjustment was used. Significant mortality was assessed, if necessary, using the

Cochran-Armitage Linear Trend Test where the data was consistent with a monotonic concentration

response, and otherwise from Fisher’s Exact test with a Bonferroni-Holm adjustment. Any treatment effect

for tubercle score was determined using the multiquantal Jonckheere-Terpstra test.

Most of the validity criteria were fulfilled, and if not and when possible, substantial measures taken to try

and meet the criteria, such as in the case of the variability in the measured test concentration. During the

exposure, signs of overt toxicity (e.g. mortality, lethargy, loss of equilibrium, bloating) were observed among

fish exposed to 0.0365 mg benfluralin/L with  38% of the fish affected; therefore, this treatment group

was removed from the study and excluded from any subsequent statistical analysis in the present study. In

the remaining control and treatment groups, there were three incidences of fish mortality during the exposure,

one in the negative control, one in the solvent control and one in the 0.000302 mg a.i./L treatment group.

There were no statistically significant differences in mortality among pooled control and benfluralin-exposed

fish at the end of the study and no indication of treatment related abnormal behaviors or appearance,

demonstrating that the remaining concentrations of benfluralin used in the present study (up to 0.00314

mg a.i./L) were not overtly toxic to fathead minnows. There were no significant differences between control

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### and benfluralin exposed fish in regard to fecundity, fertility, wet weight and length, gonado-somatic indices,

tubercle scores, or blood plasma concentrations of VTG in either male or female fish. Furthermore, there

were no treatment-related histopathologic changes in the testes or ovaries in any of the benfluralin exposed

treatment groups relative to controls. According to the study authors, since there were no significant effects

on assay endpoints observed at the two concentrations of benfluralin that did not result in overt toxicity, the

results from this assay indicate that benfluralin does not have the potential to interfere with the HPG axis

of fish.

C. Reviewer’s Analysis and Conclusions

Statistical Methods : For all biological endpoints examined in this study, there were no significant differences

between negative controls and solvent controls. However, the treatment groups were compared to the

negative control. Excluding data from the highest concentration group (0.0365 mg a.i./L) due to signs of

overt toxicity, visual interpretation of the data did not suggest benfluralin caused concentration-dependent

mortality. A single fish died in the control, solvent control, and lowest (0.000302 mg a.i./L) treatment

group (resulting in 4.2% mortality in each group). No mortality was observed among fathead minnows

exposed to 0.00314 mg a.i./L concentration. Male and female weight, male length, male and female

vitellogenin (VTG), and female gonadal somatic index (GSI), male tubercle score, fecundity and fertility of

controls were not significantly different from the means of fish exposed to low or medium concentrations of

benfluralin over the 21 day study. Male GSI was significantly different from the negative control for the mid

treatment concentration. All data were tested for normality using Shapiro-Wilks test and for homogeneity of

variance using Levene’s test (SAS 8.1). All of these parameters met the assumptions of normality and

homogeneity of variance, except for male GSI, and were then analyzed using Dunnett’s test. Male GSI did

not pass the assumptions for Levene’s test, and was therefore analyzed using the exact version of the

Mann Whitney test.

Given that there were only two treatment groups available at test termination, the reviewer concluded that

inferences about data exhibiting a trend are limited. Therefore, all endpoints were analyzed using non-trend

statistical tests. Sex steroids were not measured and histopathology and gonadal staging data were visually

assessed. Unless otherwise indicated, effects were considered statistically significant at p<0.05. The

reviewer additionally compared the negative and solvent control groups using a 2-sided t-test assuming

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### equal variances via Excel, and no significant difference between the two groups were observed for any

endpoint.

Conclusions:

There were no significant effects in this study on any analyzed endpoint (p>0.05) and no apparent

treatment-effects on any empirically-determined parameter ( e.g. , secondary sex characteristics, gonadal

stage, or histopathology), except for male GSI at the mid test concentration.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page55of91Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Benfluralin.Treatment(mg a.i./L)[TWA-measured]Tubercle ScoreGSIGonadal Stagingand Histo.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect? (Yes/No)% Diff.p% Diff.p% Diff.pNegative control(<LOQ)30 NA 0 NANo 0 NA NA NA NA NASolvent control(<LOQ)260.4944.350.657No70.940.362NA NA NA NA0.000302250.52925.00.678No-29.90.325NANANANA0.00314300.97627.20.029No-24.80.443NANANANA0.03653NANANANANANANANANANANAStatistical testDunnett’s4Mann-WhitneyNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.0000505 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequent analysis due to overt signs oftoxicity.4Mean tubercle scores: 28, 26, 26, and 29 for the negative control, solvent control, and TWA 0.000302 and 0.00314 mg a.i./L treatment levels, respectively.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page56of91Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Benfluralin.Treatment(mg a.i./L)[TWA-measured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.pMedianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p% Diff.pNegative control (<LOQ)0NA0NA0NA0NANo0NANANANANASolvent control (<LOQ)-38.80.231-0.100.7480NA-7.090.354No4-9.560.634NANANANA0.000302-29.20.602-0.350.6620NA-9.490.546No4-22.40.764NANANANA0.00314-21.00.759-0.450.4760NA1.300.987No26.50.691NANANANA0.03653NANANANANANANANANANANANANANANAStatistical testDunnett’sDunnett’sNADunnett’sNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.0000505 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded from subsequent analysis due to overt signs oftoxicity.4The incidences of grade 4 oocyte atresia were slightly higher inthe solvent control group and the 0.000302 mg a.i./L group ascompared to the negative control and the0.00314 mg a.i./L group. The reason for this is not known, and the study authors did not think it was treatment-related. Thereplicates where this atresia was observedalso had reduced fecundity.

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### Table 24: Growth Endpoints 1,2 

### in the Fish Short-Term Reproduction Assay (FSTRA) with Benfluralin.

Treatment

(mg a.i./L)

[TWA-measured] Body Weight Length

Males Females Males Females

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

Negative control (<LOQ) 0 NA 0 NA 0 NA 0 NA

Solvent control (<LOQ) -14.77 0.145 2.62 0.741 -7.12 0.060 0.00 0.970

0.000302 -10.01 0.360 -4.98 0.659 -4.10 0.286 -1.13 0.826

0.00314 -2.03 0.950 -1.12 0.977 -2.58 0.569 -1.49 0.725

0.0365 3 NA NA NA NA NA NA NA NA

Statistical test Dunnett’s Dunnett’s Dunnett’s Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.

LOQ=0.0000505 mg a.i./L

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water)

control.

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

3 The 0.0365 mg a.i./L treatment group was terminated and removed from the study on exposure Day 11 and excluded

from subsequent analysis due to overt signs of toxicity.



### E. Study Deficiencies

All replicate vessels did not met the validity criteria for the control group (spawning at least every 4 days

or ca . 15 eggs/female/reproductive day). There was one replicate in the negative control and two replicates

in the solvent control which had fecundity of 13.2, 11.1, and 11.0 eggs/female/reproductive day, respectively.

It was also noted by the study authors that spawning in these replicate tanks did not occur at least every

4 days. However, mean fecundity values for the negative control, solvent control, and pooled controls were

30.7, 18.8, and 24.7 eggs/female/reproductive day, respectively. In the preceding 7 days prior to transfer

from the pre-exposure phase to the exposure phase of the study, these same vessels exhibited mean

fecundity values of 31.3, 27.3, and 20.6 eggs/female/reproductive day, respectively. The study authors

determined that these data suggest the fish were reproductively fit and concluded that the drop in fecundity

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### for some replicates was likely a result of uncontrolled biological variability and had no impact on the

interpretation of the study results. While the groups in the negative control were exposed under the same

conditions during the exposure and pre-exposure periods ( i.e. , only dilution water), the groups in the solvent

control were now additionally exposed to a solvent (DMF) during the exposure period which is a change in

environmental conditions from the pre-exposure period. Therefore, while the exact cause for the observed

decrease in fecundity is not known and may be due to uncontrolled biological variability, the possibility of a

solvent interference cannot be conclusively ruled out. According to the raw data, five of the six reported

incidences of severe atresia in the solvent control occurred in the two replicates in which a decrease in

fecundity was observed (<15 eggs/female/day and not spawning every 4 days; two of the four females

in replicate C and all three females in replicate D), and all four females in the 0.000302 mg a.i./L replicate

that produced no eggs also reported severe oocyte atresia.

The relative standard deviation (coefficient of variation) of the measured concentrations for all of the replicate

tanks in the study (specifically in the two lowest treatments, since the high treatment was terminated on

Day 11) ranged from 21.9 to 52.5%. This reflected the expected loss of test substance in the water column

primarily during the first week of the study. These data did not meet the guideline criterion that measured

exposure concentration coefficients of variation be <20% for all replicates. This loss of benfluralin in the

water column was explained both by the absorptive (Koc = 9840 – 11,660 L/kg) and lipophilic (log Pow

= 5.29) nature of the test substance (USEPA, 2004 and Day et al., 1983, respectively). To compensate

for this expected loss of test substance, test solutions were dosed at 2.5X of target exposure concentrations

( i.e. , 0.083, 0.0083, and 0.00083 mg a.i./L of nominal); test solution flow rate (complete test vessel

turnover rate) was doubled from that suggested in the USEPA guideline ( i.e. , the suggested flow rate of

45 mL/min was doubled to 90 mL/min; complete vessel volume turnover rate of 6 was doubled to 12);

the exposure system was equilibrated for 10 days prior to exposing fish; and test vessels were cleaned

(scraped and siphoned) daily. By taking these steps, test concentrations were dramatically higher than

targeted exposure concentrations at test initiation (Day 0), but stabilized near the target concentrations by

Day 11. In addition, to better define the exposure during the study, four additional water sampling time

points and analyses ( e.g. , exposure Days 2, 4, 11, and 18) were added to the standard weekly analysis.

These measures were taken to compensate for expected uncontrollable loss of the test substance in the

water column during the exposure and to provide exposure concentrations near to target.

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### All other performance and validity criteria were met.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were in agreement for all endpoints, except for male GSI

where the reviewer’s analysis detected a significant effect at the mid test concentration whereas the study

author’s analysis did not. The difference is due to the use of different statistical test where the reviewer

used Mann-Whitney and the study author used Jonckheere-Terpstra.

While not statistically significant (p=0.231) due to the observed variability in the control replicates, the mean

fecundity in the solvent control group was reduced by 39% compared to the negative control. Therefore,

given this observed decrease there is concern that the use of a solvent may have interfered and impacted

the interpretation of the study. This decrease is due to two of the four replicates having fecundity rates of

11 eggs/female/day and these groups did not spawn every four days; the fecundity rates for these two

groups of fish 7 days prior to the exposure period were 27.3 and 20.6 eggs/female/day. Fecundity in one

replicate in the negative control was also less than was observed in the pre-exposure period and did not

spawn every four days (13.2 in the 21 day exposure period vs. 31.1 eggs/female/day in the 7 days prior

to the exposure period). While the exact reason for the observed decline is unknown, it may be due to

possible biological variation, however, the possibility of a solvent interference cannot be conclusively ruledout. Based on the observed results, there were no significant differences (p>0.05) in fecundity in the

treatment groups compared to the negative control with an observed decrease of 29 and 21% in fecundity

in the 0.000302 and 0.00314 mg a.i./L groups, respectively, and one replicate in the 0.000302 mg

a.i./L did not produce any eggs during the exposure period.

Regarding VTG measurements, a total of 9 plates were analyzed over three days. The correlation coefficient

(R2) for the calibration curves used for each plate ranged from 0.999 to 1.000 and were all greater than

0.99 as specified in the guideline. The concentrations for each standard, as calculated from the calibration

curve for each plate, all fell between 90.1 and 113% of their nominal concentrations, which was within the

recommended range of 70 to 120% nominal specified in the guideline. The average absorbance measured

for the non-specific binding assay blanks for each plate ranged from 0.044 to 0.049 and were all below

0.06 absorbance units as suggested in the guideline. The fortification sample prepared and analyzed on

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### each analysis day resulted measured concentrations that ranged from 97.6-111% of expected concentrations.

The well duplicates of all the samples analyzed (N=92) did not differ by more than 20%, supporting

acceptable method precision.

III. REFERENCES

Day, E., Saunders, D., and Loh, A. (1983). Octanol/Water Partition Coefficient of Benefin: Lab Project

Number: I0EWD-93-92. Unpublished study prepared by Lilly Research Labs. 9p.

Hollander, M. and D. A. Wolfe. (1973). Nonparametric Statistical Methods. John Wiley & Sons, Inc. New

York. Pp 120-123.

Koors B. (2009). Recertification of purity and identity of TSN100037, Benefin technical grade active

ingredient. FA&PC Number 09-227482. 13 August, 2009.

Mayer, F. M. and Ellersieck, M. R. (1986). Manual of Acute Toxicity: Interpretation and Data Base for 410

Chemicals and 66 Species of Freshwater Animals. Resource Publication 160. U. S. Department of the

Interior, Fish and Wildlife Service. Washington, D.C.

USEPA (2002). A Short-term Test Method for Assessing the Reproductive Toxicity of Endocrine-Disrupting

Chemicals Using the Fathead Minnow (Pimephales promelas). EPA/600/R-01/067, June 2002.

USEPA (2004). Reregistration Eligibility Decision for Benefin. EPA 738-R-04-012, July 2004.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page61of91APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:TABLE OF STATISTICAL INTERPRETATION: EDSP VS EFED METHODSEndpointMonotonic?1Parametric?EDSPEFEDCommentsFemale bodyweightNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body weightNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale bodylengthNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body lengthNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale VTGNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale VTGNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale GSINAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale GSINANoMann-Whitneymid dosep=0.029Mann-Whitneymid dosep=0.029EDSP and EFED same conclusionsData Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPA MRID Number 48614906Page62of91EndpointMonotonic?1Parametric?EDSPEFEDCommentsFemale tuberclescoreNANANANANo scoreMale tuberclescoreNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFecundityNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFertilityNAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectF testosteroneNANANANANAM testosteroneNANANANANAF estradiolNANANANANAM estradiolNANANANANA1Given that there were only two treatment groups available at test termination, the ability to make inferences about monotonicity and trends arelimited. Therefore, the reviewer analyzed all data using non-trend tests.

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

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

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Page 65 of 91 084301 Benfluralin 890.1350 48614906 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

0.891 0.123 0.592 0.574 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 1.91 0.20 0.10 10.46 1.59, 2.23
Dose1 4 1.82 0.12 0.06 6.54 1.63, 2.01
Dose2 4 1.89 0.18 0.09 9.63 1.60, 2.18

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

Ctrl 1.83 1.78 2.21 . .

Dose1 1.84 1.66 1.94 95.02 4.98

Dose2 1.85 1.73 2.14 98.88 1.12

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

Numerator df Denominator df F-stat P-value

2 9 0.34 0.719

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 1.91 . 1.91 . . . . . .

Dose1 1.82 0.659 1.86 0.381 . . . . .

Dose2 1.89 0.977 1.86 0.407 0.818 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.15 0.926

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 1.83 . .

Dose1 1.84 0.889 0.386

Dose2 1.85 0.889 0.442

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

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

Numerator df Denominator df F-stat P-value

2 9 0.34 0.719

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Page 66 of 91 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 -1.91 . -1.87 . . . . . .

Dose1 -1.82 0.659 -1.87 0.736 . . . . .

Dose2 -1.89 0.977 -1.89 0.691 0.818 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.15 0.926

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 -1.83 . .

Dose1 -1.84 0.889 0.614

Dose2 -1.85 0.889 0.558

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

084301 Benfluralin 890.1350 48614906 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

0.957 0.738 1.112 0.370 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 4.73 0.74 0.37 15.68 3.55, 5.91
Dose1 4 4.26 0.27 0.13 6.30 3.83, 4.69
Dose2 4 4.64 0.40 0.20 8.72 3.99, 5.28

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

Ctrl 4.86 3.72 5.51 . .

Dose1 4.31 3.90 4.53 89.99 10.01

Dose2 4.59 4.22 5.17 97.97 2.03

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

Numerator df Denominator df F-stat P-value

2 9 0.96 0.420

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

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Page 67 of 91 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 4.73 . 4.73 . . . . . .

Dose1 4.26 0.360 4.45 0.270 . . . . .

Dose2 4.64 0.950 4.45 0.288 0.570 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 2.01 0.367

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 4.86 . .

Dose1 4.31 0.343 0.123

Dose2 4.59 0.676 0.385

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

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

Numerator df Denominator df F-stat P-value

2 9 0.96 0.420

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 -4.73 . -4.50 . . . . . .

Dose1 -4.26 0.360 -4.50 0.817 . . . . .

Dose2 -4.64 0.950 -4.64 0.724 0.570 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 2.01 0.367

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 -4.86 . .

Dose1 -4.31 0.343 0.877

Dose2 -4.59 0.676 0.615

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

084301 Benfluralin 890.1350 48614906 ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

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Page 68 of 91 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

0.946 0.577 1.770 0.225 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 44.39 1.49 0.75 3.37 42.01, 46.77
Dose1 4 43.89 0.76 0.38 1.74 42.68, 45.10
Dose2 4 43.73 1.66 0.83 3.79 41.10, 46.37

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

Ctrl 44.02 43.18 46.35 . .

Dose1 43.74 43.13 44.95 98.87 1.13

Dose2 43.61 41.95 45.75 98.51 1.49

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

Numerator df Denominator df F-stat P-value

2 9 0.26 0.779

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 44.39 . 44.39 . . . . . .

Dose1 43.89 0.826 43.89 0.366 . . . . .

Dose2 43.73 0.725 43.73 0.325 0.985 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.62 0.735

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 44.02 . .

Dose1 43.74 0.889 0.386

Dose2 43.61 0.494 0.232

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

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

Numerator df Denominator df F-stat P-value

2 9 0.26 0.779

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

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Page 69 of 91 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 -44.39 . -44.00 . . . . . .

Dose1 -43.89 0.826 -44.00 0.739 . . . . .

Dose2 -43.73 0.725 -44.00 0.773 0.985 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.62 0.735

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 -44.02 . .

Dose1 -43.74 0.889 0.614

Dose2 -43.61 0.494 0.768

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

084301 Benfluralin 890.1350 48614906 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

0.969 0.905 1.031 0.395 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 57.60 3.35 1.68 5.82 52.26, 62.94
Dose1 4 55.24 1.32 0.66 2.39 53.14, 57.33
Dose2 4 56.11 1.53 0.77 2.73 53.67, 58.55

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

Ctrl 57.73 53.40 61.55 . .

Dose1 55.10 54.05 56.70 95.90 4.10

Dose2 56.15 54.40 57.75 97.42 2.58

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

Numerator df Denominator df F-stat P-value

2 9 1.12 0.369

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

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

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Page 70 of 91 Ctrl 57.60 . 57.60 . . . . . .

Dose1 55.24 0.286 55.68 0.154 . . . . .

Dose2 56.11 0.569 55.68 0.164 0.850 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 2.19 0.334

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 57.73 . .

Dose1 55.10 0.346 0.124

Dose2 56.15 0.494 0.330

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

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

Numerator df Denominator df F-stat P-value

2 9 1.12 0.369

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

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

Ctrl -57.60 . -56.32 . . . . . .

Dose1 -55.24 0.286 -56.32 0.854 . . . . .

Dose2 -56.11 0.569 -56.32 0.881 0.850 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 2.19 0.334

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 -57.73 . .

Dose1 -55.10 0.346 0.876

Dose2 -56.15 0.494 0.670

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

084301 Benfluralin 890.1350 48614906 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

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Page 71 of 91 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 0.966 2.735 0.118 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 1.0653E8 37968801 18984401 35.64 46112175,1.6695E8
Dose1 4 82671038 33239111 16619555 40.21 29780195,1.3556E8
Dose2 4 1.3477E8 78936043 39468022 58.57 9166083,2.6038E8

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

Ctrl 98524612 69516501 1.5955E8 . .

Dose1 85619901 42623282 1.1682E8 77.60 22.40

Dose2 1.2384E8 56738478 2.3467E8 126.51 -26.51

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

Numerator df Denominator df F-stat P-value

2 9 0.93 0.429

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 1.0653E8 . 1.0799E8 . . . . . .

Dose182671038 0.764 1.0799E8 0.599 . . . . .

Dose21.3477E8 0.691 1.0799E8 0.634 0.399 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.73 0.694

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 98524612 . .

Dose1 85619901 0.678 0.282

Dose2 1.2384E8 1.000 0.558

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

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

Numerator df Denominator df F-stat P-value

2 9 0.93 0.429

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

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

EPA MRID Number 48614906

Page 72 of 91 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.065E8 . -9.46E7 . . . . . .

Dose1-8.267E7 0.764 -9.46E7 0.707 . . . . .

Dose2-1.348E8 0.691 -1.348E8 0.306 0.399 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.73 0.694

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 -9.852E7 . .

Dose1 -8.562E7 0.678 0.718

Dose2 -1.238E8 1.000 0.442

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

084301 Benfluralin 890.1350 48614906 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

0.879 0.084 0.193 0.827 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 849.13 233.80 116.90 27.53 477.09, 1221.16
Dose1 4 595.13 288.47 144.23 48.47 136.11, 1054.15
Dose2 4 638.88 252.62 126.31 39.54 236.91, 1040.84

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

Ctrl 840.00 626.00 1090.50 . .

Dose1 642.00 238.02 858.50 70.09 29.91

Dose2 641.25 361.00 912.00 75.24 24.76

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

Numerator df Denominator df F-stat P-value

2 9 1.10 0.375

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 849.13 . 849.13 . . . . . .

Dose1 595.13 0.325 617.00 0.141 . . . . .

Dose2 638.88 0.443 617.00 0.150 0.969 . . . .

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

EPA MRID Number 48614906

Page 73 of 91 ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 1.88 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 840.00 . .

Dose1 642.00 0.346 0.124

Dose2 641.25 0.346 0.153

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

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

Numerator df Denominator df F-stat P-value

2 9 1.10 0.375

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 -849.13 . -694.38 . . . . . .

Dose1 -595.13 0.325 -694.38 0.863 . . . . .

Dose2 -638.88 0.443 -694.38 0.889 0.969 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 1.88 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 -840.00 . .

Dose1 -642.00 0.346 0.876

Dose2 -641.25 0.346 0.847

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

084301 Benfluralin 890.1350 48614906
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

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

EPA MRID Number 48614906

Page 74 of 91 0.928 0.360 1.364 0.304 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 14.09 1.93 0.97 13.72 11.01, 17.16
Dose1 4 12.75 2.41 1.20 18.88 8.92, 16.58
Dose2 4 14.27 1.39 0.69 9.72 12.06, 16.48

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

Ctrl 14.69 11.35 15.63 . .

Dose1 12.60 10.37 15.43 90.51 9.49

Dose2 14.22 12.95 15.70 101.30 -1.30

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

Numerator df Denominator df F-stat P-value

2 9 0.72 0.512

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 14.09 . 14.09 . . . . . .

Dose1 12.75 0.546 13.51 0.407 . . . . .

Dose2 14.27 0.987 13.51 0.435 0.538 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 1.42 0.491

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 14.69 . .

Dose1 12.60 0.346 0.124

Dose2 14.22 1.000 0.442

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

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

Numerator df Denominator df F-stat P-value

2 9 0.72 0.512

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 -14.09 . -13.42 . . . . . .

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

EPA MRID Number 48614906

Page 75 of 91 Dose1 -12.75 0.546 -13.42 0.766 . . . . .

Dose2 -14.27 0.987 -14.27 0.560 0.538 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 1.42 0.491

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -14.69 . .

Dose1 -12.60 0.346 0.876

Dose2 -14.22 1.000 0.558

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

084301 Benfluralin 890.1350 48614906
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

0.975 0.955 8.367 0.009 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 1.15 0.14 0.07 12.32 0.92, 1.38
Dose1 4 1.44 0.43 0.21 29.82 0.76, 2.12
Dose2 4 1.46 0.12 0.06 8.51 1.26, 1.66

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

Ctrl 1.14 1.01 1.32 . .

Dose1 1.41 0.99 1.94 125.00 -25.00

Dose2 1.45 1.34 1.62 127.17 -27.17

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

Numerator df Denominator df F-stat P-value

2 9 1.65 0.245

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

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

Ctrl 1.15 . 1.35 . . . . . .

Dose1 1.44 0.276 1.35 0.902 . . . . .

Dose2 1.46 0.229 1.35 0.923 0.991 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

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

EPA MRID Number 48614906

Page 76 of 91 Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 3.23 0.199

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 1.14 . .

Dose1 1.41 0.678 0.718

Dose2 1.45 0.067 0.928

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

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

Numerator df Denominator df F-stat P-value

2 9 1.65 0.245

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

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

Ctrl -1.15 . -1.15 . . . . . .

Dose1 -1.44 0.276 -1.44 0.099 . . . . .

Dose2 -1.46 0.229 -1.46 0.085 0.991 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 3.23 0.199

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 -1.14 . .

Dose1 -1.41 0.678 0.282

Dose2 -1.45 0.067 0.072

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

084301 Benfluralin 890.1350 48614906
ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

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

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

EPA MRID Number 48614906

Page 77 of 91 BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 0.00 0.00 0.00 . . , .
Dose1 4 0.00 0.00 0.00 . . , .
Dose2 4 0.00 0.00 0.00 . . , .

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

Ctrl 0.00 0.00 0.00 . .

Dose1 0.00 0.00 0.00 . .

Dose2 0.00 0.00 0.00 . .

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

Numerator df Denominator df F-stat P-value

. . . 9 . . . . . . .

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

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

Ctrl 0.00 . . . . . . . .

Dose1 0.00 . . . . . . . .

Dose2 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.00 1.000

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.00 . .

Dose1 0.00 1.000 .

Dose2 0.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL

Williams Dose1

Jonckheere Dose1

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

Numerator df Denominator df F-stat P-value

. . . 9 . . . . . . .

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

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

Ctrl 0.00 . . . . . . . .

Dose1 0.00 . . . . . . . .

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

EPA MRID Number 48614906

Page 78 of 91 Dose2 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.00 1.000

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.00 . .

Dose1 0.00 1.000 .

