Page 1 of 300
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 

Page 2 of 300
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'

Page 3 of 300
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 Unpublished study 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.

Page 4 of 300
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 

Page 5 of 300
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 Effects1,2 in the Amphibian 

Metamorphosis Assay (AMA) with Benfluralin. 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental 

Stage 

Hind Limb 

Length3

Asynchronous 

Development 

Thyroid Gross 

and 

Histopathology 

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21 

0.0027 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). 

Page 6 of 300
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, 

Page 7 of 300
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 conditions 

Page 8 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 7 of 67

DER Template Version: 22 September 2011 

B. Test Organism

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

(Xenopus laevis). Western [Africa] clawed 

frog Silurana (Xenopus) tropicalis may be 

used as an alternate species1; however, a 

list of all of the necessary protocol 

deviations to accommodate this species is 

recommended for inclusion in the study 

report. The guideline recommends that the 

performance criteria used to support the 

reliability of the test be identified.

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

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

Disruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention 

(OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 

Page 9 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 

8 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as inhouse stock? Yes Breeding pairs were ordered through Xenopus Express (Brooksville, Florida). EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimation conditions same as definitive test?

Acclimated under flowthrough conditions with comparable temperature. No further details provided.

Acclimation period for parental frogs (if applicable): 

Not specified

Details on parental feeding: Not specified Details on parental health: Not specified 

Page 10 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 

9 of 67

DER Template Version: 22 September 2011 

Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? Not specified Multiple spawns were at times available 

for use in the study. The selection of the spawning group used was dependent upon there being a sufficient 

amount of stage 51 tadpoles available from a single spawn to initiate the exposure. 

EPA and OECD recommend that the best 2 

– 3 individual spawns, with a minimum of 

1500 larvae/spawn, be evaluated to identify 

the best single spawn, and that the larvae 

selected for testing originate from the best 

single spawn (i.e., the spawns are not comixed)

Number of spawns evaluated (if applicable): 

Not specified 

Number of eggs sampled per spawn: 

Not specified 

NF stage at test initiation 51 EPA recommends that the definitive study 

be initiated with larvae at Nieuwkoop – 

Faber (NF) developmental stage 51 (17 

days post-fertilization).

Age at test initiation: 15 days postfertilization (dpf)

Page 11 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 10 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Mean total length at test initiation (if reported): 

Not reported 

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

Not reported 

Was the optional size selection method used? 

No 

Details on larval selection: Tadpoles were 

individually staged without anesthesia. Stage 51 tadpoles were externally characterized by a conical-shaped hind limb. 

Page 12 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 11 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Loading rate (rearing density): ~4 larvae/L Holding capacity of 5.2 L in test 

vessels, 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 (if known):

40 mg/kg Analyzed at A & L Great Lakes 

Laboratories, Inc.

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

least twice per day.

Page 13 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 12 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: Same as guideline 

recommendations. 

It is recommended that food rations during 

the pre-exposure period be increased along 

with larval growth to approximately 30 

mg/larva/day by test initiation. EPA and 

OECD recommend that food rations 

increase from 30 mg/larva/day at test 

initiation (Study Day 0-4) to 80 

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

(Study Day 15-21).

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Page 14 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 13 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Continuous flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

50 mL/min on Days 0-14 and 60 mL/min on Days 15-21. 

Volume turnover was approximately every 1.7 hrs on Days 0-14 and approximately 

every 1.4 hrs on Days 15-21. The higher 

flow rates were an attempt to maximize measured concentrations of benfluralin in 

the water column and reach concentrations that were closer to the nominal target doses. Study authors reported that 

increased flow rates have been demonstrated to have no deleterious effects on the rate of X. laevis tadpole development. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement 

ca. every 2.7 hrs).

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

Page 15 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 14 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Syringe pumps with gastight syringes continuously delivered a concentrated stock solution at a designated rate to a stainless steel mixing chamber where it was mixed with dilution water, then gravity fed (through splitters) to replicate test vessels. 

The diluter was calibrated prior to test initiation and was monitored at least twice daily 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 static renewal (if applicable): 

NA If static renewal is used, EPA 

recommends 24-hr renewal; renewal 

period is recommended not to exceed 72 

hours.

Page 16 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 15 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? Not reported DO levels remained above 40% saturation 

throughout 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 Lake 

Huron supplied by the City of Midland Water Treatment Plant. Water was obtained from the upper Saginaw Bay and was limed and flocculated with ferric chloride. Water was pumped to the laboratory prior to municipal treatment. 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.

Page 17 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 16 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

the test, water was sand-filtered, pHadjusted with CO2, carbon-filtered, and UVirradiated. 

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

Yes 

Iodide supplementation in water?

No Study authors reported that 

supplementation of water was not considered necessary since tadpoles developed at the expected rate and 40 mg/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 

սg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

(e.g.. in Sera Micron).

Page 18 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 17 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel type/materials: Glass aquaria sealed 

together with clear 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: ~30 x 14.5 x 20 cm 

deep. Water depth maintained at 12 cm with a screen-covered drain.

Fill volume: 5.2 LAdditional details on exposure system:

Diluter and aquaria were cleaned prior to test and all vessels were cleaned daily. Mixing cells and tubing were cleaned or replaced as needed. 

Page 19 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 18 of 67

DER Template Version: 22 September 2011 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Hardness (mg/L as CaCO3) 

56 68 62a

Weekly in replicate tanks from the 

control and highest treatment group. 

EPA recommends hardness 40 to 48 mg/L 

as CaCO3.

pH 6.9 7.9 7.4a

Weekly 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 7.9a

Weekly 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) 21.8 22.3 22a

Weekly in all replicate test vessels, 