Dose2 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL

Williams Dose1

Jonckheere Dose1

084301 Benfluralin 890.1350 48614906
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

0.917 0.261 2.024 0.188 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 28.38 4.27 2.13 15.05 21.58, 35.17
Dose1 4 25.63 2.10 1.05 8.18 22.29, 28.96
Dose2 4 28.88 4.80 2.40 16.63 21.23, 36.52

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

Ctrl 30.25 22.00 31.00 . .

Dose1 25.25 23.50 28.50 90.31 9.69

Dose2 29.50 23.00 33.50 101.76 -1.76

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

Numerator df Denominator df F-stat P-value

2 9 0.80 0.477

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 28.38 . 28.38 . . . . . .

Dose1 25.63 0.529 27.25 0.412 . . . . .

Dose2 28.88 0.976 27.25 0.439 0.494 . . . .

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

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

EPA MRID Number 48614906

Page 79 of 91 Degrees of Freedom TestStat P-value

2 1.50 0.472

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 30.25 . .

Dose1 25.25 0.346 0.124

Dose2 29.50 0.678 0.558

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

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

Numerator df Denominator df F-stat P-value

2 9 0.80 0.477

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 -28.38 . -27.00 . . . . . .

Dose1 -25.63 0.529 -27.00 0.770 . . . . .

Dose2 -28.88 0.976 -28.88 0.539 0.494 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 1.50 0.472

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -30.25 . .

Dose1 -25.25 0.346 0.876

Dose2 -29.50 0.678 0.442

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

084301 Benfluralin 890.1350 48614906 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

0.958 0.754 1.335 0.311 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

EPA MRID Number 48614906

Page 80 of 91 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 30.68 15.52 7.76 50.58 5.99, 55.36
Dose1 4 21.73 17.67 8.84 81.34 -6.39, 49.84
Dose2 4 24.23 8.39 4.19 34.63 10.88, 37.57

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

Ctrl 30.25 13.20 49.00 . .

Dose1 22.80 0.00 41.30 70.82 29.18

Dose2 21.95 17.30 35.70 78.97 21.03

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

Numerator df Denominator df F-stat P-value

2 9 0.41 0.675

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

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

Ctrl 30.68 . 30.68 . . . . . .

Dose1 21.73 0.602 22.98 0.280 . . . . .

Dose2 24.23 0.759 22.98 0.300 0.968 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.50 0.779

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 30.25 . .

Dose1 22.80 0.678 0.282

Dose2 21.95 0.678 0.330

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

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

Numerator df Denominator df F-stat P-value

2 9 0.41 0.675

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

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

Ctrl -30.68 . -25.54 . . . . . .

Dose1 -21.73 0.602 -25.54 0.772 . . . . .

Dose2 -24.23 0.759 -25.54 0.805 0.968 . . . .

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

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

EPA MRID Number 48614906

Page 81 of 91 NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 0.50 0.779

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -30.25 . .

Dose1 -22.80 0.678 0.718

Dose2 -21.95 0.678 0.670

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

084301 Benfluralin 890.1350 48614906 ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.646 <.001 8.826 0.008 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 4 99.15 0.13 0.06 0.13 98.94, 99.36
Dose1 4 74.10 49.40 24.70 66.67 -4.51, 152.71
Dose2 4 98.70 0.83 0.42 0.84 97.38, 100.02

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

Ctrl 99.15 99.00 99.30 . .

Dose1 98.65 0.00 99.10 74.74 25.26

Dose2 98.70 97.70 99.70 99.55 0.45

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

Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 99.15 . 99.15 . . . . . .

Dose1 74.10 0.393 86.40 0.324 . . . . .

Dose2 98.70 1.000 86.40 0.346 0.472 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 2.46 0.293

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

EPA MRID Number 48614906

Page 82 of 91 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 99.15 . .

Dose1 98.65 0.225 0.070

Dose2 98.70 0.346 0.119

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

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

Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 -99.15 . -86.63 . . . . . .

Dose1 -74.10 0.393 -86.63 0.807 . . . . .

Dose2 -98.70 1.000 -98.70 0.627 0.472 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups
Degrees of Freedom TestStat P-value

2 2.46 0.293

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 -99.15 . .

Dose1 -98.65 0.225 0.930

Dose2 -98.70 0.346 0.881

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

084301 Benfluralin 890.1350 48614906 ANALYSIS RESULTS FOR VARIABLE VAR13 ( F testosterone (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

. . . . NO DATA FOR TEST

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 0 . . . . . , .
Dose1 0 . . . . . , .

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

EPA MRID Number 48614906

Page 83 of 91 Dose2 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

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

Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 . . .

Dose1 . . .

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

2 9 1.01 0.402

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 . . .

Dose1 . . .

Dose2 . . .

Jonckheere Dose1

084301 Benfluralin 890.1350 48614906 ANALYSIS RESULTS FOR VARIABLE VAR14 ( M testosterone (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

. . . . NO DATA FOR TEST

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 0 . . . . . , .
Dose1 0 . . . . . , .
Dose2 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

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

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

EPA MRID Number 48614906

Page 84 of 91 Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 . . .

Dose1 . . .

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

2 9 1.01 0.402

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 . . .

Dose1 . . .

Dose2 . . .

Jonckheere Dose1

084301 Benfluralin 890.1350 48614906 ANALYSIS RESULTS FOR VARIABLE VAR15 ( F 17b-estradiol (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

. . . . NO DATA FOR TEST

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 0 . . . . . , .
Dose1 0 . . . . . , .
Dose2 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

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

Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 . . .

Dose1 . . .

Dose2 . . .

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

EPA MRID Number 48614906

Page 85 of 91 Jonckheere Dose1

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

Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 . . .

Dose1 . . .

Dose2 . . .

Jonckheere Dose1

084301 Benfluralin 890.1350 48614906 ANALYSIS RESULTS FOR VARIABLE VAR16 ( M 17b-estradiol (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

. . . . NO DATA FOR TEST

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

BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
Ctrl 0 . . . . . , .
Dose1 0 . . . . . , .
Dose2 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

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

Numerator df Denominator df F-stat P-value

2 9 1.01 0.402

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 . . .

Dose1 . . .

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

2 9 1.01 0.402

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

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

EPA MRID Number 48614906

Page 86 of 91 Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .

Dose1 . . .

Dose2 . . .

Jonckheere Dose1

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

EPA MRID Number 48614906

Page 87 of 91 

### Male GSI (Mann-Whitney exact test)



### Obs **conc endpoint** 

**1** 

## 0 1.01

**2** 

## 0 1.32

**3** 

## 0 1.21

**4** 

## 0 1.06

**5** 

## 1 1.19

**6** 

## 1 1.63

**7** 

## 1 0.99

**8** 

## 1 1.94

**9** 

## 2 1.62

**10** 

## 2 1.39

**11** 

## 2 1.50

**12** 

## 2 1.34

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

EPA MRID Number 48614906

Page 88 of 91 

## The NPAR1WAY

Procedure

**Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc** 

**conc N**  **Sum of**

**Scores Expected** 

**Under H0 Std Dev** 

**Under H0 Mean** 

**Score**

**0** 

## 4 16.0 26.0 5.887841 4.00

**1** 

## 4 28.0 26.0 5.887841 7.00

**2** 

## 4 34.0 26.0 5.887841 8.50

**Kruskal-Wallis Test Chi-Square**  

## 3.2308

**DF** 

## 2 **Asymptotic Pr > Chi-Square** 

## 0.1988
**Exact Pr >= Chi-Square** 

## 0.2118

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

EPA MRID Number 48614906

Page 89 of 91 

## The NPAR1WAY

Procedure

**Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc** 

**conc N**  **Sum of**

**Scores Expected** 

**Under H0 Std Dev** 

**Under H0 Mean** 

**Score**

**0** 

## 4 16.0 18.0 3.464102 4.0

**1** 

## 4 20.0 18.0 3.464102 5.0

**Wilcoxon Two-Sample Test**
**Statistic (S)** 

## 16.0000

**Normal Approximation**

**Z** 

## -0.4330

**One-Sided Pr < Z** 

## 0.3325
**Two-Sided Pr > |Z|** 

## 0.6650

**t Approximation**

**One-Sided Pr < Z** 

## 0.3390
**Two-Sided Pr > |Z|** 

## 0.6780

**Exact Test**

**One-Sided Pr <= S** 

## 0.3429
**Two-Sided Pr >= |S - Mean|** 

## 0.6857

**Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test Chi-Square**  

## 0.3333

**DF** 

## 1 **Pr > Chi-Square** 

## 0.5637

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

EPA MRID Number 48614906

Page 90 of 91 

## The NPAR1WAY

Procedure

**Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc** 

**conc N**  **Sum of**

**Scores Expected** 

**Under H0 Std Dev** 

**Under H0 Mean** 

**Score**

**0** 

## 4 10.0 18.0 3.464102 2.50

**2** 

## 4 26.0 18.0 3.464102 6.50

**Wilcoxon Two-Sample Test**
**Statistic (S)** 

## 10.0000

**Normal Approximation**

**Z** 

## -2.1651

**One-Sided Pr < Z** 

## 0.0152
**Two-Sided Pr > |Z|** 

## 0.0304

**t Approximation**

**One-Sided Pr < Z** 

## 0.0336
**Two-Sided Pr > |Z|** 

## 0.0671

**Exact Test**

**One-Sided Pr <= S** 

## 0.0143
**Two-Sided Pr >= |S - Mean|** 

## 0.0286 **Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test Chi-Square**  

## 5.3333

**DF** 

## 1 **Pr > Chi-Square** 

## 0.0209Data Evaluation Record onthe Fish Short-Term Reproduction Assay with BenfluralinEPAMRIDNumber48614906Page91of91Control Comparisons% Mort.(all)% Mort.(male)% Mort.(female)Fecundity% Fert.Wt(male)Length(male)Wt. (female)Length (female)Neg Control00049.099.23.7253.41.8143.300023.999.04.8657.31.7843.200013.299.15.5161.62.2146.416.702536.699.34.8658.21.8544.8Average4.20.06.25030.799.24.7457.61.9144.4SolventControl00026.999.34.1653.32.1645.900026.099.24.2654.52.0245.400011.199.53.4652.12.0044.816.702511.098.24.2654.21.6741.4Average4.20.06.25018.899.14.0453.51.9644.4t-test pvalue1.000#DIV/0!1.0000.2310.7480.1450.0600.7410.970Control ComparisonsGSI(male)GSI(female)Tubercle(male)VTG(male)VTG(Female)GSI(male)GSI(female)Tubercle(male)VTG(male)VTG(Female)Neg Control1.0111.422.00.001010159.551.0111.422.00.001010159.551.3215.630.00.00062669.521.3215.630.00.00062669.521.2214.131.00.00109095.741.2214.131.00.00109095.741.0715.230.50.000673101.311.0715.230.50.000673101.31Average1.214.128.40.000850106.531.214.128.40.000850106.53SolventControl1.113.928.50.00211094.081.113.928.50.00211094.081.312.731.00.00272090.521.312.731.00.00272090.521.313.224.00.000948117.111.313.224.00.000948117.111.112.521.00.00003383.691.112.521.00.00003383.69Average1.213.126.10.00145396.351.213.126.10.00145396.35t-test pvalue0.6570.3540.4940.3620.6340.6570.3540.4940.3620.634

# DATA EVALUATION RECORD

BENFLURALIN

Study Type: O CSPP 890.1400, Hershberger Assay in Rats

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614907)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road,

Bui ldin g 100, Suit e B

Durham, NC 27713

Primary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date : 11/ 15 / 20 11

Secondary Reviewer : Signature: Rebecca L. Bryan, B.S. Date: 11/ 15 / 20 11

Program Manager : Signature: Jack D. Early, M.S. Date: 11/ 15 / 20 11

Quality Assurance: Signature: Jack D. Early, M.S Date: 11/ 15 / 20 11

Disclaimer

This Data Evaluation Record may have been altered by the Endocrine Disruptor Review Team subsequent to signing by Dynamac Corporation personnel.

## 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 rob ustness 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

indiv idual 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).In vivo Hershberger Assay ( 2011 ) / Page 2 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## samples were taken prior to sacrifice to evaluate serum testosterone levels in animals treated with TP + 0, 50, 250 or 750 mg/kg/day benfluralin for 10 days.

In addition, a 14 

## C-t estosterone clearance experiment was conducted to examine if the decrease in androgen

- resp onsive tissue weights were attributed to enhanced clearance of exogenous testosterone secondary to hepatic enzyme induction by benfluralin. In this study, two groups of seven castrated, jugular vein

- cannulated male rats were dosed with benfluralin daily v ia oral

gavage to at dose levels of 0 (vehicle) or 750 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s ubcutaneous injection. 14 

## C- Testosterone was administered on Days 1 and 10 to measure plasma clearance of 14 

## C- te stosterone radioactivity. On Day 1, all animals had blood samples collected from the cannulae at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, and 6 h post-dosing. On Day 10, three controls and four 750 mg/kg/day rats remained patent and had blood samples collected as on Day 1, except the 6 h sample was collected via the orbital sinus. The non-patent animals only had 6 h samples co llected via the orbital sinus. Clinical signs, body weight and androgen responsive tissue weights were also evaluated.

All animals survived until scheduled termination. There were no treatment

- related gross pathological findings.

In the androgen agonist assay, 3/7 rats were noted to have decreased feces and 2/7 rats were observed with clear perioral soiling at 750 mg/kg/day. Additionally at this dose, b ody weights were decreased (p≤0.05, except not significant [NS] on Day 11) on Days 4, 7, and 11 by 7 -11%, resulting in a 15.0 g cumulative body weight loss (p≤0.05) for Days 1

- 4 and decreased (p≤0.05) cumulative body weigh t gains for Days 1

- 7 (↓76%) and Days 1

- 11 (↓49%). Cumulative body weight gains were also decreased (p≤0.05) by 61% for Days 1 -4 in the 250 mg/kg/day group; however, body weights in this group were similar to controls, as were cumulative body weight gains for Days 1 -7 and 1-11. Animals dosed with 0.4 mg/kg/day TP had 6- 7% increases (p≤0.05) in body weights on Days 7 and 11, resulting in increased (p≤0.05) body weight gains for Days 1

- 4 (↑55%), 1

- 7 (↑60%) and 1

- 11 (↑44%). During Days 1 -4, food consumption was decreased (p≤0.05) by 12 -44% at 250 mg/kg/day and above. There were no significant increases in any of the five target accessory sex organs of animals dosed with the test compound. Absolute liver weights were increased (p≤0.05) by 38 -68% in the 250 mg/kg/day and above groups. Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases in all five accessory sex organs indicating that the assay was responding properly. The %CVs for the vehicle controls, 0.4 mg/kg/day TP group, and the 750 mg/kg/day group were compared to the

pe rformance criteria values as stated in the Guideline. The %CV for the ventral prostate in the vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended ); however, is unlikely that these data would have affected the interpretation of the assay results since no increases were observed in the other tissue weights. All other %CVs were less than the

maximum recommended values.

In the anti-androgen assay , 3/7 rats at 750 mg/kg/day were observed to have decreased feces. Cumulative body weight gains were decreased (p≤0.05, except NS on Day 11) on Days 1 -4 (↓55%), 1

- 7 (↓37%) and 1

- 11 (↓23%) at 250 mg/kg/day; however, body weights in this group

were similar to controls. At 750 mg/kg/day, body weights w ere decreased (p≤0.05) on Days 4, 7, and 11 by 10- 14%, resulting in a 15.1 g cumulative body weight loss (p≤0.05) for Days 1 -4 and decreased (p≤0.05) cumulative body weight gains for Days 1

- 7 (↓69%) and Days 1 -11 (↓57%). Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05)In vivo Hershberger Assay ( 2011 ) / Page 3 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## cumulative body weight gains on Days 1- 4 (↓22%) and 1

- 7 (↓19%); however body weights and cumulative body weight gains on Days 1- 11 were similar to TP controls. During Days 1 -4, food consumption was decreased (p≤0.05) by 23 -50% at 250 mg/kg/day and above, and was decreased at 750 mg/kg/day during Days 4- 7 and 7

- 11 (↓13% both periods).

In the 750 mg/kg/day group, accessory sex organ weight were significantly decreased in two of the five target tissues: levator ani -bulbocavernosus (LABC; 33%) and seminal vesicles (27%)

. Non -significant decreases were observed in the remaining three target tissues. Absolute liver weights were increased (p≤0.05) by 39 -65% in the 250 mg/kg/day and above groups. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights in all five accessory tissues to indicate that the test system was responding properly to detect androgen antagonists. In rats treated with 750 mg/kg/day + 0.4 mg/kg/day TP, serum testosterone levels were decreased (NS) by 29% compared to TP controls. The %CVs for the vehicle controls, 0.4 mg/kg/day TP group, and the 750 mg/kg/day group were compared to the performance crit eria values according to the Guideline. The %CV for the ventral prostate in the vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended ), but was unlikely to have affected the interpretation of the assay results. All other %CV were less than

the maximum recommended values.

In the 14 

## C-testosterone clearance experiment, 6/7 rats dosed with 750 mg/kg/day had urine perineal staining, and 2/7 rats had red perinasal staining. In the 750 mg/kg/day group, body weights were decreased (p≤0.0 5) by 12-20% on Days 4, 7, and 10; resulting in body weight losses of 18.7 g and 1.0 g for Days 1-4 and 1- 7, and decreased (p≤0.05) cumulative body weight gains for Days 1

- 10 (↓85%). Animals dosed with 750 mg/kg/day of the test compound had nonsignifican t decreases in accessory sex organ weights: 17% in Cowper’s gland; 25% in glans penis; 37% in LABC; 53% in ventral prostate; and 48% in seminal vesicles. Absolute liver weights were increased (NS) by 38% in this group. Rats treated for 10 days with 750 mg/kg/day of the test compound cleared 14 

## C-radioactivity 3.7- fold faster (p≤0.05) than the TP -only controls.

This was a result of the elimination half

- life present in the 750 mg/kg/day group. Additionally, the 750 mg/kg/day animals had an AUC that was lo wer (p≤0.05) than the TP -only controls (0.014 μg×h× g -1 

## treated versus 0.039 μg×h× g -1 

## control ).

Benfluralin was negative for androgenic activity in the Hershberger assay as n o statistically significant increases in organ wei ghts were seen in two or more of the five androgen sensitive

tissues in the androgen agonist assay. However, benfluralin was positive for anti-androgenicity in the Hershberger assay as s tatistically significant decreases in organ weights were seen in two
of th e five androgen sensitive tissues in the androgen antagonist assay. At 750 mg/kg/day, a significant decrease in body weight (12%; p<0.05) and body weight gain (57%; p<0.05) were seen after 10 days of treatment indicating overt toxicity at the dose that sh owed decreases in androgen

- sensitive tissue weights . Additional data provided on testosterone clearance in benfluralin-treated rats in conjunction with the observed increase in liver weights, provide evidence that the reduced accessory sex organ weights may be secondary to increased

testosterone metabolism and clearance

.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Hershberger a ssay (OCSPP 890.1400).In vivo Hershberger Assay ( 2011 ) / Page 4 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



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

Assurance statements were provided.In vivo Hershberger Assay ( 2011 ) / Page 5 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Toxicology & Environmental Research and Consulting

Location: The Dow Chemical Company, Midland, MI Study Director: V.A. Marshall

Other Personnel: M.S. Marty, Lead Scientist S.C. Hansen, Biotransformation Specialist Y.C. Jeong, Toxicologist



## 2. Test Substance : Benfluralin (Benefin)

Description: yellow solid

Source: Dow AgroSciences LLC, Indianapolis, IN

Lot # : ACD13683 (TSN 100037) Purity: 97.5 % a.i. Stability: Stable in the corn oil vehicle for up to 23 days at room temperature

CAS #: 1861

- 40

- 1

Structure:



## 3

. Reference Androgen : Testosterone propionate (TP)

Supplier Sigma

- Aldrich, St. Louis, MO

Lot #: 048K1328 Purity: ≥ 98 %

CAS # : 57

- 82

- 5



## 4

. Reference Anti

- androgen : Flutamide (FT)

Supplier Sigma

- Aldrich, St. Louis, MO

Lot #: 099K1112 Purity: >99 %

CAS # : 1311

- 84

- 7



## 5

. Solvent/Vehicle Control : Corn oil

Supplier Sigma

- Aldrich (St. Louis, MO)

Lot/Batch #: MKBD2427
Rationale (if other than water) Corn oil was selected as vehicle based on solubility properties of the test material

Final concentration 4 ml/kg bw (benfluralin and flutamide); 0.5 mg/kg bw (s.c. TP)



## 6

. R adiolabeled a ndrogen for testosterone clearance exp. : [ 14 

## C]

- Testosterone (testosterone clearance study)

Supplier Perkin Elmer, Inc. (Boston MA)

Lot #: 3624214 Purity: 97 %

CAS # : Specific Activity

Vehicle: 58

- 22

- 0

58.8 mCi/mmol Approx. 10% Intralipid (20% Intralipid from Baxter Healthcare Corp. ) Deerfield, IL. Diluted 50:50 with sterile saline.In vivo Hershberger Assay ( 2011 ) / Page 6 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## 7

. Test Animals :
Species: Rat

Strain: Sprague Da w ley (Crl:CD[SD])

Age/weight at dose initiation: P ost

- natal day (PND) 55

– 243.7

- 283.4 g

Source: Charles River Laboratories, Inc., Portage, MI Housing: Rats were housed 2

- 3/cage in stainless steel cages with wire mesh floors, suspended above absorbent paper . Non

- woven gauze was placed in the cages to provide a cushion. For the 14 C- testosterone clearance experiment, rats were housed individually in glass Roth

- type metabolism cages.

Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO), ad libitum Phytoestrogen content ( μg of genistein equivalents/gram diet ) not provided

Water: T ap water

, ad libitum

Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22 ± 1 oC

40

- 70 %

12

- 15 /hr

12 h light/ 12 h dark Acclimation period: P rior to castration : 5

- 6 days (animals castrated by Supplier on PND 45)

P ost

- castration : 10 days prior to initiation of dosing Rats were acclimated to the performing laboratory for 7 days prior to initiation

of treatment. Jugular vein

- cannulated rats were acclimated in metabolism cages for five days prior to treatment.



## B. STUDY DESIGN

1. In -l ife D ates : Main Study: Start: October 3, 2010 End: August 11, 2011 Testosterone Clearance Experiment: Start: August 2, 2011 End: August 11, 2011

2. Study Design : Hershberger Assay: The present Hershberger a ssay was conducted to screen for both potential anti -androgenic and androgenic activity . T he test substance was administered daily via oral gavage to groups of castrated male rats both in conjunction with a daily dose of TP ( 0.4 mg/kg/day) by s ubcutaneous injection (anti -androgenic activity) and without TP (androgenic activity) . Anti- androgenic activity is indicated by a statistically significant decrease in two or more target organ weights of the treated groups (test substance + TP) compared to the TP-only control group. Positive androgenic activity is defined as a significant increase in two or more organ weights in the absence of TP compared to the vehicle control. For both assays, the animals were dosed for 10 consecutive day s and sacrificed approximately 24 hours after the final dose administration. On necropsy day,

animals were anesthetized with isofluorane/O 2 

## and blood was collected via cardiac puncture for serum testosterone measurements. Animals were euthanized by decapitation and necropsied for organ weight measurements. Two vehicle control groups were included in the androgenic portion of the study. One vehicle control group for oral corn oil only and a second vehicle control group that was administered corn oil by oral gavage and s.c. to determine that s.c. administration of corn oil did not alter baseline accessory sex organ weights. Liver, kidney and adrenal weights were also measured

.

Testosterone Clearance Experiment: Based on the resu lts of the main Hershberger assay, a testosterone clearance experiment was conducted to determine if benfluralin could enhanceIn vivo Hershberger Assay ( 2011 ) / Page 7 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## testosterone metabolism by inducing hepatic enzymes resulting in increased TP clearance and decreased accessory sex tissue weight s. This hypothesis was supported by evidence of increased liver weights and dose- dependent decreases in serum testosterone levels observed in the Hershberger assay. To evaluate the effect of benfluralin on testosterone clearance, two groups of seven castrated and cannulated rats (surgery conducted on PND 45) were dosed as described above for the Hershberger assay with 0 or 750 mg/kg/day benfluralin via oral gavage. All animals received TP as described above. On Day 1 (PND 55) and Day 10 (PND 64), animals were administered 14 

## C-testosterone by intravenous injection (approximately 13 μg/kg). Unlabeled TP was administered s.c. on Days 2-9. To measure plasma clearance of

14 

## C-testosterone, radioactivity in plasma was monitored on Day prior to administration of benfluralin or vehicle and on Day 10 after benfluralin or vehicle treatment. The benfluralin and vehicle control groups each had eight time-course blood samples scheduled at 5 min., 10 min., 15 min., 30 min., 1 hr., 2 hr., 3 hr., and 6 hr. post radiolabeled testosterone administration. Plasma samples were analyzed for radioactivity by liquid scintillation spectrometry (LSS). On Day 10, three control animals and four b enfluralin

- treated animals had patent cannulas which resulted in full blood/plasma time course. From the remaining animals, a limited number of time points were obtained by collection via the jugular vein. The testosterone clearance experiment also evaluat ed clinical signs, body weight and target organ weights as described for the Hershberger Assay.

3. Study Schedule : S exually mature male Sprague Dawley rats were castrated on PND 4 5 by

the s upplier and allowed 10 days for recovery and regression of accessory sex organ weights prior to initiation of dosing. The dose administration period was from PND 55 through

PND 6 4. Rats were euthanized on PND 65 approximately 24 hours after the last dose and necropsied for or gan weight measurements.

4. Animal Assignment : Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1 using a computer program designed to increase the probability of uniform group mean weights and standard deviations on Day 1 . Statistical analysis indicated that there were no significant differences in group means at study initiation. It was

not stated if the body weight of each animal was within 20% of the overall mean.In vivo Hershberger Assay ( 2011 ) / Page 8 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



### TABLE 1

. Study design a

Test group Dose (mg/kg/day) # of Males

Androgen Agonist Assay
Vehicle control ( oral vehicle only) 0 7

V ehicle control (oral and s.c. vehicle ) 0 7

Low 50 7

Medium 250 7
High 750 7

Anti

- Androgen Assay

Vehicle control (+ TP) 0 7
Low (+ TP) 50 7
Mid (+ TP) 250 7
High (+ TP) 750 7

F T (oral gavage) ( + TP ) 0 7

14 C

- Testosterone Clearance Assay
Vehicle control (+ TP) 0 7
High (+ TP) 750 7

a Data were obtained from Text Table 1 on page 22 of the study report.



## 5. Dose Selection Rationale: The dose levels used in the present study were selected based on the results of a probe study 1 

## conducted by the Performing Laboratory, in which groups of

4 intact male and female Crl:CD(SD) rats were administered the test compound in corn oil by oral gavage at dose levels of 0, 250, 500, 750, or 1000 mg/kg/day for 15 days. Males were dosed on PND 30-44; females were dosed on PND 22-36. Both sexes exhibited decreased body weights and food consumption at 250 mg/kg/day and above. On Day 15, males showed decreased body weights of 7.2%, 11.5%, 12.3% and 13.5% at 250, 500, 750 and 1000 mg/kg/day, respectively. At necropsy, clear facial soiling was noted in females at all dose levels, and in 2-3 males/group at 750 mg/kg/day and above. One male in each of the 750 and 1000 mg/kg/day groups had bilateral decreased epididymides and testes sizes, and 1 male in each of the 500 and 750 mg/kg/day groups and 2 males in the 1000 mg/kg/day group had bilateral decreased seminal vesicle size. Additionally, relative liver weights were

increased 17%, 30%, 53% and 60% at 250, 500, 750 and 1000 mg/kg/day , respectively in both sexes, and hepatocellular hypertrophy was noted at 10 00 mg/kg/day. Based on these findings, 750 mg/kg/day was expected to produce decreased terminal body weights and some systemic toxicity, but not mortality or morbidity, in the present study.

6. (a) Dose Preparation : Dose formulations were prepared prior to treatment by suspend ing appropriate amounts of the test compound in corn oil. Flutamide and TP were prepared as

solutions in corn oil. 14 

## C-Testosterone was prepared fresh on Days 1 and 10 as an emulsion in approximately 10% Intralipid Fat Emulsion. Homogeneity was measured prior to

administration in the 50 and 750 mg/kg/day formulations, concentration analyses were performed prior to administration on all dose levels. Stability analyses were performed on 0.25, 2.5, and 250 mg/mL formulations prior to administration following storage at room temperature for up to 23 days.

1 Marty, M.S., and Marshall, V.A. (in progress) Benefin: Hershberger, Uterotrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland,

MI.In vivo Hershberger Assay ( 2011 ) / Page 9 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## ( b ) Dose Analysis

Results

Homogeneity (% RSD) : 1.2-7.3%

Stability (% of Day 0): 94.9-114.3% following storage at room temperature for up to 23 days.

Concentration (% of nominal): 90.0-98.0%

The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable.

7. Dosage A dministration : The dose suspensions were administered to the animals daily via oral gavage (dose volume 4 mL/kg) for 10 consecutive days. Flutamide was administered daily via oral gavage (dose volume 4 mL/kg) at a dose level of 3 mg/kg/day for 10 consecutive days. TP was given via s.c. injection (dose volume 0.5 mL/kg) at a dose level of 0.4 mg/kg/day. 14 

## C-Testosterone was administered intravenously via indwelling jugular vein cannulae on Days 1 and 10 at target doses of approximately 0.85 μ Ci/rat. The actual radioactive dose administered was determined gravimetrically. Dose s were adjusted daily

based on the most recent body weight.

8. Statistics : Body weights, body weight gains, absolute organ weights, and food consumption were first analyzed by Bartlett’s test for equality of variances. Depending on the results of Bartlett’s test, either a parametric (Steel and Torrie, 1960) or non- parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was pe rformed. If the ANOVA was significant (p ≤ 0.05), either Dunnett’s test or the Wilcoxon Rank- Sum test with Bonferroni’s correction was performed, as appropriate. Coefficients of variation were calculated for organ weights from the control and 750 mg/kg/day groups and compared with the performance criteria provided in the Guidelines . It was stated that preputial separation occurred in all animals; therefore, the incidence of preputial separation was not analyzed. Statistical outliers were identified by a s equential method (Grubbs, 1969), and excluded if justified by sound scientific reasons. Descriptive statistics were used for the

14 

## C- testosterone clearance assay. Pharmacokinetic parameters were estimated using PK Solutions (v.2.0.6, Summit Research Services, Montrose, CO), and analyzed with Student’s

t- test. Significance was denoted at p ≤ 0.05. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations : Cage -side checks for mortality, moribundity, and clinical signs of toxicity were conducted twice daily. Additionally, animals were given a hand-held examination for clinical signs and alterations in behavior or demeanor once daily approximately 1 h after dosing.

2. Body Weight: A ll a nimals were weighed at randomization and daily throughout the dosing period. In the main study, body weights were reported for Days 1, 4, 7, and 11, and bodyIn vivo Hershberger Assay ( 2011 ) / Page 10 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## weight gains (relative to Day 1) were calculated for Days 4, 7, and 11. For the

14 

## C- testosterone clearance experiment , body weights were reported for Days 1, 4, 7, and 10, and body weight gains (relative to Day 1) were calculated for Days 4, 7, and 10.

3. Food Consumption : In the main study, f ood consumption (g/rat/day) was measured for Days 1 -4, 4-7, and 7-11. Food consumption was not measured in the 14 

## C- testosterone clearance experiment

.

4. Serum Hormone Measurements : Serum from animals dosed with TP alone or TP and the

test compou nd were transferred to an off -site laboratory (AniLytics Inc., Gaithersburg, MD ) for testosterone analysis. Total testosterone was measured using an RIA kit (Coat-A-Count Total Testosterone RIA kit, Siemens Diagnostics, CA).

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, anesthetized with isoflurane, and blood was collected by cardiac puncture for serum testosterone analysis as described above. The animals were then euthanized by decapitation

, exsanguinated, and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesicles with coagulating glands and fluid , LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). The liver, kidneys, and adrenals were also collected

and weighed. All tissues were preserved in neutral, phosphate-buffered 10% formalin. Additionally, a portion of the liver was frozen in liquid nitrogen for possible evaluation of liver enzyme induction.

6. 14 

## C- Testosterone Clearance Analysis : On Days 1 -10, either vehicle or 750 mg/kg/day of the test compound was administered to the rats. The rats were dosed on Days 1 and 10 with

14 

## C-testosterone via the jugular vein cannulae or by the tail vein (if the cannulae were not patent). On Days 2-9, the rats were dosed with TP as previously described. On Day 1, all animals had blood samples collected from the cannulae at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, and 6 h post-dosing. On Day 10, three controls and four 750 mg/kg/day rats remained patent and had blood samples collected as on Day 1, except the 6 h sample was collected via the orbital

sinus. The non- patent animals only had 6 h samples collected via the orbital sinus. Blood samples were centrifuged, and radioactivity was quantified by liquid scintillation counting (LSC). Counts were corrected for quench (method not provided) and converted to disintegrations per minute (dpm). Background was determined by counting blank samples, and samples with dpm less than twice background were considered to have too little radioactivity to quantify.

II. RESULTS

A. OBSERVATIONS

1. Mortality : All animals survived until scheduled termination.In vivo Hershberger Assay ( 2011 ) / Page 11 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## 2. Clinical signs of toxicity : At 750 mg/kg/day, 6/14 rats were noted to have decreased feces. Additionally, 2/7 rats dosed at 750 mg/kg/day with no TP were observed with clear perioral soiling. In the 14 

## C-testosterone clearance assay, 6/7 rats dosed with 750 mg/kg/day h ad urine perineal staining, and 2/7 rats had red perinasal staining. There were no other

treatment

- related clinical signs.

B. BODY WEIGHT AND WEIGHT GAIN : Body weight and body weight gain data for the androgen agonist assay are presented in Table 2. At 750 mg/kg/day, body weights were decreased (p≤0.05, except not significant [NS] on Day 11) on Days 4, 7, and 11 by 7 -11%, resulting in a 15.0 g cumulative body weight loss (p≤0.05) for Days 1

- 4 and decreased (p≤0.05) cumulative body weight gains for Days 1- 7 (↓76%) and Days 1

- 11 (↓49%). Cumulative body weight gains were also decreased (p≤0.05) by 61% for Days 1 -4 in the 250 mg/kg/day group; however, body weights in this group were similar to controls, as were cumulative body weight gains for Days 1-7 and 1-11. There were no effects on body weights or body weight gains in the 50 mg/kg/day group. Animals dosed with 0.4 mg/kg/day TP had 6- 7% increases (p≤0.05) in body weights on Days 7 and 11, resulting in increased (p≤0.05) body weight gains for Days 1 -4 ( ↑55%), 1

- 7 (↑60%) and 1

- 11 (↑44%).

There were no differences in body weights or body weight gains between the rats that received only oral vehicle and animals that received oral and s.c. vehicle.

TABLE 2. Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day

# Dose (mg/kg/day)

Vehicle Control TP 50 250 750

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 7 260.4 6.7 7 259.7 12.7 7 263.9 11.4 7 262.2 11.4 7 262.5 11.4

4 7 277.7 7.4 7 286.4 16.0 7 279.7 15.5 7 268.9 10.5 7 247.5* (↓11) 13.2

7 7 291.0 8.1 7 308.8* (↑6) 17.1 7 294.3 17.3 7 286.6 15.8 7 269.7* (↓7) 14.1

11 7 310.2 9.3 7 331.6* (↑7) 20.4 7 311.7 18.5 7 303.1 23.2 7 287.9 (↓7) 17.4

BWG Day

1

- 4 7 17.2 3.0 7 26.7* (↑55) 5.4 7 15.8 5.2 7 6.7* (↓61) 4.2 7

- 15.0* 11.3

Da y 1

- 7 7 30.6 7.6 7 49.1* (↑60) 8.0 7 30.4 7.2 7 24.4 5.6 7 7.2* (↓76) 10.3

Day 1

- 11 7 49.8 8.0 7 71.9* (↑44) 13.5 7 47.8 8.3 7 40.9 13.0 7 25.5* (↓49) 14.2

a Data were obtained from Table 4 on pages 52

- 53 of the study report. Percent differences from controls

, calculated by the

reviewers

, are included in parentheses.

N Number of animals in the group

SD Standard Deviation

* Significantly different from contro ls; p ≤ 0.05



## Body weight and body weight gain data for the anti

- androgen assay are presented in Table

3. At 250 mg/kg/day, cumulative body weight gains were decreased (p≤0.05, except not significant [NS] on Day 11) Days 1- 4 (↓55%), 1

- 7 (↓37%) and 1 -11 (↓23%); however, body weights in this group were similar to controls. At 750 mg/kg/day, body weights were decreased (p≤0.05) on Days 4, 7, and 11 by 10 -14%, resulting in a 15.1 g cumulative body weight loss (p≤0.05) for Days 1

- 4 and decreased (p≤0.05) cum ulative body weight gains for Days 1

- 7 (↓69%) and Days 1

- 11 (↓57%). There were no effects on body weights or bodyIn vivo Hershberger Assay ( 2011 ) / Page 12 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## weight gains in the 50 mg/kg/day group. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) cumulative body weight g ains on Days 1- 4 (↓22%)

and 1- 7 (↓19%); however body weights and cumulative body weight gains on Days 1 -11

were similar to TP controls.

TABLE 3. Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti

- Androgen Assay a

Study Day

# Dose (mg/kg/day)

Vehicle Control TP Negative

Control TP + FT

Positive Control 50 250 750

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 7 260.4 6.7 7 259.7 12.7 7 260.7 9.9 7 261.8 10.6 7 264.4 13.3 7 262.0 7.0

4 7 277.7 7.4 7 286.4 16.0 7 281.4 9.7 7 283.2 8.0 7 276.5 16.7 7 246.9* (↓14) 11.

5

7 7 291.0 8.1 7 308.8 17.1 7 300.5 13.1 7 307.3 10.3 7 295.4 19.0 7 277.0* (↓10) 14.

1

11 7 310.2 9.3 7 331.6 20.4 7 322.6 15.9 7 334.1 9.5 7 319.7 27.4 7 292.8* (↓12) 16.

7

BWG Day

1

- 4 7 17.2 3.0 7 26.7 5.4 7 20.7* (↓22) 2.0 7 21.4 4.0 7 12.1* (↓55) 8.1 7

- 15.1* 8.8

Day 1

- 7 7 30.6 7.6 7 49.1 8.0 7 39.8* (↓19) 4.6 7 45.5 5.9 7 31.0* (↓37) 7.0 7 15.0* (↓69) 11.

2
Day 1

- 11 7 49.8 8.0 7 71.9 13.5 7 61.9 7.3 7 72.3 10.2 7 55.4 (↓23) 16.7 7 30.8* (↓57) 14.

8

a Data were obtained from Table 4 on pages 54

- 55 of the study report. Percent differences from controls

, calculated by the

reviewers , are present ed in parentheses.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls ; p ≤ 0.05



## Body weight and body weight gain data for the 14 

## C- testosterone clearance study are presented in Table 4. In the 750 mg/kg/day group, body weights were decreased (p≤0.05) by 12 -20% on Days 4, 7, and 10; resulting in body weight losses of 18.7g and 1.0 g for Days

1-4 and 1- 7, and decreased (p≤0.05) cumulative body weight gains for Days 1

- 10 (↓85%).

TABLE 4. Mea n (±SD) Body Weights and Cumulative Body Weight Gains (g) in the 14 

### C

- Testosterone Clearance Study a

Study Day # Dose (mg/kg/day)

Vehicle Control 750

N Mean SD N Mean SD

1 7 274.4 29.2 7 275.5 26.1

4 7 292.3 28.6 7 256.8* (↓12) 28.1

7 7 330.9 36.1 7 274.5* (↓17) 26.0

10 7 359.7 38.5 7 288.1* (↓20) 27.7

BWG Day 1

- 4 7 18.0 8.5 7

- 18.7* 8.1
Day 1

- 7 7 56.5 9.9 7

- 1.0* 13.4
Day 1

- 10 7 85.4 10.3 7 12.6* (↓85) 13.3

a Data were obtained from Table 12 on page 74 of the study report. Percent differences from controls

, calculated by the

reviewers , are present ed in parentheses.

N Number of animals in the group

SD= Standard Deviation

* Significantly different from controls ; p ≤ 0.05In vivo Hershberger Assay ( 2011 ) / Page 13 of 19

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## C. FOOD CONSUMPTION : Food consumption data for the androgen agonist assay are presented in Table 5. During Days 1- 4, food consumption was decreased (p≤0.05) by 12-44% at 250 mg/kg/day and above. There were no effects on food consumption during days 4 -7 or 7-11 in any of the dose groups. Food consumption was incr eased (p≤0.05) in animals dosed with 0.4 mg/kg/day TP during Days 1- 4 (↑15%), 4

- 7 (↑17%), and 7 -11 (↑21%).

A nimals that received oral and s.c. vehicle had decreased (p≤0.05) food consumption on Days 1

- 4 (↓10%) compared to the oral vehicle controls

. This minor difference was

considered incidental.

TABLE 5. Mean (±SD) Food Consumption (g/rat/day) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control TP 50 250 750

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1

- 4 7 18.1 1.1 7 20.8* (↑15) 0.6 7 19.7 2.2 7 15.9* (↓12) 1.1 7 10.1* (↓44) 1.6

4

- 7 7 18.5 0.2 7 21.6* (↑17) 0.5 7 19.2 1.5 7 18.4 1.9 7 16.8 1.6

7

- 11 7 18.2 0. 7 7 22.0* (↑21) 1.0 7 18.4 1.8 7 18.9 2.5 7 19.1 2.0

a Data were obtained from Table 5 on pages 57-58 of the study report. Percent differences from controls

, calculated by the

reviewers

, are included in parentheses.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls ; p ≤ 0.05



## Food consumption data for the anti -androgen assay are presented in Table 6. During Days

1- 4, food consumption was decreased (p≤0.05) by 23 -50% at 250 mg/kg/day and above, and was decreased at 750 mg/kg/day during Days 4-7 and 7- 11 (↓13% both periods). There were no effects on food consumption at 50 mg/kg/day at any time point and at 250 mg/kg/day during Days 4 -7 and 7-11. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) food consumption during Days 7

- 11 (↓7%). Food consumption

was not measured in the 14 

## C-t estosterone clearance study.In vivo Hershberger Assay ( 2011 ) / Page 14 of 19

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### TABLE 6. Mean (±SD) Food Consumption (g/rat/day) in the Anti

- Androgen Assay a

Study Day

# Dose (mg/kg/day)

Vehicle Control TP Negative

Control TP + FT

Positive Control 50 250 750

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1

- 4 7 18.1 1.1 7 20.8 0.6 7 20.0 1.4 7 19.8 0.7 7 16.1* (↓23) 1.1 7 10.4* (↓50) 1.0

4

- 7 7 18.5 0.2 7 21.6 0.5 7 20.4 1.7 7 22.7 2.7 7 20.2 1.2 7 18.8* (↓13) 0.8

7

- 11 7 18.2 0. 7 7 22.0 1.0 7 20.5* (↓7) 1.3 7 21.4 1.5 7 20.7 2.9 7 19.1* (↓13) 0.9

a Data were obtained from Table 5 on pages 59

- 60 of the study report. Percent differences from controls

, calculated by the

reviewers , are present ed in parentheses.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls ; p ≤ 0.05



## D. SERUM HORMONE CONCENTRATIONS : Serum testosterone level data are presented in Table 7. In rats treated with 750 mg/kg/day + 0.4 mg/kg/day TP, serum testosterone levels were decreased (NS) by 29% compared to TP controls. Serum testosterone levels at 50 and 250 mg/kg/day were similar to controls.

TABLE 7. Serum Testosterone Levels (ng/mL) in the Anti

- Androgen Assay a

Hormone Dose (mg/kg/day)

TP 50 250 750

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Serum T 7 0.24 0.18 75 7 0.28 0.16 57 7 0.25 0.14 56 7 0.17 (↓29) 0.12 71

a Data were obtained from Table 10 on page 72 of the study report.

N N umber of animals in the group

SD Standard Deviation

CV Coefficient of Variation (calculated by the reviewers from data presented in this table)

* Significantly different from controls ; p ≤ 0.05



## E. ORGAN WEIGHTS : Accessory sex organ weights and liver weights for the androgen agonist assay are presented in Table 8. There were no significant increases in any of the

accessory sex organ weights of animals dosed with the test compound. Absolute liver weights were increased (p≤0.05) by 38 -68% in the 250 mg/kg/day and above groups. Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increase s (p≤0.05)

as follow s: 399% in Cowper’s gland; 52% in glans penis; 96% in LABC; 493% in ventral prostate; and 855% in seminal vesicles. Liver weights were unchanged in this group. %CVs for the vehicle controls, 0.4 mg/kg/day TP group, and the 750 mg/kg/day group were compared to the performance criteria value s as stated in the Guidelines. The %CV for the ventral prostate in the vehicle controls exceeded the criteria value (70% vs . 45% maximum

recommended ); however, it was stated that as none of the accessory sex tissues (including ventral prostate) were increased, it was unlikely that these data would have affected the interpretation of the assay results. All other %CV values wer e less than the maximum

recommended values indicated in the test guideline

.In vivo Hershberger Assay ( 2011 ) / Page 15 of 19

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### TABLE 8. Accessory Sex Organ Weights (mg) and Liver Weights (g) in the Androgen Agonist Assay a

Organ Dose (mg/kg/day)

Vehicle control TP 50 250 750

N Mean SD % CV N Mean SD % CV N Mean SD N Mean SD N Mean SD % CV

Cowper ’ s

gland 7 8.3 1.9 23 7 41.4* (↑399) 4.9 12 7 8.6 1.1 7 10.0 1.9 7 8.6 0.8 9

Glans penis 7 59.4 6.9 12 7 90.0* (↑52) 8.5 9 7 55.7 5.7 7 58.3 9.0 7 59.7 3.1 5

LABC 7 149.8 11.6 8 7 294.2* (↑96) 42.9 15 7 135.1 29.4 7 146.2 34.8 7 131.5 30.6 23

Ventral

prostate 7 23.5 16.4 70 7 139.3* (↑493) 21.6 16 6 17.5 4.7 7 16.1 5.0 7 16.7 3.7 22

Seminal

vesicles 7 59.8 12.0 20 7 571.2* (↑855) 79.2 14 7 51.7 7.9 6 56.0 11.8 7 57.3 14.0 24

Liver 7 11.5 0.7 NA 7 12.4 1.1 NA 7 12.6 1.7 7 15.9* (↑38) 2.6 7 19.3* (↑68) 2.8 NA

a Data were obtained from Tables 6 and 7 on pages 62

- 63 and 66 of the study report. Percent differences from controls

, calculated by the reviewers , are included in parentheses.

N Number of animals in the group SD Standard Deviation

%CV Percent Coefficient of Variation

* Significantly different from controls; p≤0.05

NA Not applicable



## Accessory sex organ weights and liver weights for the anti

- androgen assay are presented in

Table 9. In the 750 mg/kg/day group, statistically significant (p<0.05) decreases in accessory sex organ weight s were seen in the LABC (33%) and seminal vesicles (27%). Nonsignificant decreases were observed in the other three accessory sex tissues. Absolute liver weights were increa sed (p≤0.05) by 3 9-65% in the 250 mg/kg/day and above groups Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as follows: 51% in Cowper’s gland; 23 % in glans penis; 36% in LABC; 73% in ventral prostat e; and 78% in seminal vesicles. Liver weights were unchanged in this

group.

The %CVs for the vehicle controls, 0.4 mg/kg/day TP group, and the 750 mg/kg/day group were compared to the performance criteria values as stated in the Guidelines. As previousl y discussed, t he %CV for the ventral prostate in the vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended ). All other %CV values were less than the

maximum recommended values.In vivo Hershberger Assay ( 2011 ) / Page 16 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



### TABLE 9. Accessory Sex Organ Weights (mg) and Liver Weights (g) from Anti

- Androgen Agonist Assay a

Organ Dose (mg/kg/day)

Vehicle Control TP Negative Control TP + FT

Positive Control 50 250 750

N Mean SD % CV N Mean SD % CV N Mean SD N Mean SD N Mean SD N Mean SD % CV

Cowper ’ s

gland 7 8.3 1.9 23 7 41.4* 4.9 12 7 20.3* (↓51) 4.1 7 46.2 9.2 7 43.8 4.3 6 34.5 (↓17) 6.7 19

Glans penis 7 59.4 6.9 12 7 90.0 8.5 9 7 69.7* (↓23) 7.6 7 92.2 6.5 7 83.9 7.7 7 83.0 (↓8) 3.6 4

LABC 7 149.8 11.6 8 7 294.2 42.9 15 7 187.1* (↓36) 27.9 7 323.3 35.5 7 259.5 54.7 7 196.2* (↓33) 37.4 19

Ventral

prostate 7 23.5 16.4 70 7 139.3 21.6 16 7 37.1* (↓73) 13.5 7 177.9 54.3 7 148.4 46.7 7 108.8 (↓22) 26.1 24

Seminal

vesicles 7 59.8 12.0 20 7 571.2 79.2 14 7 124.4* (↓78) 32.4 7 568.3 101.1 7 533.5 97.1 7 419.8* (↓27) 91.4 22

Liver 7 11.5 0.7 NA 7 12.4 1.1 NA 7 12.8 1.0 7 14.2 0.6 7 17.2* (↑39) 2.8 7 20.5* (↑65) 2.8 NA

a Data were obtained from Tables 6 and 7 on pages 64

- 65 and 67 of the study report. Percent differences from controls

, calculated by the reviewers , are included in parentheses.

N N umber of animals in the group

SD Standard Deviation

%CV Percent Coefficient of Variation

* Significantly different from controls ; p ≤ 0.05In vivo Hershberger Assay ( 2011 ) / Page 17 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## Accessory sex organ weights and liver weights for the 14 

## C- testosterone clearance study are presented in Table 10. Animals dosed with 750 mg/kg/day of the test compound had similar decreases (NS) in accessory sex organ weights as rats in the anti -androgen assay: 17 % in Cowper’s gland; 25% in glans penis; 37% in LABC; 53% in ventral prostate; and 48 % in seminal vesicles. Absolute liver weights were increased ( NS ) by 3 8 % in this group.

Accessory Sex Organ Weights (mg) and Liver Weights (g) in the 14 

### C

- Testosterone Clearance Study a

Organ Dose (mg/kg/day)

TP Control 750

N Mean SD N Mean SD
Cowper’s gland 7 33.5 13.3 7 27.7 (↓17) 5.6

Glans penis 7 90.2 6.5 7 68.1 (↓25) 10.8

LABC 7 478.1 52.0 7 301.6 (↓37) 45.7
Ventral prostate 7 236.0 53.7 7 110.6 (↓53) 28.5

Seminal vesicles 7 590.9 105.6 7 306.6 (↓48) 78.3

Liver 7 14.8 2.1 7 20.4 (↑38) 2.6

a Data were obtained from Table 13 on page 75 of the study report. Percent differences from controls

, calculated by the

reviewers , are included in parentheses.

N Number of animals in the group

SD Standard Deviation



## F. GROSS PATHOLOGY : There were no treatment

- relate d gross pathological findings.

G. 14 

## C- TESTOSTERONE CLEARANCE : Pharmacokinetic parameters for

the 14 

## C- testosterone clearance study are presented in Table 11 . Rats treated for 10 days with 750 mg/kg/day of the test compound cleared 14 

## C-radioactivity 3.7- fold faster (p≤0.05) than

the TP -only controls. This was a result of the elimination t 1⁄2α 

## present in the 750 mg/kg/day group. Additionally, the 750 mg/kg/day animals had an AUC that was lower (p≤0.05) than

the TP -only controls (0.014 treated vs. 0.039 controls).

TABLE 11. Kinetic Parameters of Plasma 14 

### C

- Testosterone a

Parameters Dose (mg/kg/day)

TP Control 750

N Mean SD N Mean SD
Day 1

Elimination t 1⁄2 (h) 7 2.679 1.379 7 2.420 0.581

AUC 0 → t ( μ g h g

- 1 ) 7 0.030 0.015 7 0.024 0.006

Clearance (mL kg

- 1 h

- 1 ) 7 510.559 380.608 7 504.502 188.884 Day 10

Elimination t 1⁄2 α (h) b 3 3.641 1.094 4 0.290* 0.056

Elimination t 1⁄2 β (h) b 3

--- --- 4 3.217 1.357

AUC 0 → t ( μ g h g

- 1 ) 3 0.039 0.010 4 0.014* 0.004 Clearance (mL kg

- 1 h

- 1 ) 3 251.470 92.432 4 930.596* 322.487

a Data were obtained from Table 14 on page 76 of the study report. Percent differences from controls

, calculated by the

reviewers , are included in parentheses.

b Elimination t 1⁄2 was not biphasic in the TP animals on Day 10

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls; p≤0.05In vivo Hershberger Assay ( 2011 ) / Page 18 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS : Based on the lack of statistically significant,

treatment -related increases in two AST weights in the androgenic portion of the assay, the test compound at doses ≤ 750 mg/kg bw/day was deemed negative for androgenic activity in the Hershberger assay. Ba sed on significant decreases in two AST weights in the

anti -androgenic portion of the assay, the highest dose level of the test compound was deemed positive for anti-androgenic potential, although this dose level slightly exceeded the MTD. Furthermore, th e significant increase in liver weight, decrease in serum testosterone levels, enhanced clearance of 14 

## C-testosterone-derived radioactivity at this dose level, coupled with previous toxicity and mode-of- action data, indicate that the anti

- androgenic

effect s of the test compound are likely secondary to enhanced testosterone metabolism and clearance, resulting in decreased AST weights. Anti- androgenic potential was not seen at

doses of ≤ 250 mg/kg/day.

B. AGENCY COMMENTS : All animals survived until scheduled termination. There were

no treatment

- related gross pathological findings.

In the androgen agonist assay, clinical signs included 3/7 rats had decreased feces and 2/7

rats were observed with clear perioral soiling at 7 50 mg/kg/day. Additionally at this dose,

b ody weights were decreased (p≤0.05 ) on Days 4 and 7 by 7 -11%. Also at 750 mg/kg/day, decreased (p≤0.05) cumulative body weight gains were seen for Days 1

- 7 (↓76%) and Days

1- 11 (↓49%). Cumulative body weight gains were also decreased (p≤0.05) by 61% for Days 1-4 in the 250 mg/kg/day group; however, body weights in this group were similar to controls, as were cumulative body weight gains for Days 1-7 and 1-11

There were no significant increases in any of the accessory sex organ weights of animals dosed with the test compound. Absolute liver weights were increased (p≤0.05) by 38 -68% in the 250 mg/kg/day and above groups. The performance criteria indicated that this assay was performing as expected.

In the anti-androgen assay, 3/7 rats at 750 mg/kg/day were observed to have decreased

feces. Cumulative body weight gains were decreased (p≤0.05 ) on Days 1

- 4 (↓55%), 1 -7 (↓37%) and 1

- 11 (↓23%) at 250 mg/kg/day; however, body weights in this group we re similar to controls. At 750 mg/kg/day, body weights were decreased (p≤0.05) on Days 4, 7, and 11 by 10-14%, and decreased cumulative body weight gains were seen for Days 1-7 (↓69%) and Days 1

- 11 (↓57%). During Days 1 -4, food consumption was decreased ( p≤0.05) by 23 -50% at 250 mg/kg/day and above, and was decreased at 750 mg/kg/day during Days

4-7 and 7- 11 (↓13% both periods). In the 750 mg/kg/day group, significant (p≤0.05) decreases in were seen in two of the five accessory sex organ weight s: glans pe nis (33%)

and seminal vesicles (27%). Non

- significant organ weight decreases were observed in the remaining three accessory sex tissues. Absolute liver weights were increased (p≤0.05) by 39-65% in the 250 mg/kg/day and above groups. In rats treated with 7 50 mg/kg/day + 0.4 mg/kg/day TP, serum testosterone levels were decreased (NS) by 29% compared to TP

controls. The %CV for the ventral prostate in the vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended), but was unlikely to have affected the interpretation of the assay results since two of the five target tissues showed a positive response. All other %CV values were less than the maximum recommended values.In vivo Hershberger Assay ( 2011 ) / Page 19 of 19

BENFLU RALIN / 084301 O CSPP 89 0. 1400/ OECD None



## In the 14 

## C- testosterone clearance assay, 6/7 rats dosed with 750 mg/kg /day had urine perineal staining, and 2/7 rats had red perinasal staining. In the 750 mg/kg/day group, body weights were decreased (p≤0.05) by 12 -20% on Days 4, 7, and 10; resulting in body weight losses of 18.7 g and 1.0 g for Days 1-4 and 1-7, and decre ased (p≤0.05) cumulative body weight gains for Days 1- 10 (↓85%). Animals dosed with 750 mg/kg/day of the test compound had similar decreases (NS) in accessory sex organ weights as rats in the anti

androgen assay: 17 % in Cowper’s gland; 25% in glans penis; 37% in LABC; 53 % in ventral prostate; and 48 % in seminal vesicles. Absolute liver weights were increased ( NS ) by 3 8 % in this group. Rats treated for 10 days with 750 mg/kg/day of the test compound

cleared 14 

## C- radioactivity 3.7

- fold fast er (p≤0.05) than the TP -only controls. This was a

result of the elimination 1⁄2α 

## present in the 750 mg/kg/day group. Additionally, the 750 mg/kg/day animals had an AUC that was lower (p≤0.05) than the TP -only controls (0.014 treated vs. 0.039 controls).

Benfluralin was negative for androgenic 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. However, benfluralin was pos itive was anti

androgenicity in the Hershberger assay as statistically significant decrease s in organ weights

were seen in two of the five androgen sensitive tissues in the androgen antagonist assay. At 750 mg/kg/day, a significant decrease in body weight (12%; p<0.05) and body weight gain (57%; p<0.05) were seen after 10 days of treatment indicating that there was overt toxicity at the dose that resulted in decreases in androgen

- sensitive tissue weights . Additional data provided on testosterone clearance in benfluralin

- treated rats in conjunction with the

observed increase in liver weights , provide evidence that the reduced accessory sex organ weights may be secondary to increased testosterone metabolism and clearance.

C. STUDY DEFICIENCIES : None

# DATA EVALUATION RECORD

BENFLURALIN (BENEFIN)

Study Type: O CSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-45-2012 (MRID 48669301)

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: Joseph P. Rieth, Ph.D., D.A.B.T. Date: 4/13/2012

Secondary Reviewer : Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 4 / 20 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4 / 30 /2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4 / 30 /2012

This Data Evaluation Record may have been altered 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 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 i s 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).Female Pubertal Assay (2012)/ Page I of Error! Bookmark not d e fined.
BENFLURALIN/084301



## Primary Reviewer: John Liccone, Ph

. D.



## Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D.



## Health Effects Division

DAT A EVALUATION RECORD OCSPP 890. 50 I CD None



## STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None

.



## PC CODE: 084301 

## DP BARCODE: D399309



## TXR#: 0054123 

## CAS No: 1861-40-1



## TEST MATERIAL (PURITY): Benfluralin (97.5%)



## SYNONYMS: Benefin; N-Butyl-N -eth yl-2,6-dinitro -4-(trifluoromethyl)benzenamine;

Benfluralin



## CITATION: Marty, M.S., Zablotny, C.L., Hukkanen, R.R. (2012). Benefin : pubertal development and thyroid function in intact juvenile/peripubertal female Crl:CD(SD) rats. Toxicology & 

## Environmental Research and Consulting, The

Dow Chemical Company, Midland, MI. Laboratory Study No: 111073

. January 25, 2012

. MRID 48669301. Unpublished.



## SPONSOR: Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, 

## IN

TEST ORDER#: EDSP-084301-15



## EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48669301), groups of 16
Crl:CD(SD) rats were treated daily via oral gavage with benfluralin (97.5% a.i.; Lot#
ACD13683) in corn oil at doses of 0, 25

, 75 or 300 mg/kg / day from post-natal day (PND) 22 to

42

. Vaginal opening (VO) was evaluated daily, and age and weight at attainment of VO were
recorded. Once VO was completed

, vaginal lavage samples were collected daily (including the day of necropsy) to evaluate estrus cycle length and pattern

. After sacrifice on PND 42, blood

was collected and a gross necropsy was conducted

. Clinical chemistry parameters were evaluated, including serum thyroxine (T 4 ) 

## and thyroid stimulating hormone (TSH) levels using a radioimmunoassay assay (TSH) or chemiluminescent immunoassay (T 4). 

## The following organs

were weighed: liver, kidneys, adrenals (weighed as pairs), ovaries (without oviducts, weighed as pairs)

, imbibed and blotted uterus (with oviducts and cervix)

, thyroid

, and pituitary. Microscopic

evaluations were performed on thyroid

, kidneys, uterus

, and typically the right ovary.

All animals survived until scheduled termination. One 75 mg / kg/day female was removed from

the study after being dosed twice on one day

. All of the 300 mg / kg / day animals had dark yellow

urine

, a typical finding following dinitroaniline herbicide exposure. There were no significant

treatment-related effects on mean estrous length, percent cycling, percent regularly cycling or body weight at VO. There were no gross findings at necropsy and no histopathological lesions

in the uterus, ovaries

, kidneys or thyroid.Female P ubertal Assay ( 2012)/ Page 2 of Error! Bookmark not defined.

B ENFLURALIN /084301 O CSPP 89 0.1450 / OECD None



## A nimals treated at 300 mg/kg/day had decreased (p<0.05) body weights ( ↓ 8- 10%) and

cumulative body weight gains ( ↓ 15- 42%) through PND 35; terminal body weights l argely recovered by necropsy at PND 42 ( ↓ 4% ; NS ) with a 6% decrease (p<0.05) in overall body weight gain ( PND 22 -42). Both unadjusted and adjusted m ean age s at VO w ere increased (p<0.05) in the 300 mg/kg/day group (PND 37.2-37.3 treated vs. PND 34.4 controls) but not at

lower dose level s. Age at first estrus was also increased (p<0.05) at 300 mg/kg/day (PND 37.2 treated vs. PND 35.4) due to the delay in VO

.

Serum T 4 

## levels w ere decreased (p<0.05) by 13% at 75 mg/kg/day and 44% at 300 mg/kg/day,

and TSH levels were increased (p<0.05) by 109% at 300 mg/kg/day; however

, there were no associated changes observed in thyroid weight or histopathology. Other clinical chemistry changes in the 300 mg/kg/day animals included: decrease d (p<0.05) alkaline phosphatase ( ↓ 16%) and increase d (p< 0.05) gamma glutamyl transpeptidase ( GGT, 47%) , cholesterol ( ↑ 12%), and creatinine ( ↑ 44%). Unadjusted , adjusted

, and relative liver weights were increased (p<0.05) by 19-23% at 75 mg/kg/day and 32-37% at 300 mg/kg/day, and relative liver weight was increased (p <0.05) by 8% at 25 mg/kg/day. At 300 mg/kg/day, d ecreases (p<0.05) were also observed in pituitary weight (unadjusted, adjusted , and relative ; ↓ 12-19%), ovary weights (unadjusted and adjusted ; ↓ 17-18%) and blotted uterus weight (adjusted; ↓ 21%)

.

The high dose of 300 mg/kg/day is considered adequate based on decreased b ody weight gains over the course of the treatment period and increased liver weights with concomitant significant decreases in alkaline phosphatase, and increases in GGT and choleste rol.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Female Pubertal Assay (OCSPP 890.1450).

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

statements were provided.Female P ubertal Assay ( 2012)/ Page 3 of Error! Bookmark not defined.

B ENFLURALIN /084301 O CSPP 89 0.1450 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Toxicology & Environmental Research and Consulting, The Dow Chemical Company

Location: Midland, M I Study Director: C. L. Zablotny

Other Personnel: M.S. Marty, Ph.D., Lead Scientist R. R. Hukkanen, D.V.M., Pathologist Study Period: June 2 0 , 2011

, to January 2 5, 201 2



## 2

. Test S ubstance : Benfluralin

Description: Technical, molecular weight 335.3

Source: Dow AgroSciences, LLC, Indianapolis, I N

Lot/Batch #: Lot # ACD13683 Purity : 97.5 % Stability: Not provided

CAS #: 1861

- 40

- 1

Structure:

N N +

N + O O

O

O CH 3

F

F F CH 3



## 3

. Vehicle : Corn oil

4

. Test A nimals : Species: Rat

Strain: Sprague Dawley Crl:CD(SD) Age/Weight at Study Initiation: 22 days/ 37.3

- 61.7 g
, females only

Source: Charles River Laboratories, Ltd., Portage M I Housing: 2/cage in solid bottom plastic cages with heat

- treated laboratory

- grade wood shavings as bedding.

Diet: Tekland Diet #2016 (Harlan Laboratories, Inc., Indianapolis, IN ), ad libitum Phytoestrogen content <325 μg of genistein equivalents/gram diet

Water: Deionized tap water, ad libitum

Environmental

Conditions: Temperature: Humidity: Air changes: Photoperiod: 22 ± 1°C

40

- 70%

12

- 15/hr 12 hrs light/ 12 hrs dark



## B. ST UDY DESIGN

1. In -L ife D ates : Start: June 24, 2011 End: July 15, 2011

2. Mating : Nulliparous female rats were bred by the supplier and shipped to arrive on gestation days (GD) 7-10. The day an animal was found to be sperm positive was designated GD 0. Litters born on GD 21 or 22 were used in the assays; study endpoints

were based on pup age relative to birth date. Litters were culled to 10 pups on PND 4 usingFemale P ubertal Assay ( 2012)/ Page 4 of Error! Bookmark not defined.

B ENFLURALIN /084301 O CSPP 89 0.1450 / OECD None



## 5 males and 5 females where possible; males were used in a concurrently reviewed male pubertal assay (MRID 48669302) and females were used in th is female pubertal assay.

3. Animal A ssignment : Offspring were weaned on PND 21 and weighed. The rats were ranked by weight and randomized to achieve a mean body weight and variance across dose groups at study initiation. Animals were assigned to the test groups noted in Table 1. Care was taken so that litter mates were not assigned to the same treatment group. The treatment groups included vehicle controls and benfluralin- treated rats with three dose levels.

Test group Dose (mg/kg/day) # of Females

Control 0 16

Low 25 16

Mid 75 16
High 300 16

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



## 4. Dose S election Rationale : Prior to the start of the current assay, a probe study 1 

## was conducted in which groups of 4 female Crl:CD(SD) rats were administered benfluralin by

gavage at dose levels of 0, 250, 500, 750, or 1000 mg/kg/day in corn oil for 15 days. The

fe male rats were treated from PND 22-36. By PND 36, females weighed 12.2%, 9.0%, 13.5%, and 12.9% less than the controls at 250, 500, 750, and 1000 mg/kg/day , r espectively. Treated animals had lower food consumption at all doses and time intervals. At necropsy, all treated females displayed clear facial staining, and had increased GGT and globulin at

≥ 500 mg/kg/day, and increased cholesterol at all dose levels. Albumin/globulin ratios were decreased and liver weights were increased at all dose levels. Some hepatocellular hypertrophy was observed at 1000 mg/kg/day. The study author stated that the effects in the 250 mg/kg/day group were more severe than expected, and were probably due to the small sample sizes used in the probe study (n=4/ dose group). Based on these data, 300 mg/kg/day was selected as the high dose for the current study.

5. Dose Preparation and Analysis : The test material was suspended in corn oil to yield the desired concentrations (further information not provided). Concentration and homogeneity ( sampling not specified) analyses w ere conducted on the first preparation prior to dose

initiation . Concentration analyses were performed on all dose levels; homogeneity analyses were performed on the 25 and 300 mg/kg/day formulations. It was stated that the stability of benfluralin in corn oil was confirmed for up to 22 days at room temperature in a previously reviewed Hershberger study (MRID 48614907).

Results of Dose Analysis

Homogeneity (% RSD) : 2.4-3.2%

1 

### Marty, M. S. and Marshall, V. A. (in progress). Benefin: Hershberger, Uterotrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI.Female P ubertal Assay ( 2012)/ Page 5 of Error! Boo kmark not defined.

B ENFLURALIN /084301 O CSPP 89 0.1450 / OECD None



## Stability (% of Day 0): 94.9-114.3% following storage at room temperature for up to 22 days.

Concentration (% of nominal): 94.6-96.2%

The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage A dministration : All doses were administered once daily at approximately the same time each day by oral gavage from PND 22 -42. A dose volume of 4.0 mL/kg was used; dose volumes were adjusted using the most recently determined individual body weight

.

7. Statistics : For continuous variables, Bartlett’s test was used to assess homogeneity of variance (alpha = 0.01). Variables that were transformed based on the outcome of the Bartlett’s test were blood urea nitrogen (square root tr ansformed) and aspartate

aminotransf erase (log transformed). Initial body weight , body weight gain, clinical

chemistr y , mean estrus cycle length, age and body weight at VO , age of first estrus, organ weights, organ weight ratios (liver, kidney, adrenal , and pituitary weights) and serum

hormones were analyzed with a two

- way analysis of variance (AN OVA) . When the dose effect was significant (alpha = 0.05), individual dose groups were compared to controls using Dunnett’s test (alpha = 0.05). Age and body weight at VO , and organ weights (thyroid, ovaries and uterus) were tested using an analysis of covariance ( AN C OVA

, alpha = 0.05) using body weight on PND 22 as the covariate. W hen dose effects were observed (alpha = 0.05), least square means with Dunnett’ s correction was used (alpha = 0.05). A

chi

- square analysis was used to analyze estrus cycle status and percent regularly cycling; when significant, pairwise comparisons to the controls were made using a Fisher’s exact test

with Bonferroni- Holm adjustment . Certain histopathological findings were analyzed using the pairwise Chi

- square test using Yates ’ continuity correction ( two -sided; alpha = 0.05). The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and C linical Examinations : Cageside examinations and observations for mortality and morbidity were performed on a ll animals at least twice daily

. Hand -held

c linical examinations were conducted each treatment day at approximately the same time following dosing (no additional details included in the report).

2. Body W eight : Animals were weighed daily prior to dosing. Body weights were reported and statistically analyzed on PND 21, 22, 25, 28, 32, 35 and 42. Body weight gains were reported and statistically analyzed for all reported weight intervals , beginning with PND 22- 25 and including the entire dosing period (PND 22 -42).

3. Vaginal O pening (VO) : Beginning on PND 22 , all animals were examined daily for onset

of VO . Age and weight on the day of completion of VO were recorded. If VO was not completed by PND 42, PND 43 was used for determining the mean age at puberty onset for the dose group.Female P ubertal Assay ( 2012)/ Page 6 of Error! Bookmark not defined.

B ENFLURALIN /084301 O CSPP 89 0.1450 / OECD None



## 4. Estr o us C yclicity : Beginning on the day of VO , and up to and including the day of necropsy, daily vaginal smears were obtained. Vaginal lavage slides were examined microscopically to determine the interva l between VO and fi rst estrus, and to evaluate the estrus cycle pattern.

5. Sacrifice and Pathology : On the day of termination (PND 42) approximately 2 hours postdosing, the rats (unfasted) were removed from their home cages one at a time

, anesthetized

with isoflurane, and blood was collected by cardiac puncture. Hormone analyses for T 4 

## and

TSH were conducted at WIL Research Laboratories , LLC (Ashland, Ohio). The rats were euthanized by exsanguination followed by decapitation. Animals were euthanized beginning at least 2 hours following dose administration that day, and all sacrifices were completed by 1300 hours. A complete necropsy was conducted on all animals, and it was

stated that the groups were counterbalanced during necropsy.

a. Hormone Analysis : Serum samples were stored at

− 80° C and shipped on dry ice. T 4 

## and TSH levels were analyzed using chemiluminescent immunoassay (T 4 

## ) or radioimmunoassay ( TSH).

b. Clinical C hemistry : 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 Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALK) X Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH) X A/G ratio

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

Sorbitol dehydrogenase

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in female rats based on G uideline 890.1 45 0.



## c. Organ W eights and Histopathology : The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subjected to histological

examination

.

UROGENITAL OTHER

X X Ovaries ( paired, without oviducts) * + X X Thyroid * + (after fixation)

X X Uterus* + (imbibed , with uterus & cervix ) X Liver *

X X Kidneys (paired)*+ X Adrenals (paired) *

X Pituitary *

* Weights required based on G uideline 890.1 4 50

+ Histopathological examination required based on guideline 890.1 4 50Female P ubertal Assay ( 2012)/ Page 7 of Error! Bookmark not defined.

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## All collected organs, except the thyroid and pituitary , were weighed prior to fixation. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus and cervix were separated from the vagina and weighed. The uterus was weighed again following removal

of the fluid in the lumen (blotted we ight)

.

The kidneys, right ovary and uteri were fixed in 10% buffered formalin for at least 24 hrs and then rinsed in graded ethanol until fixation in paraffin , sectioning and stai ning with hematoxylin and eosin. The histopathological assessment of the uterus and ovary took into account the stage of estrus cycle at the time of necropsy. The thyroid was fixed in 10% buffered formalin for at least 24 hrs. Following fixation, the thyroid w as dissected from the trachea, processed into paraffin blocks, sectioned, and sta ined with hematoxylin and eosin. The thyroid sections were subjectively evaluated for follicular cell height and colloid area using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest). Uterine histology

documented cases of uterine hyper - or hypotrophy as characterized by changes in uterine horn diameter and myometrial stromal, or endometrial gland development.

II. RESULTS

A. Mortality : All animals survived until scheduled termination. One animal in the 75 mg/kg/day group (#7026) was removed from the study on PND 34 due to a dosing error (animal was dosed twice on this day).

B. Clinical S igns of Toxicity : All rats treated with benfluralin at 300 mg/kg/day were observed to have dark yellow/orange urine. It was stat ed that this is a common finding following dinitroaniline herbicide treatment . No other clinical signs were reported

.

C. General G rowth and Vaginal O pening : Mean body weights and body weight gains over the course of the treatment period are shown for the control and 300 mg/kg/day animals in

Table 2. Mean body weights were the same across all groups on PND 21. At 300 mg/kg/day, body weights were decreased (p <0.05) by 8-10% during PND 22-35, resulting

in cumulative decreases in body weight g ain (p<0.05) of 15-42% for each interval up to

PND 22 -35.Female P ubertal Assay ( 2012)/ Page 8 of Error! Bookmark not defined.

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### TABLE 2. Mean (±SD) body weights and gains in control and high dose animals (g). a

Parameter Treatment Group

Vehicle Control Benfluralin 300 mg/kg/day
Body weight PND 22 50.6±5.9 50.6±4.7

PND 25 63.7±7.4 58.3±6.5* (↓ 8)

PND 28 76.6±8.7 70.7±8.4* (↓8)

PND 32 101.5±12.5 91.5±11.2* (↓10)

PND 35 120.4±14.5 110.1±13.9* (↓9) Terminal body weight PND 42 160.6±19.1 154.5±17.1 (↓4)
Body weight gain PND 22

- 25 13.2±2.1 7.7±2.5* (↓42)

PND 22

- 28 26.1±5.6 20.1±4.4* (↓23)

PND 22

- 32 50.9±7.3 40.9±7.0*(↓20)

PND 22

- 35 69.9±9.4 59.5±9.7* (↓15)

PND 22

- 42 110.0±14.4 103.9±13.3 (↓6)

a Data were obtained from page 56 of the study report. Percent difference s from control , presented

in parentheses, were calculated by the reviewer s. * Significantly different from controls at p< 0.05

.



## Body weights, body weight gains, age of attainment of VO and weight at day of attainment are presented in Table 3. Although body weights and cumulative bod y weight gains were decreased (p<0.05) during the period PND 22-35 at 300 mg/kg/day, terminal body weights and overall ( PND 22 -42) body weight gains were similar to the controls for all treated groups. A ge at VO was delayed (p<0.05) in the 300 mg/kg/day females for both unadjusted (PND 37.3 treated vs. PND 34.4 controls) and adjusted for body weight on PND 22 ( PND 37.2 treated vs. PND 34.4 controls). Both unadjusted and adjusted body weights at VO in the 300 mg/kg/day group were similar to controls

. Age at VO was not si gnificantly affected
at doses ≤ 75 mg/kg/day. All animals in the study achieved VO by PND 42.Female P ubertal Assay ( 2012)/ Page 9 of Error! Bookmark not defined.

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### TABLE 3

. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Benfluralin 25 mg/kg/day) Benfluralin 75 mg/kg/day Benfluralin 300 mg/kg/day

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean b SD/

SE b CV (%)
Initial body weight (PND 2 2 ; g) U 16 50.56 5.88 11.63 16 50.45 5.51 10.91 16 50.63 4.74 9.37 16 50.60 4.66 9.2

Body weight at VO (g) U 16 116.46 15.01 12.89 16 118.03 15.42 13.06 15 121.79 18.55 15.23 16 123.44 10.99 8.90

A 16 116.62 3.262 NA 16 118.35 3.262 NA 15 121.17 3.371 NA 16 123.53 3.262 NA
Final body weight (g) U 16 160.57 19.12 11.91 16 162.28 18.80 11.58 15 166.01 18.04 10.87 16 154.43 17.05 11.04

A 16 160.87 2.775 NA 16 162.88 2.776 NA 15 164.86 2.868 NA 16 154.71 2.775 NA
Final body weight (% of control) U NA NA NA NA 16 101.1 11.7 11.6 15 103.4 11.2 10.9 16 96. 2 10.6 11.0

A NA NA NA NA NR NR NR NR NR NR NR NR NR NR NR NR
Body weight gain (final

– initial; g) U 16 110.01 14.43 13.12 16 111.83 15.23 13.62 15 114.94 14.86 12.93 16 103.93 13.33 12.83

A NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
Age at VO (PND) U 16 34.4 1.1 3.2 16 34.8 1.8 5.3 15 35.3 2.5 7.0 16 37.3* 2.6 7.0

A 16 34.4 0.50 NA 16 34.8 0.50 NA 15 35.4 0.51 NA 16 37.2* 0.50 NA
Proportion unopened (#/N) 0/16 NA 0/16 NA 0/15 NA 0/16 NA

a Data were obtained from page 57 of the study report.

b Standard deviation reported for unadjusted values and standard error of the mean reported for adjusted values.

U Unadjusted for body weight on PND 22

A Adjusted for body weight on PND 22

N Number of animals examined

SD Standard d eviation

SE Standard e rror of the m ean CV Coefficient of v ariation

* Significantly different from controls at p<0.05.

NA Not applicable

NR Not requiredFemale P ubertal Assay ( 2012)/ Page 10 of Error! Bookmark not defined.

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## D. Organ W eights : Organ weights at necropsy are presented in Table 4. Dose

- related increases in liver weights were observed at all dose levels. Unadjusted, adjusted , and

relative (to body) liver weights were increased (p<0.05) by 19-23% at 75 mg/kg/day and 32- 37% at 300 mg/kg/day . Relative liver weight was also increased (p<0.05) by 8% at 25 mg/kg/day. Unadjusted, adjusted and relative pituitary weights were decrea sed (p <0.05) at 300 mg/kg/day by 12-19% ; at lower doses

, pituitary weigh ts were not affected by treatment. Absolute and adjusted ovary weights were decreased (p <0.05) by 17 -18% at 300 mg/kg/day. Adjusted, blotted uterine weights were decreased (p<0.05) by 21% in the 300 mg/kg/day animals; it was stated that unadjusted blotte d uterine weights showed a significant difference by ANOVA, but pairwise comparisons were not significant.

The control and 300 mg/kg/day groups had similar numbers of animals in estrus and diestrus at necropsy so the decreases in ovary and uterus weights were not due to differences in stage of estrus at termination. Ovary and uterine weights were una ffected by treatment at doses

≤ 75mg/kg/day. There were no effects of treatment on weights of kidneys , adrenal s, thyroid

or wet uterine at any dose level in this study.Female P ubertal Assay ( 2012)/ Page 11 of Error! Bookmark not defined.

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### TABLE 4

. Organ Weights at Necropsy a

Organ Vehicle Control Benfluralin 25 mg/kg/day) Benfluralin 75 mg/kg/day Benfluralin

300 mg/kg/day

N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/

SE b CV (%)

Li ver (g) U 16 7.26 1.07 14.71 16 7.92 0.85 10.75 1 5 8.90* (↑23 ) 0.99 11.16 16 9.57* (↑32 ) 1.24 12.98

A 16 7.27 0.190 NA 16 7.95 0.190 NA 1 5 8.84* (↑22 ) 0.196 NA 16 9.58* (↑32 ) 0.190 NA

R 16 4.51 0.24 5.42 16 4.89* (↑8 ) 0.24 4.88 1 5 5.36* (↑19 ) 0.19 3.46 16 6.19* (↑37 ) 0.33 5.28

Kidneys (g) U 16 1.27 0.13 9.88 16 1.30 0.12 9.40 1 5 1.30 0.16 12.13 16 1.19 0.15 12.51

A 16 1.27 0.024 NA 16 1.30 0.024 NA 15 1.29 0.025 NA 16 1.19 0.024 NA

R 16 0.80 0.04 5.49 16 0.80 0.05 6.75 15 0.78 0.04 5.40 16 0.77 0.04 4.77
Pituitary (mg) U 16 8.1 1.4 17.2 16 7.9 1.0 12.8 15 7.6 1.0 13.6 16 6.8* (↓19 ) 1.2 18.3

A 16 8.1 0.23 NA 16 8.0 0.23 NA 15 7.6 0.24 NA 16 6.8* (↓19 ) 0.23 NA

R 16 5.0 0.6 11.9 16 4.9 0.6 11.5 15 4.6 0.5 11.4 16 4.4* (↓12) 0.6 13.2

Adrenals (mg) U 16 34.4 6.3 18.4 16 33.8 5.5 16.2 15 34.0 4.9 14.5 16 31.1 5.0 15.9

A 16 34.5 1.12 NA 16 33.9 1.12 NA 15 33.8 1.16 NA 16 31.2 1.12 NA

R 16 21.4 3.1 14.6 16 20.8 2.7 13.0 15 20.5 2.4 11.7 16 20.2 2.5 12.6
Ovaries (mg) U 16 57.4 8.5 14.9 16 55.9 9.8 17.5 15 52.5 11.3 21.6 16 47.7* (↓17 ) 10.6 22.2

A 16 57.6 2.40 NA 16 56.2 2.40 NA 15 52.4 2.48 NA 16 47.4* (↓18 ) 2.48 NA

Uterus, wet (mg) U 16 320.7 133.4 41.6 16 289.3 102.2 35.3 15 376.4 184.3 48.9 16 248.9 90.9 36.5

A 16 321.6 31.38 NA 16 291.1 31.38 NA 15 373.0 32.43 NA 16 249.4 31.38 NA

Uterus, blo tted (mg) U 16 290.4 67.1 23.1 16 271.7 75.4 27.8 15 300.9 89.4 29.7 16 230.6 55.1 23.9

A 16 291.1 16.61 NA 16 273.0 16.61 NA 15 2 98.5 17.16 NA 16 231.0* (↓21 ) 16.61 NA

Thyroid, fi xed (mg) U 16 9.0 1.1 12.4 16 9.4 0.9 9.2 15 9.8 1.4 14.0 16 9.5 1.4 15.0

A 16 9.0 0.29 NA 16 9.4 0.29 NA 15 9.7 0.30 NA 16 9.5 0.29 NA

a Data were obtained from page 61 of the study report

. Percent difference from control , presented in parentheses, were calculated by the reviewer s. b Standard deviation reported for unadjusted values and standard error of the mean reported for adjusted values.

U Unadjusted for body weight on PND 22

A Adjusted for body weight on PND 22

R Organ

- to

- body weight ratio (relative to body weight)

* Significantly different from controls at p<0.05.Female P ubertal Assay ( 2012)/ Page 12 of Error! Bookmark not defined.

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## E. Estr o us C yclicity : Estr o us cycle data are provided in Table 5. The mean age at first estrus was PND 35.4 in controls compared with PND 35.3, 36.1 and 37.2 (p ≤ 0.05) in the 25, 75 and 300 mg/kg/day groups , respectively

. The delay in first estrus was consistent with the delay in VO ; the mean age at first estrus was th e same as the mean adjusted age of VO ( PND 37.2). In the control group, the first estrus was 1.0 days later than the day of VO

. The number of animals that did not have first estrus during the monitoring period w as 0, 1, 2 and 4 for the control, 25, 75 and 300 mg/kg/day groups, respectively. There were no inter

group differences in mean cycle length which ranged from 3.7 to 4.9 days in treated animals and was 4.3 days in controls. However, the overall ANOVA was significant (p<0.05). There were no differences in the percent cycling or the percent regularly cycling females at any dose of benfluralin

.

TABLE 5

. Age at Vaginal Opening and Estrus Cycling a

Dose Level (mg/kg/day) N Mean Age at First Vaginal Estrus (PND) N Mean Cycle Length (days) N Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling

Vehicle 16 35.4 13 4.3 16 100 81 12 0 4 0

Benfluralin ( 25 ) 15 35.3 10 4.9 16 88 75 12 1 3 0

Benfluralin (7 5 ) 13 36.1 8 4.3 15 80 73 10 0 5 0

Benfluralin (300) 12 37.2* (↑5) 7 3.7 16 94 81 13 0 3 0

a Data were obtained from page 58 of the study report

. Percent difference from control , presented in parentheses, were calculated by the reviewer s. N Number of animals examined

* Significantly different from controls at p<0.05.



## F. Clinical C hemistry and H ormone L evels : Mean hormone levels are presented in Table 6.

There were dose- dependent decreases (p <0.05) in serum T 4 

## of 13% at 75 mg/kg/day and 44% at 300 mg/kg/day . TSH levels were increased (p <0.05) by 109% at the high dose only. These findings are reportedly consistent with the effects of benfluralin on thyroid hormones observed in previous studies and are possibly due to treatment- related liver enzyme induction and subsequently enhanced T 4 

## hormone clearance. Animals given 300 mg/kg/day

benfluralin showed a 16% decrease (p<0.05) in alkaline phosphatase activity ; a decreasing trend across treatment groups was also observed. The 300 mg/kg/day animals also had

inc reased (p<0.05 ) levels of GGT (↑47%) and cholesterol (↑12%) that are consistent with the higher liver weights observed in the study. Creatinine was increased (p <0.05) by 44% at 300 mg/kg/day and was considered to be treatment- related although there was no histopathological correlate in the kidney.Female P ubertal Assay ( 2012)/ Page 13 of Error! Bookmark not defined.

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### TABLE 6

. Hormone Levels and Clinical Chemistry a



### Parameter

Evaluated Vehicle Control Benfluralin

25 mg/kg/day Benfluralin

75 mg/kg/day Benfluralin

3 00 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 16 3.58 0.54 14.96 16 3.21 0.42 12.98 15 3.11* ( ↓13 %) 0.57 18.29 16 1.98* (↓44 %) 0.31 15.42

Serum TSH (ng/mL) 16 3.3 1.7 51.6 16 4.1 2.2 52.1 15 4.7 2.4 52.3 16 6.9* (↑109%) 4.0 58.8

Clinical Chemistry

Creatinine (μmol/L) 16 0.100 0.000 0.0 16 0.106 0.025 23.5 15 0.120 0.041 34.5 16 0.144* (↑44%) 0.051 35.6

Serum urea nitrogen ( mg/dL ) 16 12 2 18 16 14 2 17 15 13 3 19 16 14 4 27

Alkaline phosphatase (U /L) b 16 301 54 18 16 297 33 11 1 5 278 51 18 16 250* (↓ 16 %) 45 18

Alanine aminotrans

ferase (U /L) b 16 55 8 15 16 59 9 15 1 5 59 6 11 16 57 12 21

Aspartate amino

transferase (U /L) b 16 101 24 24 16 105 50 47 15 97 27 28 16 116 23 20

Gamma glutamyl transferase (U /L) b 16 1.5 0.0 0.0 16 1.5 0.0 0.0 15 1.5 0.0 0.0 16 2.2* (↑47%) 1.3 60.4

Albumin (g/dL) 16 3.9 0.2 5.2 16 3.9 0.3 6.9 15 3.8 0.3 6.9 16 3.8 0.3 7.9

A/G ratio 16 3.2 0.4 11.3 16 3.1 0.4 11.8 15 2.8 0.3 9.3 16 2.5 0.2 9.6 Cholesterol (mg/dL) 16 97 14 15 16 99 12 12 15 100 10 9 16 109* (↑12%) 16 15

Globulin (g/dL) 16 1.2 0.2 12.1 16 1.3 0.2 13.9 15 1.4 0.1 9.7 16 1.5 0.1 8.0

Total bilirubin (mg/dL) 16 0.05 0.00 0.00 16 0.05 0.00 0.00 15 0. 0 5 0.00 0.00 16 0.05 0.00 0.00

Total protein (g/dL) 16 5.2 0.3 5.6 16 5.2 0.4 7.5 15 5.2 0.3 6.6 16 5.3 0.3 6.5

Sodium (mmol/L) 16 140 2 2 16 141 2 1 15 141 3 2 16 141 3 2
Potassium (mmol/L) 16 6.2 0.9 15.3 16 6.0 0.9 14.9 15 6.1 0.8 12.7 16 5.8 0.9 15.3
Chloride (mmol/L) 16 100 2 2 16 100 2 2 15 100 2 2 16 99 2 2
Calcium (mg/dL) 16 10.9 0.3 2.9 16 10.9 0.5 4.3 15 11.2 0.3 2.8 16 11.1 0.4 3.4
Phosphorus (mg/dL) 16 8.0 0.9 11.7 16 7.9 0.8 9.5 15 8.2 1.0 12.4 16 8.6 1.3 15.0

a Data were obtained from page s 59 and 60 of the stud y report

. Percent difference from control, presented in parentheses, were calculated by the reviewers.

b The unit is presented in the text as μ /L; review of the individual clinical chemistry tables (p. 102 of the report) shows the correct unit: U/L.

N – N umber of animals examined ; SD

– Standard d eviation ; SE

– Standard error; and CV

– Coefficient of variation

* Significantly different from controls at p<0.05.Female P ubertal Assay ( 2012)/ Page 14 of Error! Bookmark not defined.

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## G. Gross and Histopathology : There were no treatment -related gross pathology findings as a result of exposure to benfluralin. Renal cysts were observed in all treatment groups; these are common findings unassociated with treatment. One 300 mg/kg/day animal (#7042) was found with blood tinged urine; this was an isolated finding and considered to be

unassociated with treatment. Two other 300 mg/kg/day animals (#7041 & 7050) showed gastric mucosal inflammation and ulceration, respectively; t hese are common lesions in experimental animals and relationship to treatment was considered equivocal.

The incidences of histopathological findings of the thyroid gland are presented in Table 7.

There were no treatment

- related findings for the thyroid gland. There were no statistical differences in thyroid grades for follicular cell height or colloid area among treatment

groups.

TABLE 7

. Incidence of Histopathological Lesions of the Thyroid Gland a



### Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height

Grade b 

### Incidence Grade b 

### Incidence

O E O E

Vehicle Control 1 0 16 1 1 16

2 0 16 2 14 16

3 1 16 3 1 16

4 14 16 4 0 16

5 1 16 5 0 16

Benfluralin

25 mg/kg/day 1 NA 0 1 NA 0

2 NA 0 2 NA 0

3 NA 0 3 NA 0

4 NA 0 4 NA 0

5 NA 0 5 NA 0

Benflu r alin 75 mg/kg/day 1 NA 0 1 NA 0

2 NA 0 2 NA 0

3 NA 0 3 NA 0

4 NA 0 4 NA 0

5 NA 0 5 NA 0

Benflu r alin 300 mg/kg/day 1 0 16 1 0 16

2 1 16 2 10 16

3 5 16 3 5 16

4 10 16 4 1 16

5 0 16 5 0 16

a Data were obtained from page 67 of the study report.

b Thyroid histopathology is graded on a 1

– 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 1 = most colloid, 5 = least colloid.

O Number o bserved

E Number e xamined

NA Not applicableFemale P ubertal Assay ( 2012)/ Page 15 of Error! Bookmark not defined.

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## The incidence of histopathological findings of the ovaries, uterus and kidneys are presented

in Table 8

. There were no treatment

- related findings for the kidney. Evaluations of the uterus and ovaries were performed with knowledge of estrus stage and there were no

treatment -related findings. Three animal demonstrated discor dance between estrus staging (comparison of vaginal cytology and histopathology). One control a nimal (#6996) showed estrus histologically in both the ovary and uterus while vaginal cytology demonstrated diestrus. One 300 mg/kg/day a nimal (#7043) showed proestrus with histology and estrus with vaginal cytology; ovarian histology in this animal was lagging indicating diestrus. Another 300 mg/kg/day animal (#7049) had ovarian histology that indicated diestrus while vaginal cytology demonstrated estrus; howe ver, uterine tissue for this animal was lost in processing. Fin ally, one 300 mg/kg/day female (#7040) showed juvenile morphology as evidenced by lack of corpora lutea.

TABLE 8. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidney a

Findings Dose Level (# mg/kg bw/day)

Vehicle Control Benflu r alin ( 25 ) Benfluralin (7 5) Benflu r alin ( 300 )

# Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined

Ovaries

Within normal limits 16 16 NA 0 NA 0 15 16
Juvenile appearance 0 0 NA 0 NA 0 1 16

Uterus

Within normal limits 16 16 NA 0 NA 0 15 15

Not examined

missing 0 0 NA 0 NA 0 1 15

Kidney

Within normal limits 1 16 NA 0 NA 0 3 16
Degeneration, tubule, focal, very slight 5 16 NA 0 NA 0 3 16

Degeneration, tubule, multifocal, very slight 10 16 NA 0 NA 0 10 16

Dilitation, tubule, focal, very slight 2 16 NA 0 NA 0 0 16

Dilitation, tubule, multifocal, very slight 3 16 NA 0 NA 0 2 16

Inflamation, chronic, interstitium, focal,

very slight 2 16 NA 0 NA 0 2 16

Inflamation, chronic, interstitium, multifocal, very slight 3 16 NA 0 NA 0 0 16

a Data were obtained from page 68 of the study report.

NA Not a pplicable

, tissues not examined.Female P ubertal Assay ( 2012)/ Page 16 of Error! Bookmark not defined.

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## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS : All mean and CV values for the control animals met the performance criteria with the exception of the mean value for adrenal weight (lower than the acceptable range) and the CVs for final body weight and liver weight (higher th an the acceptable range). The adrenal weights were similar to the mean control values reported in previous pubertal assays conducted in the performing laboratory. The CV for terminal body weight (11.9%) was similar to the CV for body weights in the control groups on PND 22 indicating that the variance was stable during the study. Also, 3 of 4 laboratories in the

interla boratory validation study did not meet the performance criteria for terminal body weight and liver weight CVs.

There was no treatment

- re lated mortality during the study. Treated animals had dark yellow urine which is a typical finding following dinitroaniline herbicide exposure. There were no significant treatment

- related effects on mean estrous length, percent cycling, percent regularly cycling or body weight at VO . There were no gross findings at necropsy and no histopathological lesions in the uterus, ovaries, kidneys or thyroid.

At 300 mg/kg/day, treated animals had significantly lower body weights and body weight gains until PND 35; however, terminal body weights largely recovered by necropsy ( PND 42, ↓ 3.8%) with an overall body weight gain PND 22-42 of ↓ 5.5%. There were no

treatment

- related effects on body weight or body weight gain at doses ≤ 75 mg/kg/day. Mean age at VO was significantly increased at 300 mg/kg/day (+2.9 days) but not at lower dose levels. Age at first estrus was increased in the high dose animals due to the delay in

VO . Serum T4 levels w ere significantly decreased in at ≥ 75 mg/kg/day, and TSH levels were was significantly increased at 300 mg/kg/day; however

, there were no associated changes in thyroid weight or histopathology. High dose animals had significant decreases in alkaline phosphatase and increases in GGT, cholesterol and creatinine. There were no other

treatment

- related changes in clinical chemistry. Absolute, relative and adjusted liver weights were statistically significantly increased in a dose- dependent manner . Signi ficant decreases in pituitary weights (absolute and adjusted) and blotted uterine weight were also observed only at 300 mg/kg/day.

B. AGENCY COMMENTS : All animals survived until scheduled termination . One 75 mg/kg/day female was removed from the study after being dosed twice on one day. All of the 300 mg/kg/day animals had dark yellow urine, a typical finding following dinitroaniline herbicide exposure There were no significant treatment

- related eff ects on mean estrous length, percent cycling, percent regularly cycling or body weight at VO . There were no gross findings at necropsy and no histopathological lesions in the uterus, ovaries, kidneys or thyroid.

Treated animals had decreased body weights and cumulative body weight gains at 300 mg/kg/day until PND 35; however, terminal body weights largely recovered by necropsy at

PND 42 with a slight decrease in overall body weight gain. There were no treatment- related effects on body weight or body weight gain at doses ≤ 75 mg/kg/day. Mean unadjusted and adjusted age s at VO w ere increased in the 300 mg/kg/day group, but not at lower dose levels. Age at first estrus was also delayed at 300 mg/kg/day, which is consistent with the delay in VO . Serum T 4 

## levels were decreased in a dose-dependent manner at ≥ 75Female P ubertal Assay ( 2012)/ Page 17 of Error! Bookmark not defined.

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## mg/kg/day, and TSH was increased at 300 mg/kg/day; however, there were no associated changes in thyroid weight or histopathology. High dose animals had decreases in alkaline phosphatase and increases in GGT, cholesterol and creatinine. There were no other

treatment

- related changes in clinical chemistry. Unadjusted, adjusted, and relative liver weights were increased in a dose- dependent manner at ≥ 75 mg/kg/day, and relative liver weight was increased at 25 mg/kg/day . D ecreases in pituitary ( unadjusted, adjusted , and

relative ) ovaries (unadjusted and adjusted), and blotted uterus (adjusted) weight s were also observed at 300 mg/kg/day; these changes were not observed at ≤ 75 mg/kg/day.

The high dose of 300 mg/kg/day is considered to be adequate based on decreased b ody weight gains over the course of the treatment period and increased liver weights with concomitant significant decreases in alkaline phosphatase, and increases in GGT and

cholesterol. Treatment

- related effects seen at the high dose included delayed VO , decreased

serum T 4 

## and increased serum TSH.

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

• The following parameters differed from the performance criteria for controls: mean adrenal weight was 34.4 mg (acceptable range 38.34 -48.84 mg ), and %CVs were higher than recommended for final body weight 11.91% (maximum 8.93%), and liver weight 14.71% (maximum 13.13% ).

• The following parameters differed from the performance criteria for controls: mean adrenal weight was 34.4 mg (acceptable range 38.34-48.84 mg), % CV for final body weight was 11.91% (maximum 8.93%), and %CV for liver weight was 14.71% (maximum 13.13%).

# DATA EVALUATION RECORD

BENFLURALIN (B ENEFIN )

Study Type: O CSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-45-2012 (MRID 48669302)

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: Joseph P. Rieth, Ph.D., D.A.B.T. Date: 4 / 16 /2012

Secondary Reviewer : Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 4 / 26 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5 / 01 /2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 5 / 01 /2012

This Data Evaluation Record may have been altered 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 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 i s 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).Male P ubertal Assay ( 2012)/ Page 1 of Error! Bookmark not defined.

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## Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D.

_____ Signature : Health Effects Division Date: Secondary Reviewer John Liccione, Ph.D. Signature : Health Effects Division Date:

Template version 0 8/ 2011



## DATA EVALUATION RECORD

STUDY TYPE : Male Pubertal Assay ; O CSPP 890.1500

PC CODE : 084301 DP BARCODE : D399309

TXR# : 0054123 CAS No. : 1861-40-1

TEST MATERIAL (PURITY) : Benfluralin (97.5% a.i. )

SYNONYMS : Benefin; N

- Butyl

- N

- ethyl -2,6- dinitro -4- (trifluoromethyl)benzenamine

CITATION : Marty, M.S., Andrus, A.K., Stebbins, K.E. (2012) Benefin: p ubertal development and thyroid function in intact juvenile/peripubertal male Crl:CD(SD) rats. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Labora tory Study N o.: 111074. January 25, 2012. Unpublished.

SPONSOR : Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, IN

TEST ORDER # : EDSP -084301-15

EXECUTIVE SUMMARY : In a male pubertal assay (M RID 48669302), groups of 16 Crl:CD(SD) rats were treated daily via oral gavage with benfluralin (97.5% a.i.; Lot #ACD13683) in corn oil at doses of 0, 25, 100 or 400 mg/kg/day from post- natal day ( PND) 23

to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and

the age and weight of each animal on the day of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, thyroxine (T 4 

## ), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassay (RIA) or chemiluminescent immuno assay. The following organs were weighed: liver, kidneys and adrenals (weighed as pairs), testes and epididymides (weighed individually), thyroid and pituitary (weighed after fixation), ventral prostate, dorsolateral prostate, seminal vesicle with c oagulating glands , and the levator ani plus bulbocavernosus muscles ( LABC ). Microscopic evaluations were performed on testes, epididymides, thyroid, and kidneys.

All rats survived to scheduled termination. There were no treatment related effects on mean age and weight at completion of PPS, mean weights of adrenal s, kidneys, and testes, and gross and microscopic examinations of the testes, epididymides, and kidneys.

Systemic findings were observed at 400 mg/kg/day. D ark yellow/orange urine , a common finding following dinitroaniline herbicide treatment , was observed in all rats. Unadjusted and adjusted final body weights were decreased (not significant [NS]) by 8%, resulting in a 9%Male P ubertal Assay ( 2012)/ Page 2 of Error! Bookmark not defined.

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## decrease (p<0.05) in overall body weight gains (PND 23 -53). Unadjust ed , adjusted

, and relative (to body) liver weights were increased (p<0.05) by 40-52%, unadjusted and adjusted pituitary weights were decreased ( p<0.05 ) by 13 -14%, and unadjusted and adjusted thyroid weights were increased ( p<0.05, except unadjusted NS ) by 15%. Serum alkaline phosphatase levels were decreased (p<0.05) by 40%, and s erum total protein

, creatinine, and gamma glutamyl transferase

were increased (p<0.05) by 7%, 50%, and 60%, respectively. Globulin was increased (not statistically analyzed) by 2 2%. Phosphorus, chloride, and potassium levels were decreased (p<0.05) by 9%, 2%, and 6%, respectively.

At 100 mg/kg/day, unadjusted, adjusted and relative (to body) liver weights were increased (p<0.05) by 22-23%. Serum alkaline phosphatase levels were decreased (p<0.05) by 17%. Phosphorus was decreased (p<0.05) by 9%.

A t 25 mg/kg/day , only relative liver weight was increased ( p<0.05), by 7 %

.

There was a dose -dependent decrease (p<0.05) in serum T 4 

## levels of 12%, 29% and 53% at 25, 100 and 400 mg/kg/day, respectively, and a dose-dependent increase (p<0.05) in serum TSH levels of 53% and 105% at 100 and 400 mg/kg/day, respectively. Serum testosterone was also decreased (NS) by 52% at 400 mg/kg/day. When one animal with a testosterone level well above the group average was excluded from the analysis, the d ecrease in testosterone at 400 mg/kg/day was 69% (p<0.05). At 400 mg/kg/day, decreased (p<0.05) unadjusted and adjusted accessory sex tissue weig hts were observed as follows: (i) seminal vesicle + coagulating gland with fluid by 32%; (ii) seminal vesicle + coagulating gland without fluid by 25-26%; (iii) ventral

prostate by 26%; (iv) dorsolateral prostate by 21- 22 %; (v) LABC by 20%; and (vi) epidi dymis by 13-18%. At 100 mg/kg/day, only adjusted right epididymis weight was decreased (p <0.05), by 9% . A nimals treated with 400 mg/kg/day had increase s in thyroid follicular cell height with concomitant decreases in colloid area. A slight decrease in spermatic elements in the right epididymis was observed in two animals (#6976 & #6982) at 400 mg/kg/day.

The dose levels used in this study were considered to be adequate based on markedly increased liver weights and significant changes in thyroid hormone levels and histopathology, as well as a significant decrease in body weight gain, at 400 mg/kg/day.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Male Pubertal A ssay (OCSPP

890.1500 ).

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

statements were pro vided.Male P ubertal Assay ( 2012)/ Page 3 of Error! Bookmark not defined.

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## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Toxicology & Environmental Research and Consulting, The Dow Chemical Company

Location: Midland, Michigan Study Director: A . K. Andrus , M.S

Other Personnel: M.S. Marty, Ph.D., Lead Scientist K.E Stebbins, D.V.M., Pathologist Study Period: 6/20/2011 to 1/2 5 /2012



## 2

. Test S ubstance : Benfluralin

Description: T echnical, molecular weight 335.3

Source: Dow AgroSciences, LLC, Indianapolis, I N

Lot/Batch #: Lot # ACD13683 Purity: 97.5 % Stability: Not provided

CAS #: 1861

- 40

- 1

Structure:

N N +

N + O O

O

O CH 3

F

F F CH 3



## 3

. V ehicle : Corn oil

4

. Test A nimals : Species: Rat

Strain: Sprague Dawley Crl:CD(SD) Age/Weight at Study

Initiation: 23 days/ 44.4

- 68.8 g
, males only

Source: Charles River Laboratories, Ltd., Portage Michigan Housing: 2 /cage in solid bottom plastic cage s with heat

- treated laboratory

- grade wood shavings as bedding.

Diet: Tekland Diet #2016 (Harlan Laboratories, Inc., Indianapolis, IN ) , ad libitum Phytoestrogen content <325 μg of genistein equivalents/gram diet

Water: Deionized tap water, ad libitum

Environmental

Conditions: Temperature: Humidity:

Air changes: Photoperiod: 22 ± 1 °C

40

- 70 %

12

- 15/hr

12 hrs light/ 12 hrs dark



## B. STUDY DESIGN

1. In -L ife D ates : Start: 6/25/2011 End: 7/26/2011

2. Mating : Nulliparous female rats were bred by the supplier and shipped to arrive on gestation days (GD) 7 -10. The day an animal was found to be sperm positive was designated GD 0. Litters born on GD 21 or 22 were used in the assays; study endpoints were based on pup ageMale P ubertal Assay ( 2012)/ Page 4 of Error! Bookmark not defined.

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## relative to birth date. Litters were culled to 10 pups on PND 4 using 5 males an d 5 females

where possible; males were used in this male pubertal assay and females were used in a concurrently reviewed female pubertal assay (MRID 48669301).

3. Animal A ssignment : Offspring were weaned on PND 21 and weighed. The rats from the extreme ends of the weight range were eliminated to achieve a mean body weight and variance across dose groups at study initiation. Animals were assigned to the test groups

noted in Table 1. Care was taken so that litter m a tes were not assigned to the same treatment group. The treatment groups included vehicle controls and benfluralin

- treated rats

with three dose levels.

TABLE 1. Study Design a


### Test group Dose (mg/kg/day) # of Males

Control 0 16

Low 25 16

Mid 100 16
High 400 16

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



## 4. Dose S election Rationale : Prior to the start of the current assay, a probe study 1 

## was conducted in which groups of 4 male and 4 female Crl:CD(SD) rats were administered benfluralin by gavage at dose levels of 0, 250, 500, 750, or 1000 mg/kg /day in corn oil for 15 days. The male rats were treated from PND 30 -44. Body weights were decreased in a dose-dependent manner beginning on D ay 9 through Day 15. On PND 44, body weight

d ecreases were 5.8 %, 11.1 %, 11.6% and 16.1 % at 250, 500, 750, and 1000 mg/kg/day respectively, and were also observed at Day 15. All treated animals had decreased feed consumption at all dose levels and time points. Facial soiling was observed at necropsy (2- 3/ group) at 750 and 1000 mg/kg /day. On male in each of the 750 and 1000 mg /k g/day groups had bilateral decreased size of the epididymides and testes. One male in each of the 500 and 750 mg/kg/day groups and 2 males in the 1000 mg/kg/day group had decreased

bilateral seminal vesicle size. L iver weights were increased at all dos e levels

. Some

h epatocellular hypertrophy was observed at 1000 mg/kg/day . Based on these data, 400 mg/kg/day was selected as the high dose for the prepubertal assay

.

5. Dose P reparation and Analysis : The test material was suspend ed in corn oil to yield the desired concentrations (further information not provided). Concentration and homogeneity ( sampling not specified) analyses w ere conducted on the first preparation prior to dose

initiation . Concentration analyses were performed on all dose levels; ho mogeneity analyses were performed on the 25 and 400 mg/kg/day formulations. It was stated that the stability of benfluralin in corn oil was confirmed for up to 22 days at room temperature in a previously reviewed Hershberger study (MRID 48614907).

Results of D ose Analysis

1 

### Marty, M. S. and Marshall, V. A. (in progress). Benefin: Hershberger, Uterotrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemica l Company, Midland, MI.Male P ubertal Assay ( 2012)/ Page 5 of Error! Bookmark not defined.

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## Homogeneity (% RSD) : 2.3-4.5%

Stability (% of Day 0): 94.9-114.3% following storage at room temperature for up to 22 days.

Concentration (% of nominal): 94.7-97.6%

The analytical data indicated that the mixing procedure was adequate and that the varia tion between nominal and actual dosage to the animals was acceptable.

6. Dosage A dministration : All doses were administered once daily at approximately the same time each day by oral gavage from PND 23 -53, in a volume of 4 mL/kg of body weight. Dose volumes were adjusted using the most recently determined individual body weight

.

7. Statistics : For continuous variables, Bartlett’s test was used to assess homogeneity of variance (alpha = 0.01). Two of the variables were log transformed based on the outcome of the Bartlett’s test: body weight gain and GGT. Age and body weight at PPS and organ weights were analyzed with analysis of covariance (ANCOVA) using body weight at PND

23 as the covariate ; when the dose effect was significant at alpha = 0.05, least square means

with a Dunnett’s correction was used to determine differences. Oth er parameters were tested using ANOVA (alpha = 0.05); when dose effects were observed then Dunnett’s test was used (alpha = 0.05). Certain histopathological findings were analyzed using the pairwise Chi

- square test using Yates ’ continuity correction ( two -sided; alpha = 0.05). The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and C linical Examinations : Cageside examinations and observations for mortality and morbidity were performed on a ll animals at least twice daily

. Hand -held

c linical examinations were conducted each treatment da y at approximately the same time post-dosing (no additional details included in the report).

2. Body W eight : Animals were weighed on PND 21 and then daily prior to dosing. Body weights were reported and statistically analyzed on PND 21, 23, 26, 30, 37, 42, 45, and 53. Body weight gains were reported and statistically analyzed for all reported weight intervals

, beginning with PND 23-26 and including the entire dosing period (PND 23 -53).

3. Preputial Separation (PPS) : Beginning on PND 30 , all animals were examined daily for

onset of PPS . Age and weight at on the day of completion of PPS were recorded. If PPS was not completed by PND 53, PND 54 was used for determining the mean age at puber ty onset for the dose group.

4. Sacrifice and Pathology : On the day of termination (PND 53) approximately 2 hours postdosing, the rats (unfasted) were removed from their home cages one at a time

, anesthetized

with isoflurane, and blood was collected by cardiac puncture. A nalyses for testosterone, T 4 

## , and TSH were conducted at WIL Research Laboratories, LLC (Ashland, Ohio). The rats were then euthanized by exsanguination followed by decapitation. A complete necropsyMale P ubertal Assay ( 2012)/ Page 6 of Error! Bookmark not defined.

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## was conducted on all animals , and it was stated that the groups were counterbalanced during

necropsy.

a. Hormone Analysis : Serum samples were stored at

− 80° C and shipped on dry ice. Total
testosterone, T 4 

## , and TSH levels were analyzed using chemiluminescent immunoassay (testosterone and T 4 

## ) or radioimmunoassay ( TSH ).

b. Clinical C hemistry : 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 Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALK) X Total protein

Cholinesterase (ChE) Triglycerides
Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH)

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)
Sorbitol dehydrogenase

X Gamma glutamyl transferase (GGT)
Glutamate dehydrogenase

* Recommended based on guideline 890.1500.



## c. Organ W eights and Histopathology : The following CHECKED (X) tissues were collected and weighed . The (XX) organs, in addition, were subjec ted to histological

examination.Male P ubertal Assay ( 2012)/ Page 7 of Error! Bookmark not defined.

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UROGENITAL OTHER

X X Testes (left and right separately) * + X X Thyroid * +

X X Epididymides (left and right separately) * + X Liver *

X Seminal vesicle plus coagulating glands (with and without fluid) * X Adrenals (paired) *

X Ventral prostate * X Pituitary *

X Dorsolateral prostate *

X Levator ani

- bulbocavernosus (LABC) muscle complex *

X X Kidneys (paired) * +

* Weights required based on G uideline 890.1500

+ Histopathological examination required based on G uideline 890.1500



## All collected organs, except the thyroid and pituitary, were weighed prior to fixation. Kidney and adrenals were weighed as pairs, and testes and epididymides were weighed individually. Seminal vesicles with coagulating glands were weighed, then cut sagittally, blotted, and reweighed.

Following weighing, the test es and epididymi des were fix ed in Bouin’s solution overnight

for 24 -72 hours ; t he duration of fixation for these tissues does not conform to the Guideline but the performing laboratory stated in the report that this fixation duration does not impact the quality of the tissues for his topathological evaluation. The other tissues were fixed in 10% buffered formalin for at least 24 hrs. Following fixation, the thyroid wa s dissected free

of the trachea and weighed . All collected tissues were routinely processed into paraffin blocks, sectioned, stained with hematoxylin and eosin, and examined microscopically. The exception was the testes which was processed and stained with modified periodic acidSchiff’s/hematoxylin. Tissues from the 400 mg/kg/day groups and the controls were

examined microscopically , except for the thyroid and epididymides, where all dose groups

were examined

. The thyroid sections were subjectively evaluated for follicular cell height and colloid area using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest).

II. RESULTS

A. Mortality : All animals survived until scheduled termination.

B. Clinical S igns of Toxicity : All rats treated with benfluralin at 400 mg/kg/day were

observed to have dark yellow/orange urine. It was stated that this is a common finding following dinitroaniline herbicide treatment . One 400 mg/kg/day male (#6987) had dermatitis on the left side of its neck during PND 48 -53, but this single finding was considered incidental to treatment. No other clinical signs were reported

.

C. General G rowth and P reputial Separation : Body weights, body weight gains, age of

attainment of PPS and weight at day of PPS are presented in Table 2. There were no significant differences in initial (PND 23) body weights at any dose level of benfluralin. At 400 mg/kg/day, unadjusted and adjusted final body weights were decreased (not significant [NS]) by 8%, resulting in a 9% decrease (p<0.05) in overall (PND 23-53) body weight gains. Final body weights and final body weight gains were similar to controls at 25 and 100 mg/kg/day. All study animals completed PPS during the study period (Table 3) . The mean ages at PPS were 45.4, 44.7, 45.2 and 45.7 d ays for the 0, 25, 100 and 400 mg/kg/d ay groups respectively. There were no significant diffe rences in either age or body weight at PPS in any of the treated groups.Male P ubertal Assay ( 2012)/ Page 8 of Error! Bookmark not defined.

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### TABLE 2

. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Benfluralin

25 mg/kg/day Benfluralin

100 mg/kg/day Benfluralin

400 mg/kg/day

N Mean SD /

SE b CV (%) N Mean SD /

SE b CV (%) N Mean SD /

SE b CV (%) N Mean SD /

SE b CV (%)
Initial body weight (PND 23; g) U 16 56.16 4.52 8.05 16 56.32 5.96 10.58 16 56.54 6.80 12.03 16 56.29 6.20 11.02

Body weight at PPS (g) U 16 220.84 23.61 10.69 16 207.53 29.55 14.24 16 221.94 28.25 12.73 16 207.73 19.13 9.21

A 16 221.20 5.587 NA 16 207.54 5.586 NA 16 221.48 5.587 NA 16 207.81 5.586 NA

Final body weight (g) U 16 284.45 22.87 8.04 16 273.89 30.04 10.97 16 285.33 32.02 11.22 16 262.99 (↓8) 29.53 11.23

A 16 285.05 5.002 NA 16 273.92 5.002 NA 16 284.57 5.003 NA 16 263.13 (↓8) 5.002 NA

Final body weight (% of control) U NA NA NA NA 16 96.3 10.6 11.0 16 100.3 11.3 11.2 16 92.5 10.4 11.2

A NA NA NA NA

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

Body weight gain (final

– initial; g) U 16 228.29 19.73 8.64 16 217.58 26.48 12.17 16 228.79 26.47 11.57 16 206.71* (↓ 9 ) 26.09 12.62

A

-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Age at PPS (PND) U 16 45.4 2.3 5.1 16 44.7 2.2 4.8 16 45.2 2.1 4.7 16 45.7 2.0 4.4

A 16 45.4 0.52 NA 16 44.7 0.52 NA 16 45.2 0.52 NA 16 45.7 0.52 NA

a Data were obtained from Table 7 on page 56 of the study report

. Percent difference s from control, calculated by the reviewers, are presented in parentheses.

b Standard deviation reported for unadjusted values and standard error of the mean reported for adjusted values.

U Unadjusted for body weight on PND 2 3

A Adjusted for body weight on PND 2 3

N Number of animals examined

SD Standard d eviation

CV Coefficient of v ariation

* Significantly different from controls at p<0.05.

NA Not applicable

-- The report states this is n ot required as part of the test guideline.Male P ubertal Assay ( 2012)/ Page 9 of Error! Bookmark not defined.

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### TABLE 3

. Preputial Separation

Vehicle Control Benfluralin 25 mg/kg/day Benfluralin 100 mg/kg/day Benfluralin 400 mg/kg/day



### Number of

Animals

Examined 16 16 16 16

Incidence Unseparated 0 0 0 0

a Data were obtained from Table 7 on page 56 of the study report.



## D. Organ W eights : Organ weights at necropsy are presented in Table 4 below. Liver weights

were increased in a treatment -related manner in all dose groups relative to controls. Unadjusted, adjusted, and relative (to body) liver weights were increased (p<0.05) by 22- 23% at 100 mg/kg/day and 40-52% at 400 mg/kg/day. A t 25 mg/kg/d ay , only the relative liver weight was increased ( p<0.05, 7%). Unadjusted and adjusted pituitary weights were decreased ( p<0.05 ) by 13 -14 % at 400 mg/kg/day, while relative weights showed no apparent change and pituitary weights at lower doses were unaffected by treatment

. Adjusted thyroid weights were increased (p<0.05 ) by 1 5 % at 400 mg/kg/day; unadjusted thyroid weights were also increased (NS) by the same magnitude . Thyroid weight was not affected by treatment at lower dose levels.

At 400 mg/kg/day, decreased (p<0.05) unadjusted and adjusted accessory sex tissue weights

were observed as follows: (i) se minal vesicle + coagulating gland with fluid by 32%; (ii)

seminal vesicle + coagulating gland without fluid by 25-26%; (iii) ventral prostate by 26%; (iv) dorsolateral prostate by 21-22%; (v) LABC by 20%; and (vi) epididymis by 13-18%. At 100 mg/kg/day, o nly adjusted right epididymis weight was decreased (p <0.05) by 9%

. Kidney, adrenals and testis weights were unaffected by treatment with benfluralin at any of

the dose levels tested.Male P ubertal Assay ( 2012)/ Page 10 of Error! Bookmark not defined.

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### TABLE 4

. Organ Weights at Necropsy a

Organ Vehicle Control Benfluralin 25 mg/kg/day Benfluralin 100 mg/kg/day Benfluralin 400 mg/kg/day

N Mean SD / SE b CV (%) N Mean c SD/ SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/ SE b CV (%)

L iver (g) U 16 12.46 1.25 10.06 16 12.79 1.69 13.18 16 15.26* (↑23 ) 1.79 11.75 16 17.46* ( ↑40 ) 1.78 10.17

A 16 12.48 0.353 NA 16 12.79 0.353 NA 16 15.23* (↑22 ) 0.353 NA 16 17.46* (↑40 ) 0.353 NA

R 16 4.38 0.25 5.76 16 4.67* (↑ 7 ) 0.23 5.02 16 5.35* (↑22 ) 0.28 5.28 16 6.66* (↑52 ) 0.46 6.98

Kidneys (g) U 16 1

. 96 0.17 8.72 16 1.92 0.15 7.76 16 2.00 0.21 10.51 16 1.87 0.19 10.01

A 16 1.96 0.040 NA 16 1.92 0.040 NA 16 2.00 0.040 NA 16 1.87 0.040 NA

R 16 0.69 0.04 5.57 16 0.70 0. 0 4 6.35 16 0.70 0.06 8.07 16 0.71 0.03 4.35

Pituitary (mg) U 16 9.6 1.1 11.2 16 8.9 1.1 12.1 16 9.3 1.1 11.5 16 8.3* (↓13 ) 0.9 11.4

A 16 9.7 0.25 NA 16 8.9 0.25 NA 16 9.3 0.25 NA 16 8.3* (↓14 ) 0.25 NA

R 16 3.4 0.3 8.8 16 3.3 0.3 10.4 16 3.3 0.3 8.7 16 3.2 0.3 9.7

Adrenals (mg) U 16 45.2 7.2 15.9 16 44.1 8.6 19.5 16 45.7 9.2 20.2 16 42 7 16.6

A 16 45.3 1.90 NA 16 44.1 1.90 NA 16 45.6 1.90 NA 16 42.0 1.90 NA

R 16 15.9 2.1 13.1 16 16.1 2.3 14.1 16 16.1 3.0 18.4 16 16.0 2.4 14.7

Seminal vesicle + coagulating gland, with fluid (mg) U 16 463.9 100.7 21.7 16 444.2 107. 0 24.1 16 439.6 137.8 31.4 16 315.2* (↓32 ) 108.0 34.3

A 16 465.0 27.2 0 NA 16 444.3 27.20 NA 16 438.2 27.21 NA 16 315.4* (↓32 ) 27.20 NA

Seminal vesicle + coagulating gland, without fluid (mg) U 16 230.5 39.5 17.1 16 213.6 42.3 19.8 16 219.4 49.8 22.7 16 171.0* (↓25 ) 49.5 29

A 16 230.9 10.81 NA 16 213.6 10.81 NA 16 218.8 10.81 NA 16 171.1* (↓ 26 ) 10.81 NAMale P ubertal Assay ( 2012)/ Page 11 of Error! Bookmark not defined.

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### TABLE 4

. Organ Weights at Necropsy a

Organ Vehicle Control Benfluralin 25 mg/kg/day Benfluralin 100 mg/kg/day Benfluralin 400 mg/kg/day

N Mean SD / SE b CV (%) N Mean c SD/ SE b CV (%) N Mean SD/

SE b CV (%) N Mean SD/ SE b CV (%)

Ventral prostate (mg) U 16 216.9 41.4 19.1 16 194.2 40.3 20.8 16 202.6 55.4 27.3 16 159.9* (↓26 ) 52.0 32.5

A 16 217.5 10.69 NA 16 194.3 10.69 NA 16 201.8 10.69 NA 16 160.1* (↓26 ) 10.69 NA

Dorsolateral prostate (mg) U 16 158.6 31.8 20.1 16 153.3 38.9 25.4 16 150.4 29.1 19.3 16 124.7* (↓21 ) 35.4 28.4

A 16 159.2 6.64 NA 16 153.3 6.64 NA 16 149.6 6.64 NA 16 124.9* (↓22 ) 6.64 NA

LA BC (mg) U 16 483.4 63.5 13.1 16 477.8 61.9 12.9 16 468.1 105.6 22.6 16 388.6* (↓20 ) 87.9 22.6

A 16 485.1 14.12 NA 16 477.9 14.12 NA 16 465.9 14.12 NA 16 389.0 * (↓20 ) 14.12 NA

Epididymis , left (mg) U 16 213.6 22.7 10.6 16 214.5 24.4 11.4 16 203.4 32.9 16.2 16 185.8* (↓13) 21.2 11.4

A 16 214. 2 4.68 NA 16 214.6 4.68 NA 16 202.8 4.68 NA 16 186.0* (↓13 ) 4.68 NA

Epididymis, right (mg) U 16 226.7 20.2 8.9 16 210.1 20.3 9.7 16 206.7 30.5 14.7 16 185.9* (↓18) 29.3 15.7

A 16 227.1 5.04 NA 16 210.2 5.04 NA 16 206.1 * (↓ 9 ) 5.04 NA 16 186.0* (↓18 ) 5.04 NA

Testis, left (mg) U 16 1405 129.3 9.2 16 1414 115.6 8.2 16 1431 199.9 14 16 1356 160.2 11.8

A 16 1407 32.8 NA 16 1414 32.8 NA 16 1428 32.8 NA 16 1357 32.8 NA

Testis, right (mg) U 16 1418 124.3 8.8 16 1441 154.1 10.7 16 1418 199.6 14.1 16 1374 162.1 11.8

A 16 1420 36.2 NA 16 1441 36.2 NA 16 1415 36.2 NA 16 1374 36.2 NA

Thy roid, fixed (mg) U 16 12.0 1.5 12.8 16 12.6 2.2 17.7 16 13.0 1.7 12.8 16 13.8 2.7 19.5

A 16 12.0 0.52 NA 16 12.6 0.51 NA 16 12.9 0.51 NA 16 13.8* (↑15) 0.51 NA

a Data were obtained from Table 9 on page s 59

- 61 of the study report

. Percent differences from controls, calculated by the reviewer s. are presented in parentheses

. b Standard deviation reported for unadjusted values and standard error of the mean reported for adjusted values.

U Unadjusted for body weight on PND 2 3

A Adjusted for body weight on PND 2 3

R Organ

- to

- body weight ratio (relative to body weight)Male P ubertal Assay ( 2012)/ Page 12 of Error! Bookmark not defined.

BENFLURALIN /084301 O CSPP 89 0. 1 500 / OECD None

N Number of animals examined

SD Standard d eviation

CV Coefficient of v ariation

NA Not a pplicable

* Significantly different from controls at p<0.05.Male P ubertal Assay ( 2012)/ Page 13 of Error! Bookmark not defined.

BENFLURALIN /084301 O CSPP 89 0. 1 500 / OECD None



## E. Clinical Chemistry and Hormone Levels : Mean hormone and clinical chemistry levels are presented in Table 5. There was a dose

- dependent decrease (p<0.05) in t otal serum T 4 

## of 12%, 29% and 53% at 25 , 100 and 400 mg/kg/day , respectively; and a dose

- dependent increase (p<0.05) in TSH of 53% and 105% at 100 and 400 mg/kg/day , respectively. Serum testosterone was decreased (NS) by 52% at 400 mg/kg/day. It was stated that one 400 mg/kg/day rat (#6984) had a serum testosterone level of 8.86 ng/mL, which was considerably higher than the mean value for the group (1.41 ng/mL). When the value for this rat was removed from calculations, the mean testosterone level was 0.91 ng/mL, which is a 69% decrease (p<0.05) compared to controls.

There was also a dose -dependent decrease (p<0.05) in s erum alkaline phosphatase levels of

17% and 40% at 100 and 400 mg/kg/day , respectively . Serum total protein

, creatinine , and gamma glutamyl transferase were increased (p<0.05) at 400 mg/kg/day ( ↑7%

, ↑50%, and ↑60%, respectively ). Globulin showed an increasing trend with dose, increasing (not statistically analyzed) by 22% at 400 mg/kg/day . Phosphorus was decreased (p<0.05) by

9% at ≥ 100 mg/kg/day, and chloride and potassium were decreased (p<0.05) by 2% and 6%, respectively, at 400 mg/kg/day. Calcium was increased (p<0.05) by 3% at 100 mg/kg/day, but this increase was not dose-dependent.Male P ubertal Assay ( 2012)/ Page 14 of Error! Bookmark not defined.

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### TABLE 5

. Hormone Levels and Clinical Chemistry a



### Parameter

Evaluated Vehicle Control Benfluralin 25 mg/kg/day Benfluralin

100 mg/kg/day Benfluralin 400 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 16 4.61 0.52 11.35 16 4.05* (↓12 ) 0.44 10.80 16 3.27* (↓29 ) 0.43 13.18 16 2.15* (↓53 ) 0.47 21.98

Serum TSH (ng/mL) 16 8.3 2.9 34.6 16 8.7 3.0 33.9 16 12.7* (↑53) 5.4 42.9 16 17.0* (↑105) 5.2 30.6

Serum testosterone (ng/mL) 16 2.916 2.399 82.26 16 2.983 1.612 54.05 16 2.122 1.30 61.44 16 1.410 (↓5 2)
[ 0.91* ] b (↓69 ) 2.124 150.7

Clinical Chemistry

Creatinine (μmol/L) 16 0.2 0.1 25.8 16 0.2 0.0 18.4 16 0.2 0.0 20.7 16 0.3* (↑50) 0.1 23.6

Serum urea nitrogen (mmol/L) 16 15 1 10 16 15 2 13 16 15 3 19 16 15 4 23

Alkaline phosphatase (μmol/L) 16 405 78 19 16 383 59 15 16 33 8* (↓17 ) 59 18 16 245* (↓40 ) 44 18

Alanine amino transferase (μmol/L) 16 65 12 18 16 60 13 21 16 62 15 24 16 54 9 17

Aspartate

aminotransferase (μmol/L) 16 115 20 18 16 104 14 14 16 120 36 30 16 114 21 18

Gamma glutamyl transferase (μmol/L) 16 1.5 0.0 0.0 16 1.5 0.0 0.0 16 1.5 0.0 0.0 16 2.4* (↑60) 2.0 81.4

Albumin (g/dL) 16 3.8 0.2 4.9 16 3.8 0.2 5.3 16 3.8 0.2 4.6 16 3.8 0.2 4.4

A/G ratio 16 2.2 0.3 12.2 16 2.1 0.3 13 16 1.9 0.2 12.9 16 1.8 0.1 6.4

Cholesterol (mg/dL) 16 103 17 16 16 107 13 12 16 102 19 19 16 106 15 14

Globulin (g/dL) 16 1.8 0.2 9.5 16 1.9 0.2 10.9 16 2.0 0.3 13.7 16 2.2 0.2 8.1

Total bilirubin (mg/dL) 16 0.05 0.02 27.91 16 0.05 0.00 0.00 16 0.06 0.02 30.36 16 0.05 0.00 0.00

Total protein (g/dL) 16 5. 6 0.2 4.0 16 5.6 0.2 4.2 16 5.8 0.3 5.3 16 6.0* (↑7 ) 0.3 5.2

Sodium (mmol/L) 16 143 2 1 16 143 2 1 16 143 1 1 16 144 1 1

Potassium (mmol/L) 16 5.3 0.3 5.2 16 5.3 0.2 4.0 16 5.1 0.3 6.8 16 5.0* ( ↓ 6 ) 0.4 8.0

Chloride (mmol/L) 16 101 1 1 16 101 1 1 16 101 2 1 16 99* (↓2 ) 1 1

Calcium (mg/dL) 16 11.1 0.3 2.5 16 11.1 0.3 2.6 16 11.4* (↑3 ) 0.3 3.0 16 11.3 0.3 2.7

Phosphorus (mg/dL) 16 9.3 0.7 8.1 16 9.1 0.6 6.7 16 8.5* (↓9 ) 0.7 8.4 16 8.5* (↓9 ) 0.7 7.6

a Data were obtained from Table 8 on page s 57

- 58 of the study report. Percent difference s from control

, calculated by reviewer

, are presented in parentheses

. b [ Mean ] when an animal with an atypically high value (animal # 6984) is excluded.

N Number of animals examined

SD Standard d eviation

CV Coefficient of v ariation

* Significantly different from controls at p<0.05.



## F. Gross and Histopathology : The incidence s of h istopathological findings of the thyroidMale P ubertal Assay ( 2012)/ Page 15 of Error! Bookmark not defined.

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## gland are presented in Table 6. There were no treatment

- related gross pathological

observations. Statistical analysis of the thyroid did not reveal any significant effects. However, animals treated with 400 mg/kg/day had i ncrease s in follicular cell height (increased in Grades 3 and 4 ) with a relate d de crease in colloid area (increase in Grades 2 and 3). Other dose groups were unaffected by treatment.

TABLE 6

. Incidence of Histopathological Lesions of the Thyroid Gland a



### Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height Dilatation, duct, focal; moderate Dilatation , duct multifocal; moderate

Grade b 

### Incidence Grade b 

### Incidence Incidence Incidence

O E O E O E O E

Vehicle Control 1 0 16 1 3 16

0 16 0 16 2 1 16 2 11 16

3 1 16 3 1 16

4 11 16 4 1 16

5 3 16 5 0 16

Benfluralin

25 mg/kg/day 1 0 16 1 1 16

0 16 0 16 2 0 16 2 13 16

3 2 16 3 2 16

4 13 16 4 0 16

5 1 16 5 0 16

Benfluralin 100 mg/kg/day 1 0 16 1 0 16

0 16 0 16 2 1 16 2 11 16

3 4 16 3 4 16

4 11 16 4 1 16

5 0 16 5 0 16

Benfluralin 400 mg/kg/day 1 0 16 1 0 16

1 16 1 16 2 4 16 2 7 16

3 5 16 3 5 16

4 7 16 4 4 16

5 0 16 5 0 16

a Data were obtained from Table 11 on page 67 of the study report.

b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height ,1 = lowest, 5 = highest; and Colloid area, 1 = most colloid, 5 = least colloid.

O N umber o bserved

E N umber e xamined



## The incidence of histopathological findings of the testes, epididymides

, and kidneys are presented in Table 7

. A slight decrease in spermatic elements in the right epididymis was observed in two animals (#6976 & #6982) at 400 mg/kg/day. There were no treatment

related histopathological effects observed in the kidneys or testis of the control or treated

animals.Male P ubertal Assay ( 2012)/ Page 16 of Error! Bookmark not defined.

BENFLURALIN /084301 O CSPP 89 0. 1 500 / OECD None



### TABLE 7. Incidence of Histopathological Lesions of the Testes, Epididymides and Kidney a

Findings Dose Level (# mg/kg bw/day)

Vehicle Control Benfluralin ( 25 ) Benfluralin ( 100 ) Benfluralin ( 400 )

# Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined

Testes

Within normal limits 16 16 NA NA NA NA 16 16
Epididymides

Within normal limits 16 16 16 16 16 16 14 16
Decreased spermatic elements. slight 0 16 0 16 0 16 2 16

Kidney

Within normal limits 5 16 NA NA NA NA 2 16
Cyst, tubule, cortex,

focal 1 16 NA NA NA NA 3 16

Cyst, tubule, medulla, focal 0 16 NA NA NA NA 3 16

Degeneration, tubule, focal, very slight 1 16 NA NA NA NA 5 16

Degeneration, tubule, multifocal, very slight 7 16 NA NA NA NA 9 16

Inflammation

, chronic, interstitium, focal, very slight 4 16 NA NA NA NA 2 16

Inflammation

, chronic, interstitium, multifocal, very slight 1 16 NA NA NA NA 2 16

a Data were obtained from page 68 of the study report.

NA Not a pplicable, tissues not examined.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : A ll mean values met the performance criteria except the thyroid and kidney mean weights. M ean kidney and thyroid control weights

were consistent with the historical control dat a; therefore, these differences were unlikely to

have affected the study outcome. Several parameters had slightly elevated CVs relative to the performance criteria: seminal vesicle weight, body weight at PPS and final body weight.

T hese variables were increased in the inter -laboratory validation studies in 2 of 4 laboratories (seminal vesicle weight and final body weight) or in all 4 laboratories (body weight at PPS). T hese variables we re likely to have little or no impact on the outcome of the study. Mortality, b ody weights, age and body weight at PPS , kidney weights, adrenal weights and testes weight were unaffected by treatment with benfluralin up to the highest dose tested of 400 mg/kg/day. Body weight gain was significantly decreased at the high dose (9.5%). There were no gross or histopathological findings in the testis or kidneys.

At 400 mg/kg/day, clinical signs consisted of dark colored urine which is consistent with exposure to dinitroaniline herbicides; no other treatment

- related clinical signs were reported in the study. Serum testosterone and T4 levels were decreased , and TSH was increased.

G GT, globulin and total protein were increased; other clinical chemistry parameters were unaffected by treatment. Liver weights were significantly increased (52%) . Absolute and adjusted pituitary weights were decreased but relative weights were not statis tically differentMale P ubertal Assay ( 2012)/ Page 17 of Error! Bookmark not defined.

BENFLURALIN /084301 O CSPP 89 0. 1 500 / OECD None



## suggesting an effect of decreased growth rate. Absolute and/or adjusted thyroid weights were significantly increased. Seminal vesicles plus coagulating glands with or without fluid, ventral and dorsolateral prostate, LABC , and epididyma l weights were significantly

decreased. There was a higher incidence of increased thyroid follicular cell height and decreased colloid area, and two animals were found with slightly decreased spermatic elements in the epididymis.

At 100 mg/kg/day, serum TSH was significantly increased and T4 was significantly decreased. Liver weights were significantly increased and the adjusted right epididymis weight was significantly decreased.

At 25 mg/kg/day, relative liver weights were significantly increased.

At 400 mg/kg/day, there was evidence of altered thyroid function that warrants consideration during a weight- of -evidence assessment for thyroid perturbation. The decreases in serum testosterone levels and androgen sensitive-organ weights at the high dose, coupled with epididymal histopathology should be considered in the context of the androgen pathway. Previous benfluralin mode-of-action studies have shown hepatic enzyme induction and enhanced thyroxine and testosterone clearance. At 100 mg/kg/day,

benf luralin produced minimal changes in endocrine-sensitive endpoints which were considered to be adaptive or of questionable toxicological significance.

B. AGENCY COMMENTS : All rats survived to scheduled termination. Mean age and weight at completion of PPS, mean adrenal, kidney, and testes weights, and gross and microscopic examinations of the testes, epididymides, and kidneys were unaffected by

treatment.

Systemic finding s were observed at 400 mg/kg/day. D ark yellow/orange urine , a common finding following dinitroaniline herbicide treatment , was observed in all rats. Unadjusted and adjusted final body weights were decreased (NS) by 8%, resulting in a 9% decrease (p<0.05) in overall body weight gains (PND 23 -53). Unadjusted, adjusted, and relative (to body) liver weights were increased (p<0.05) by 40-52%, unadjusted and adjusted pituitary weights were decreased ( p<0.05 ) by 13 -14%, and unadjusted and adjusted thyroid weights were increased ( p<0.05, except unadjusted NS ) by 1 5%. Serum alkaline phosphatase levels

were decreased (p<0.05) by 40%, and s erum total protein

, creatinine , and gamma glutamyl

transferase were increased (p<0.05) by 7%, 50%, and 60%, respectively. Globulin was increased (not statistically analyzed) by 22% . Phosphorus, chloride, and potassium were

decreased (p<0.05) by 9%, 2%, and 6%, respectively.

At 100 mg/kg/day, unadjusted, adjusted and relative (to body) liver weights were increased (p<0.05) by 22 -23%. Serum alkaline phosphatase levels were decreased (p<0.05) by 17%. Phosphorus was decreased (p<0.05) by 9%.

A t 25 mg/kg/day

, relative liver weight was increased ( p<0.05) by 7 %

.

There was a dose -dependent decrease (p<0.05) in serum T 4 

## levels of 12%, 29% and 53% at 25, 100 and 400 mg/kg/day , respectively; and a dose -dependent increase (p<0.05) in serum TSH levels of 53% and 105% at 100 and 400 mg/kg/day , respectiv e ly. S erum testosteroneMale P ubertal Assay ( 2012)/ Page 18 of Error! Bookmark not defined.

BENFLURALIN /084301 O CSPP 89 0. 1 500 / OECD None



## was also decreased (NS) by 52% at 400 mg/kg/day. It was stated that one 400 mg/kg/day rat (#6984) had a serum testosterone level of 8.86 ng/mL, which was considerably higher than the mean value for the group (1.41 ng/mL). When the value for this rat was removed from calculations, the mean testosterone level was 0.91 ng/mL, which is a 69% decrease (p<0.05) compared to controls. At 400 mg/kg/day, decreased (p<0.05) unadjusted and adjusted accessory sex tissue weights were observed as follows: (i) seminal vesicle + coagulating gland with fluid by 32%; (ii) seminal vesicle + coagulating gland without fluid by 25 -26%; (iii) ventral prostate by 26%; (iv) dorsolateral prostate by 21- 22 %; (v) LABC by 20%; and (vi) epididymis by 13-18%. At 100 mg/kg/day, only adjusted right epididymis weight was decreased (p <0.05) by 9% . Compared to controls, a nimals treated with 400 mg/kg/day had an increases in thyroid follicular cell height with a concomitant decrease in colloid area. A slight decrease in spermatic elements in the right epididymis was observed in two animals (#6976 & #6982) at 400 mg/kg/day.

The dose levels used in this study were considered to be adequate b ased on markedly increased liver weights and significant changes in thyroid hormone levels and histopathology, as well as a significant decrease in body weight gain, at 400 mg/kg/day.

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

• Several parameters had slightly elevated or reduced values relative to the performance

criteria: : CV for seminal vesicle weight was 21.7% (maximum 21.06%); CV for body weight at PPS was 10.69% (maximum 7.57%); CV for final body weight was 8.04% (maximum 7.47%); Kidney weight was 1.96 g (minimum 2.242 g).

# DATA EVALUATION RECORD

B ENFLURALIN

Study Type: O CSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614908)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Scott Studenberg, Ph.D., D.A.B.T. Date: 11/13/2011

Secondary Reviewer Signature: Michelle J. Sharpe

- Kass, M.S

. Date: 11/16/2011

Program Manager: Signature: Jack D. Early, M.S. Date: 11/18/2011

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 11/18/2011

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel

.

## 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).

### BENFLURALIN/084301



## Primary Reviewer: Gregory Akerman



## Health Effects Division Steroidogenesis Assay (2011) I 

### Page I of 13

OCSPP 890.1550/ OECD None



## Signature:**-7"-~~~'£;;.g:=:.-**



## Secondary Reviewer: --""""J-"'o=hn=-=L=i-=-cc=i=o=ne=·c...::P'"""'h=.D=------ 

## Health Effects Division Datet:

# =:;~~~~====---

Template version 08/2011



## DAT A EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



## PC CODE: 084301 

## DP BARCODE: D399309



## TXR#: 0054123 

## CAS No.: 1861-40-1



## TEST MATERIAL (PURITY): Benefin (97

. 5% a.i.)



## SYNONYMS: Benfluralin; N-Butyl-N-ethyl-2,6-dinitro-4-(trifluoromethyl)benzenamine



## CITATION: Lebaron

, M.J.; Kan

, H.L.; Perala, A.W. (2011)

. Evaluation ofBenefin in the In

Vitro Steroidogenesis Assay. Toxicology & 

## Environmental Research and Consulting, The Dow Chemical Company, Midland

, Michigan, 48674. Study No.

101183

, October 14

, 2011. 48614908. Unpublished.



## SPONSOR: Dow AgroSciences LLC

, 9330 Zionsville Rd, Indianapolis, IN 46268



## TEST ORDER#: EDSP-084301-15



## EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48614908), H295R cells

cultured 

## in vitro in 24-well plates were incubated with benfluralin (97.5% purity

, Lot#
ACD13683, TSN100037) at logarithmic concentrations from 10- 10 

## to 10- 4 

## Min triplicate for 48

hours. Dimeth y l sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1 %

.

Testosterone and estradiol levels were measured with LC / APPI-MS / MS. Three independent experiments were performed. A Quality Control (QC) plate was run concurrently with each independent run of the test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels ; positive controls included the known inducer (forskolin) and inhibitor (prochloraz) of estradiol and testosterone production.

Benfluralin was tested at concentrations up to 10- 4 

## M with no evidence of precipitation or cytotoxicity. Although the final assay run was conducted with H295R cells from passage 11.5 (one passage higher than the recommended maximum of 10), the guideline requirements for CV

:S3 0% between replicate wells within a plate and bet ween plates were met for all three assay

runs. Estradiol production was statistically increased 1.2-fold in two of three independent assays

at 10- 4 

## M and by 1.3-fold in one assay at 10- 5 

## M, but no dose-response was observed

. Slight

decreases in testosterone and estradiol production in a single run were also not considered of biological relevance as statistically significant differences at concentrations that do not follow a dose-response curve may be due to random effects.Steroidogenesis Assay ( 2011 ) / Page 2 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## Based on the hormone responses in three independent runs, b enfl uralin treatment did not result in statistically significant and reproducible alteration s in testosterone or estradiol production.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Steroidogenesis a ssay (OCSPP 890.1550).

COMPLIANCE : Signed and dated GLP Compliance and Quality Assurance statements were provided.Steroidogenesis Assay ( 2011 ) / Page 3 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Toxicology & Environmental Research and Consulting, The Dow Chemical Company

Location: Midland, MI 48674 Study Director: H.L. Kan

Other Personnel: M.J. Lebaron, Lead Scientist; A.W. Perala, Analytical Chemist Study Period: 2/17/2011

– 10/11/2011



## 2. Test Substance : Benfluralin (Benefin TGAI)

Description: Yellow solid

Lot / Batch #: Lot No. ACD13683, TSN100037 Purity: 97.5 % Solubility (in Solvent): DMSO ≤ 0.1M; Water < 0.001 g/L Stability: 3.5 years shelf life Storage conditions: Ambient

CAS #: 1861

- 40

- 1 Molecular weight: 335.3

Structure:



## 3. Positive Control : Forskolin

Description: White powder

Source: Sigma, St. Louis, MO

Lot / Batch #: Lot No. 097K50653 Purity: 99% Solubility (in Solvent): DMSO ≤ 0.1M Storage conditions: Ambient

CAS #: Molecular weight: 66428

- 89

- 5 (66575

- 29

- 9)

410.5



## 4. Negative Control : Prochloraz

Description: Off

- white powder

Source: Sigma, St. Louis, MO

Lot / Batch #: Lot No. SZE6220X Purity: 99.1% Solubility (in Solvent): DMSO ≤ 0.1M Storage conditions: Ambient

CAS #: Molecular weight: 67747

- 09

- 5

376.67Steroidogenesis Assay ( 2011 ) / Page 4 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## 5. Solvent/Vehicle Control : Dimethyl Sulfoxide (DMSO)

Description: Colorless liquid

Source: Sigma Aldrich, Cat. No. 276855

Lot / Batch #: Lot No. 68996LMV Purity: ≥ 99.9% Storage conditions: Room temperature

CAS #: Molecular weight: 67

- 68

- 5

78.13

Justification for choice of solvent: Not provided

Final concentration: (% volume in assay) 0.1%



## 6. Stock Medium : Dulbecco’s modified Eagle’s medium/Nutrient Mixture F

- 12 Ham (DMEM:F

- 12 Ham)

Description:

Source: Cat. No. D 6434, Sigma, St. Louis, MO

Lot / Batch #: RNBB 2720

Sodium bicarbonate: Component of DMEM:F

- 12 Ham

Nu

- Serum: Lot No. 81515; tested for background hormone concentrations ITS + premix: Lot No. 84337



## 7. Test Cells : H295R human adrenocortical carcinoma cells (ATCC CLR

- 2128; lot number not reported)

. H295R cells were cultured for five passages from the original ATCC source cells. Cells from passage number five were cryogenically stored in liquid nitrogen. Cells

from these frozen batches were cultured for four additional passages prior to initiation of testing. The passage number in Assay No. 3 was 11.5. This was one passage higher than the recommendation listed in the guideline (10 passages); however, the QC criteria for basal hormone production, induction, and inhibition indicated acceptable responsiveness of the assay system in Assay No. 3.

The following performance criteria were met (indicated by an “x”):

x Cell passage identifier. Cell Passage #: 5 10.5

– 11.5

x Cells frozen down at passage 5

x Frozen cells cultured for 4 additional passages Total number of passages does not exceed 10



## B. METHODS

1. Pre -Test Information:

a. Hormone Assay Interference Test : A chemical interference assay was not conducted

because a LC/APPI

- MS/MS analytical method was used to determine hormone concentrations. This method included internal spiked controls to measure extraction and quantitation efficiency, therefore, no interference was expected.

b. Hormone Extraction : A liquid -liquid extraction (LLE) system was used to prepare the culture medium for hormone analysis. Standards and samples were vortexed with 3.5 mL of methylene chloride containing 0.01 ng/mL D 3 

## -testosterone and 13 

## C 3



## - estradiol as internal standards. After vortexing, the organic phases were transferred to clean vials, and the portions (~3 mL) were evaporated to dryness. Residues in each vial was derivitized by the addition of 100 μL of sodium bicarbonate buffer (100 mM, pH 10.5) and 100 μL of dansyl

chlo ride solution (2.0 mg/mL in acetone, freshly prepared). The vials containing theSteroidogenesis Assay ( 2011 ) / Page 5 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## derivitization mixture were heated in a water bath at 60°C for 15 min, cooled to room temperature, and transferred to vials for LC/APPI- MS/MS analysis. Because of the selected method of analysis with internal spiked controls, tritiated -hormone recovery for the determination of extraction efficiency was not considered necessary.

c. Laboratory Proficiency Test : A validated hormone measurement system (LC

- APPI

MS/MS) was used f or the analysis of testosterone and estradiol produced by H295R cells. Validation was reported to have been determined by conformance to method detection limits

of ≥25 pg/mL and ≥ 10 pg/mL for testosterone and estradiol, respectively, and spiked sample re covery with deviation from nominal concentrations of ≤ 30% for testosterone (500 and 2500 pg/mL) and estradiol (50 and 250 pg/mL). No data and study date/reference information were included in MRID 48614908 to support the statement of laboratory proficienc y. EC 50 

## data were not provided for comparison with the acceptable ranges for testosterone and estradiol (μM). The investigators stated that the large dynamic range for linearity of hormone detection and quantitation of 0-2500 pg/mL observed with the LC/MS method ensured the validity of hormone quantitation results.

2. Test Solutions : A 100 mM stock solution was prepared by dissolving an appropriate amount of the test compound in 100 μL DMSO. Six serial 1:10 dilutions were additionally prepared to yield final stock solutions ranging from 100 nM to 100 mM. Forskolin and prochloraz solutions are assumed to have been prepared according to the guideline to yield three (1 -100 mM) and four (0.1-100 mM) serial dilutions, respectively. No issues regarding solubility were reported.

3. Cell Plating and Preincubation : H295R cells were grown for five passages, and subsequently frozen in liquid nitrogen. Cells from the frozen stock were thawed, combined with the H295R growth medium (DMEM:F-12 Ham), and cultured for a minimum of four additional passages prior to testing. The cells were seeded in 24- well plates with H295R cells at a concentration of 200,000 – 300,000 cells/mL, yielding approximately 50- 60% confluency at 24 hours. The seeded plates were incubated for 24 hours at 37°C in a 5% CO 2 

## atmosphere. Although the number of 24-well plates and the volume of media were not reported specifically, it was stated that the plating methodology followed the OPPTS 890.1550 guidelines.

4. Exposure : The cells were checked microscopically for good attachment and proper morphology, and the medium was removed and replaced. The cells were then exposed to each serial dilution of the test compound or 0.1% DMSO in triplicate according to the schematic presented in Table 1.

TABLE 1

. Dosing Schematic for the Exposure of H295R Cells to Benfluralin (Final Concentrations in μM). a

1 2 3 4 5 6

A DMSO 0.1% DMSO O.1% DMSO O.1% 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 17 of the study report. Dosing was calculated based on a total volume of 1 mL per well.



## A concurrent quality control (QC) plate was included with each of the three independent runs of the test chemical plates to demonstrat e the assay’s response to forskolin (an inducerSteroidogenesis Assay ( 2011 ) / Page 6 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## of testosterone and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol production). The QC plate was prepared and dosed in the same manner with either forskolin or prochloraz a ccording to the schematic presented in Table 2.

TABLE 2

. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM). a

1 2 3 4 5 6

A Blank b Blank Blank Blank + MeOH c Blank + MeOH Blank + MeOH

B DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO + MeOH DMSO + MeOH DMSO + MeOH

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM

a Data were obtained from page 16 of the study report. Dosing was calculated based on a total volume of 1 mL per well.

b Blank wells received medium only.

c MeOH = methanol was added to these wells for 30 minutes at room temperature following medium removal

.



## Following dosing, the plates were incubated for 48 hours under the conditions previously described. The medium from each well was removed, split into two 0.45 mL aliquots, and

frozen at −80°C until hormone measurements were conducted.

5. Cell Viability/Cytotoxicity Assay : Cell viability was determined with the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, Wisconsin, Catalog No. G3580) immediately after removal of the culture medium. This cell prolif eration assay kit is a colorimetric modified MTT cell viability assay. The Aqueous One Solution contains a tetrazolium compound [3 -(4,5- dimethylthiazol -2- yl)

- 5(3

- carboxymethoxyphenyl)-2- (4 -sulfophenyl)-2H-tetrazolium, inner salt] and an electron coupling reagent (phenazine ethosulfate). The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of viable cells in culture. The plates were read at 490 nm with a 96- well plate reader (Molecular Devices VMax Microplate Reader). The minimum cell viability required per well was ≥ 80%; wells with lower viability were excluded in the final data analysis. Hormone concentration data were not normalized for variability in cell viability.

6. Hormone Measur ement System : Test samples were extracted and derivatized as described above in Section 1.b. Derivatized samples were analyzed and quantified by

reverse

- phase LC/APPI -MS/MS with an Applied Biosystems Sciex API 4000 tandem mass spectrometer (Concord, Onta rio, Canada) equipped with an Agilent HP 1100 HPLC system. The mass spectrometer was equipped with a TurboIonspray interface. The system used an Agilent SB -C18 (4.6 mm x 50 mm, 1.8 μ M) analytical column. The HPLC eluent consisted of Milli Q water + 0.1% acetic acid and acetonitrile + 0.1% acetic acid with a flow rate of 0.45 mL/min, and the method had a sample run time of 20 min. As described previously, this method included internal spiked controls to monitor extraction and quantitation efficiency. A tr itiated

- hormone recovery test for the determination of extraction efficiency was not considered necessary. Hormone cross- reactivity was not examined because an antibody-based hormone measurement system was not used. The determination of the positions of the solvent control (DMSO) basal hormone concentrations on the hormone concentration standard curves was not reported.

The following performance criteria were met (indicated by an “x”):

x Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

x Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount from triplicate samples ≤30% of nominal concentration)

NA Hormone cross

- reactivity (antibody

- based assays only; ≤30% of basal produc tion of the respective hormone)Steroidogenesis Assay ( 2011 ) / Page 7 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None

NR Solvent control within 75% range below maximum response on standard curve Test compound tested for interference with measurement system



## C. DATA ANALYSIS : Hormone concentrations after test chemical exposure were normalized to the mean solvent control (SC) value for each assay to evaluate the relative increase or decrease in concentration. Results were expressed as the fold

- change relative to the SC in each exposure plate. All data were expressed as the mean ± standard deviation (SD), and doses that exhibited cytotoxicity greater than 20% were omitted from further evaluation per guidelines. Relative changes in hormone concentration were calculated as

follo ws:

Relative Fold

- Change = (Hormone concentration in each well) ÷ (Mean solvent hormone concentration)

Prior to conducting statistical analyses, the assumptions of normality and variance homogeneity were evaluated. Homogeneity of variance was evaluated by Bartlett’s test and normality by Shapiro-Wilk’s test at alpha = 0.01. If the data were not homogeneous or normally distributed, the data were transformed to approximate these conditions. If the data were homogeneous and approximately normally distrib uted, differences between chemical treatments and SC were analyzed by using a parametric analysis of variance followed by Dunnett’s test, if significant. If the data were not homogeneous or normally distributed, a non- parametric test was used (Kruskal Wal lis). This analysis, if significant,

was followed by the Wilcoxon rank sum test with a Bonferroni-Holm correction. Differences were considered significant at p ≤ 0.05. The statistical methods are considered appropriate.

II. RESULTS

A. TEST COMPOUND : No reproducible alterations in testosterone or estradiol production were demonstrated by the results of the three independent runs with benfluralin in the steroidogenesis assay (Table 3 and Figures 1 – 6) . Slight, but statistically significant,

decreases in testosterone (0.001 μM) and estradiol (0.0001

- 0.1 μM) production were observed in a single assay run, and were not considered biologically relevant. Although estradiol production was statistically increased 1.2-fold in two of three independent runs at 10 μM and 1.3

- fold in one run at 100 μM, no dose -response was observed. The slight 1.2- to 1.3-fold increase in estradiol production (with 10 and 100 μM benfluralin) did not meet a 1.5-fold cut-off criterion established in the validation program for th is assay

.

Between plate SC CV values were within the guideline recommended level of ≤30% based on absolute concentration; the value for testosterone assay plates was 5% and the value for estradiol assay plates was 9%.Steroidogenesis Assay ( 2011 ) / Page 8 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



### TABLE 3

. Mean (±SD) Hormone Concen trations Following Treatment with Benfluralin for 48 Hours. a

Nominal

Concentration (μM) Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Mean ± SD Statistical Significance b

Testosterone (pg/mL) Fold Difference

DMSO 556.7 586.7 528.3 1.0 1.0 1.0 1.0 0.0

--- 0.0001 520.7 536.3 472.0 0.9 0.9 0.9 0.9 0.0 None

0.001 433.3 535.7 451.0 0.8 0.9 0.9 0.8 0.1 Trial #1

0.01 537.3 579.7 482.3 1.0 1.0 0.9 1.0 0.0 None

0.1 525.7 595.3 472.3 0.9 1.0 0.9 0.9 0.1 None

1 519.3 587.7 490.0 0.9 1.0 0.9 1.0 0.0 None

10 548.0 599.3 475.7 1.0 1.0 0.9 1.0 0.1 None

100 542.3 605.7 485.7 1.0 1.0 0.9 1.0 0.1 None
Estradiol (pg/mL) Fold Difference

DMSO 56.3 58.7 67.0 1.0 1.0 1.0 1.0 0.0

--- 0.0001 48.5 51.2 61.5 0.9 0.9 0.9 0.9 0.0 Trial #1

0.001 47.4 52.5 63.6 0.8 0.9 0.9 0.9 0.0 Trial #1

0.01 47.0 53.1 58.7 0.8 0.9 0.9 0.9 0.0 Trial #1

0.1 47.5 56.5 59.7 0.8 1.0 0.9 0.9 0.1 Trial #1

1 49.4 56.5 61.6 0.9 1.0 0.9 0.9 0.0 None

10 69.7 67.3 78.6 1.2 1.1 1.2 1.2 0.1 Trial #1 & #2

100 71.2 68.4 75.1 1.3 1.2 1.1 1.2 0.1 Trial #1

a Data were obtained from page 29 of the study report.

b p ≤ 0.05



## F IGURE 1. Changes in Testosterone Production Relative to Benfluralin Concentration

, Assay 1.

* Significantly different from the solvent control at p ≤ 0.05.Steroidogenesis Assay ( 2011 ) / Page 9 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## F IGURE 2. Changes in Testosterone Production Relative to Benfluralin Concentration, Assay 2.

* Significantly d ifferent from the solvent control at p ≤ 0.05.



## F IGURE 3. Changes in Testosterone Production Relative to Benfluralin Concentration, Assay 3.

* Significantly different from the solvent control at p ≤ 0.05.Steroidogenesis Assay ( 2011 ) / Page 10 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## F IGURE 4. Change in Estradiol Production Relative to Benfluralin Concentration, Assay 1.

* Significantly different from the solvent control at p ≤ 0.05.



## F IGURE 5. Change in Estradiol Production Relative to Benfluralin Concentration, Assay 2.

* Significantly different from the solvent control at p ≤ 0.05.Steroidogenesis Assay ( 2011 ) / Page 11 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## F IGURE 6. Change in Estradiol Production Relative to Benfluralin Concentration, Assay 3.

* Significantly different from the solvent control at p ≤ 0.05.



## B. CYTOTOXICITY : It was reported in MRID 48614908 that treatment with benfluralin did not induce cytotoxicity of greater than 20% (the maximum permitted by the guideline) during any of the three steroidogenesis assays. Cell viability on the test and QC plates (relative to control) ranged from 107.7 – 119.7% and 96.0 – 123.7%, respectively. The inclusion of methanol with the cells in media only or DMSO only yielded cell viability

measurements of 29.9 – 35.9%.

C. QC PLATE : The hormone concentrations after exposure 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 presented in Table 4. The QC criteria for basal hormone production, induction, and inhibition were deemed by the study authors to indicate acceptable responsiveness of the assay system for all three steroidogenesis assays.

Basal hormone production was 491 pg/mL for testosterone and 51.7 pg/mL for estradiol in Assay No. 1. The concentration of testosterone was lower than the guideline criterion of 500 pg/mL, however, the minimum basal production limits are criterion based on the method detection limits for each hormone as described in the guideline. Because this study used LC/MS detection with a testosterone method detection limit (lower limit of quantitation) of 25 pg/mL, a minimal basal production of >125 pg/mL was deemed

a cceptable. The Reviewer agrees that this decrease in the acceptable basal hormone production level is acceptable based on the guideline recommendations (OPPTS 890.1550). In Assay No. 2, basal hormone production was 503 pg/mL for testosterone and 55.6 pg/ mL for estradiol. Basal hormone production was 454 pg/mL for testosterone and 58.2 pg/mL for estradiol in Assay No. 3. The passage number in Assay No. 3 was higher than theSteroidogenesis Assay ( 2011 ) / Page 12 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## recommended 10 passages described in the guideline; however in Assay No. 3, the Q C criteria for basal hormone production, induction, and inhibition indicated acceptable responsiveness of the assay system.

A comparison of the QC data between the three assay runs indicates that the data were generally similar for each cell treatment, with no apparent difference in hormone production. The variability (%CV) between the runs (calculated by the reviewer) based o n the absolute hormone concentrations in the SC were 5% for testosterone and 6% for estradiol, within the guideline criteria of ≤30% for the assay. The %CVs within each run for the QC plates were 6.0 to 8.3% for testosterone and 5.7 to 8.3% for estradiol..

Cells incubated with 10 μ M forskolin exhibited a mean fold induction in testosterone production of 2.5 ± 0.4 (range: 2.1- to 2.8-fold) and a mean fold induction in estradiol production of 13.3 ± 1.4 (range: 11.8- to 14.5- fold). Cells incubated with 1 μM prochloraz exhibited a mean fold inhibition in testosterone production of 0.2 ± 0.1 (range: 0.2- to 0.3- fold) and a mean fold inhibition in estradiol production of 0.4 ± 0.0 (range: 0.4- to 0.5-fold). These induction and inhibition results met the gu ideline requirements.

TABLE 4

. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for

48 Hours. a

Concentration (μM) Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Mean ± SD

Testosterone (pg/mL) Fold Difference

Blank 589 667 554

--- --- --- --- --- DMSO 491 503 454 1.0 1.0 1.0 1.0 0.0
1 μM Forskolin 1009 1008 866 2.1 2.0 1.9 2.0 0.1
10 μM Forskolin 1243 1433 934 2.5 2.8 2.1 2.5 0.4
0.1 μM Prochloraz 211 251 258 0.4 0.5 0.6 0.5 0.1
1 μM Prochloraz 91.7 123 133 0.2 0.2 0.3 0.2 0.1

Estradiol (pg/mL) Fold Difference

Blank 55.2 56.0 67.3

--- --- --- --- --- DMSO 51.7 55.6 58.2 1.0 1.0 1.0 1.0 0.0
1 μM Forskolin 442 454 416 8.5 8.2 7.1 7.9 0.7
10 μM Forskolin 700 809 686 13.5 14.5 11.8 13.3 1.4
0.1 μM Prochloraz 32.9 30.7 38.7 0.6 0.6 0.7 0.6 0.1
1 μM Prochloraz 22.2 23.1 26.2 0.4 0.4 0.5 0.4 0.0

a Data were obtained from page 30 of the study report.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS : The collective responses of three independent assays with benfluralin in the steroidogenesis assay demonstrated no reproducible alterations in testosterone or estradiol production. Although estradiol production was statistically increased 1.2

- fold in two of three independent assays at 10 μM and 1.3 -fold in one assay at 100 μM, there was no demon strated dose -response. Therefore, these data do not support a positive response. Based on the combined hormone responses to benfluralin in each of three independent H295R steroidogenesis assay runs, it was determined that benfluralin administration did not alter testosterone or estradiol production. The slight 1.2-

to 1.3- fold increase in estradiol production (with 10 to 100 μM benfluralin) did not meet aSteroidogenesis Assay ( 2011 ) / Page 13 of 13

BENFLURALIN / 084301 O CSPP 89 0. 1550/ OECD None



## 1.5-fold cut-off criterion established in the validation program for this assay, and was not interp reted to be biologically relevant. Therefore, under the conditions of this study, benfluralin was not considered to be positive in the steroidogenesis assay.

B. AGENCY COMMENTS : Benfluralin was tested at concentrations up to 100 μ M with no evidence of pr ecipitation or cytotoxicity in the in vitro steroidogenesis assay. Three individual assay runs were completed, and testosterone and estradiol concentrations were determined after H295R cells were incubated with benfluralin (test compound), forskolin (inducer), and prochloraz (inhibitor) for 48 hours. Although the final assay run was conducted with H295R cells from passage 11.5 (one passage higher than the recommended maximum of 10), the guideline requirements for CV ≤30% between replicate wells within a plate and between plates were met for all three assay runs. Basal hormone production was acceptable based on the method detection limits determined with the LC/APPI

- MS/MS method used for this study. Estradiol production was statistically increased 1.2- fol d in two of three independent assays at 10 μM and 1.3

- fold in one assay at 100 μM, but no dose

response was observed. Slight decreases in testosterone and estradiol production in a single run are also not considered of biological relevance as statistically significant differences at concentrations that do not follow a dose-response curve may be due to random effects.

Under the conditions of this study, benfluralin did not alter testosterone or estradiol production and, therefore, gave a negative response in the steroidogenesis assay.

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

• Expiration dates for benfluralin, forskolin, and prochloraz were not included.

• Storage conditions, method and frequency of preparations for test substances, reagents,

and controls were not included.

• Lot number of the H295R cells used in the assays was not included.

• Standard and calibration curve data for all analytical assays conducted were not reported.

• Details of test (incubation) parameters such as volumes for media, test article,

inducer/inhibitor were not included.

• Documentation/details supporting proficiency testing/training for perti nent staff involved in the study were not reported.

• EC 50 

## ranges for forskolin and prochloraz were not reported.

• Validity of the solvent control in terms of its position within the hormone standard curve ranges was not reported.

• Three out of eight standards and two out of four spiked controls for benfluralin in DMSO concentration assay were excluded due to matrix effect interferences.

• The passage number for assay #3 was 11.5, one passage higher than the required number of passages. However, the responsiveness of the cells to positive and negative controls was adequate.

• The determination of the positions of the solvent control (DMSO) basal hormone concentrations on the hormone concentration standard curves was not reported.

# DATA EVALUATION RECORD

BENFLURALIN (BENEFIN)

Study Type: O CSPP 890.1600, Uterotrophic Assay in Rats

EPA Contract No. EP10H001452 Task Assignment No. 2-1-2012 (MRID 48614909)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Rebecca Bryan, B.S

. Date: 11/09/2011

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 11/13/2011

Program Manager: Signature: Jack D. Early, M.S. Date: 11/15/2011

Quality Assurance: Signature: Jack D. Early, M.S. Date: 1 2 /1 9 /2011

Disclaimer

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel.

## 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 rob ustness 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

indiv idual 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).

### BENFLURALIN/084301



## Primary Reviewer: Sheila Healy, Ph

. D.

Health Effects Division Secondary Reviewer: Jess Rowland, M.S

Health Effects Division In 

### vivo Uterotrophic Assay (2010/ Page 1 of 7

OCSPP 890.1600/ OECD 440



## Sig

Template version 09/2011



## DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat) ; OCSPP 890.1600 ; OECD 440

PC CODE: 084301 DP BARCODE: D399309

TXR#: 0054123 CAS#: 1861-40-1

TEST MATERIAL (PURITY): Benefin (97

. 5% a.i.)

SYNONYMS: Benfluralin ; N-butyl-N-ethyl-2

, 6-dinitro-4-(trifluoromethyl)benzenamine

CITATION: Marty

, M

. S

., Brooks, K.J., and Jeong

, Y.C. (2011). Benefin: uterotrophic assay
in the immature female Crl : CD(SD) rat. Toxicology & 

## Environmental Research

and Consulting

, The Dow Chemical Company

, Midland, MI. Laboratory Project Study ID: 101116

, June 23, 2011. MRID 48614909. Unpublished.

SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN

TEST ORDER#: EDSP-084301-15

EXECUTIVE SUMMARY: In an uterotrophic assay (MRID 48614909) conducted to screen for potential estrogenic activity, Benefin (97.5% a.i.

, lot# ACD13683) in com oil was administered daily via oral gavage to groups of six immature female Sprague-Dawley rats at

dose levels ofO (vehicle), 0 (untreated)

, 25

, 75

, or 200 mg / kg / day on post-natal day (PND) 19-

21. A positive control group was treated with 17a-ethynyl estradiol (EE) by daily oral gavage at

a dose level of 10 μg / kg / day. All animals were terminated and necropsied approximately 24

hours after the final dose on PND 22 to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in

animals for any dose groups. No precocious vaginal opening was observed in the test animals. Body weights in the dose groups were comparable to the controls throughout the duration of the

assay

. At a dose of 200 mg / kg / day, body weight gains were decreased by 30% compared to the

controls

. Uterine weights in the dose groups were comparable to the controls. Absolute wet and

blotted uterus weights for the 10 μg EE group were increased (p < 0

. 05) by 811 % and 466%, respectively.

No statistically significant changes were seen in uterine weight in this study. Benfluralin was negati v e in the uterotrophic assay.

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 890.1600).In vivo Uterotrophic Assay (2010/ Page 2 of 7

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440



## COMPLIANCE : Signed and dated Data Co n fidentiality, GLP Compliance , Flagging, and Quality Assurance statements were provided.In vivo Uterotrophic Assay (2010/ Page 3 of 7

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : Benfluralin (Benefin)

Description: Not provided

Source: Dow AgroSciences LLC, Indianapolis, IN

Lot/Batch #: ACD13683 Purity: 97.5% a.i. Stability: Stable in the vehicle for up to 23 days at room temperature (See MRID 48614907)

CAS #: 1861

- 40

- 1

Structure:



## 2. Reference E strogen : 17α

- ethynyl estradiol (EE)

Supplier : Sigma

- Aldrich (St. Louis, Missouri)

Lot/ Batch #: 028K1411 Purity: Not provided

CAS # : 57

- 63

- 6



## 3

. Solvent/ V ehicle C ontrol : Corn Oil

Supplier : Sigma

- Aldrich ( St. Louis, Missouri )

Lot/Batch #: Not provided Rationale (if other than water) : Solubility properties, and does not exert any estrogenicity or toxicity in i mmature
female rats.

Final concentration : Not a pplicable



## 4

. Test A nimals : Species : Rat (females only)

Strain: Sprague

- Da w ley (Crl:CD[SD] ) Age/weight at dose initiation : PND 19 ; 39.1

- 4 9.1 g Source: Charles River Laboratories, Inc.

, Portage, Michigan

Housin g: 6/cage in solid bottom cages with paper pulp bedding (7089 Tekland Diamond Soft)

Diet : Tekland Diet #2016 (Harland Laboratories, Inc., Indianapolis, Indiana ) in meal

form

, a d li bitum ; low phytoestrogen with total gen i stein equivalents <325 μg/g

diet

. Water: Tap water

, ad libitum

Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22 ± 3 oC

40

- 70 %

12

- 15 /hr (average)

12 hrs light/ 12 hrs dark Acclimation period: 7 days, immature females housed with their dam.In vivo Uterotrophic Assay (2010/ Page 4 of 7

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440



## B. METHODS

1. In L ife D ates : Start: 9/17/10 End: 9 /2 0/10

2. Study Design : Following an initial 7- day acclimation period, immature, intact female rats

were administered the test substance from PND 19-21 . Rats were euthanized approximately

24 hours after the last dose and necropsied for uterine weight measurements. The pups were housed with their dam prior to weaning.

3. Animal A ssignment : Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1 using a computer program designed to increase the probability of uniform group mean weights and standard deviations on Day 1. Statistical analysis indicated that there were no significant differences in group means at study initiation. It was not stated if the body weight of each animal was within 20% of the overall mean.

TABLE 1. Study Design a



### Test G roup Dose (mg/kg/day) # of Females
Estrogen Agonist Assay

Untreated control 0 6

Vehicle control 0 6

Low 25 6

Mid 75 6
High 200 6

17 α

- ethynyl estradiol (EE), Reference Estrogen 10 μ g/kg/day 6

a Data were obtained from Text Table 1 on page 14 of the study report.



## 4. Dose S election Rationale : The dose levels in the present study were selected based on the

results from a probe study 1 

## conducted by the Performing Laboratory in which four fe male Crl:CD(SD) rats/dose group received the test substance in corn oil via gavage at doses of 0, 250, 500, 750, or 1000 mg/kg/day for 15 consecutive days (PND 22 -36). F emale ra ts given 250 mg/kg/day had 42% lower body weight gains during days 1 through 3; body weights

were more affecte d at higher dose levels, reaching a 20% decrease in the 1000 mg/kg/day dose group. By day 15 (PND 36), females weighed 12.2%, 9.0%, 13.5% and 12.9% less than controls in the 250, 500, 750 and 1000 mg/kg/day dose groups, respectively. For the males, body weights were decreased in a dose

- dependent manner at day 9 (decreases were 7.2%, 11.5%, 12.3%, and 13.5% at 250, 500, 750 and 1000 mg/kg/day, respectively) and these decreases in body weight were sustained to day 15 (PND 44) (i.e., 5.8%, 11.1%, 11.6% and 16.1% at 250, 500, 750 and 1000 mg/kg/day, respectively). Based on these findings, dose levels of 0, 25, 75 or 200 mg/kg/day were selected for the main study.

5. Dose P reparation and Analysis : Dose formulations were prepared once prior to initiation

of t reatment by initially dissolving the a ppropriate amounts of test substance in corn oil

. Homogeneity and concentration analyses were performed prior to administration on all dose

levels. For the Hershberger study (MRID 48614907), stability analyses were performed on

1 

### Marty, M.S., and Marshall, V.A. (in progress) Benefin: Hershberger, Uterotrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. No MRID found or notedIn vivo Uterotrophic Assay (2010/ Page 5 of 7

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440



## 0.25, 2.5, and 250 mg/kg/day formulations prior to administration following storage at room temperature for up to 23 days.

Results of D ose Analysis

Homogeneity (%RSD) : 2.3-13.4% ; the 50 mg/mL sample was reanalyzed and found to be

3.1%

Stability (% of Day 0): 94.9-114.3% following storage at room temperature for up to 23 days (MRID 48614907).

Concentration (% of nominal): 93.5-100.0%

The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage A dministration : Animals were administered the test formulations

, EE

, or vehicle daily via gavage for three consecutive days in a dose volume of 4 mL/kg body weight . Dose volumes were adjusted daily based on the concurrent body weight measurement.

7. S tatistics : Body weights and body weight gains were analyzed by a forced parametric test. The blotted and wet uterine weights were first analyzed by Bartlett’s test for homogeneity of

variance . Analysis of covariance (AN C OVA) was performed with terminal body weight as

the covariate . If the ANC OVA was significant (p ≤ 0.05), either the least square means with

Dunnett’s correction or the Wilcoxon Rank-Sum test was performed, as appropriate. Significance was denoted at p ≤ 0.05. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations : Cage -side checks for mortality, moribundity and clinical signs of toxicity were conducted at least twice daily.

2. Body W eight : Animals were weighed at randomization, daily throughout the dosing period

and at termination.

3. Food Consumption (Optional) : Food consumption was not measured

.

4. Necropsy and Measurement of Uterine Weight : On PND 22 (approximately 24 hours after final administration of the test substance), all surviving animals were anesthetized by

CO 2 

## inhalation, euthanized by cervical dislocation , and subjected to a gross necropsy.

Dissecti on 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 “wet” uterus (i.e., containing the luminal fluid) was weighed. Subsequently, the uterine horns were cut longitudinally and gently blotted with moist filter paper to remove the luminal fluid while preventing desiccation and the blotted uterus was weighed. After weighing, the uteri were fixed in 10% neutral phosphate- buffered form alin for potential

future examination.In vivo Uterotrophic Assay (2010/ Page 6 of 7

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440



## 5. Microscopic Examination (Optional) : Microscopic examinations were n ot conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality : All animals survived until scheduled termination.

2. Clinical S igns of Toxicity : No clinical signs of toxicity were observed in animals for any dose groups.

No precocious vaginal opening was observed in test animals.

B. BODY WEIGHT AND WEIGHT GAIN : Selected body weight and body weight gain data are presented in Table 2. Body weights in the dose groups were comparable to the controls throughout the duration of the assay. In the 200 mg/kg/day dose group, b ody weight gains ( TD 1 -4) were decreased by 30 % compared to the controls.

Table 2. Selected Group Body Weights an d Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day

# Dose (mg/kg/day)

Untreated

Control Vehicle

Control Low (25) Mid (75) High (200) Reference Estrogen

EE ( 10 μ g/kg/day )

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 6 45.0 2.5 6 44.5 2.6 6 43.8 1.8 6 44.1 2.7 6 43.7 1.8 6 43.6 2.4

2 6 48.8 3.2 6 47.3 3.5 6 47.3 1.9 6 45.4 2.6 6 43.3 2.5 6 46.4 2.0

3 6 52.9 3.3 6 52.1 3.3 6 51.5 2.4 6 49.5 3.3 6 47.5 3.8 6 50.5 2.6

4 6 57.0 3.4 6 55.9 3.5 6 55.6 2.3 6 53.4 3.4 6 51.7 3.2 6 55.1 2.8

BWG Days

1

- 4 6 12.0 1.1 6 11.4 1.3 6 11.8 1.3 6 9.3 1.1 6 8.0 * (↓30) 2.1 6 11.5 1.6

a Data were obtained from Table 3 on page s 28

- 29 of the study report.

N No. of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05.



## C. FOOD CONSUMPTION (Optional) : Food consumption was not measured.

D. PATHOLOGY

1. Uterine Weights : U terine weight data are presented in Table 3. Uterine weights in the dose groups were comparable to the controls. Additionally, it was stated that the mean blotted vehicle control uterine weight was 0.04% of the mean vehicle control terminal body weight, which also meets the guideline requirement (<0.09%).

Absolute wet and blotted uterus weights for the 10 μg EE group were increased (p<0.05) by 811% and 466%, respectively. The positive control elicited the expected response.In vivo Uterotrophic Assay (2010/ Page 7 of 7

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440



### Table 3. Uterine Weights from Estrogen Agonist Assay in SD Rats a

Parameter Dose (mg/kg/day)

Untreated

Control Vehicle

Control Low (25) Mid (75) High (200) Reference Estrogen

EE ( 10 μ g/kg/day )

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

Terminal BW (g) 6 57.0 3.4 6 55.9 3.5 6 55.6 2.3 6 53.4 3.4 6 51.7 3.2 6 55.1 2.8

Wet, absolute (mg) 6 25.3 3.6 6 24.1 2.3 6 26.5 1.9 6 23.7 2.5 6 20.9 1.3 6 219.6 * (↑811) 21.1

Wet, relative (%) b 6 0.044 0.007 6 0.043 0.006 6 0.048 0.004 6 0.044 0.004 6 0.040 0.003 6 0.398 0.023

Blotted, absolute (mg) 6 24.1 3.5 6 22.7 2.5 6 25.3 1.5 6 22.9 2.6 6 19.9 1.4 6 128.5* (↑466) 8.3

Blotted, relative (%) b 6 0.042 0.006 6 0.041 0.006 6 0.046 0.003 6 0.043 0.004 6 0.039 0.004 6 0.233 0.015

a Data were obtained from Appendix Table 2 on pages 38

- 40 of the study report.

b Relative wet and blotted uterine weights were calculated by the reviewers from the individual data

. BW B ody weight

N No. of animals in the group ; SD Standard Deviation

* Significantly different from controls at p<0.05.



## 2. Microscopic Examination (Optional) : M icroscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS : There were no treatment

- related effects on uterine weights in any benefin treatment group. In the positive control group, a significant increase in uterine weight was observed without any significant change in body weights or body weight gains. There were no significant differences in body weights, body weight gains or uterine weights between the vehicle control group and the untreated control group. None of the animals in this study exhibited precocious vaginal opening. Vehicle- treated

control uterine weights met the performance criteria outlined in the applicable test guidelines, indicating acceptable assay sensitivity. Under the conditions of this study, there was no indication of estrogenicity from benefin at doses <200 mg/kg bw/day, the highest

dose level tested in female immature rats.

B. AGENCY COMMENTS : All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any dose groups. No precocious vaginal opening was observed in test animals. Body weights in the dose groups were comparable to

the controls throughout the duration of the assay. In the 200 mg/kg/day dose group, body weight gains (TD 1- 4) were decreased by 30% compared to the controls indicating that the dose levels tested were adequate . Uterine weights in the dose groups were comparable to

the controls. It was stated that all of the blotted uterine weights were <40 mg and the mean blotted vehicle control uterine weight was 0.04% of the mean vehicle control terminal body weight . Absolute wet and blotted uterus weights for the 10 μg EE group were increased (p<0.05) by 811% and 466%, respectively. These increased uterine weights were in the expected range. No statistically significant changes were seen in uterine weight in this stud y. Benfluralin was negative in the uterotrophic assay.

C. STUDY DEFICIENCIES : None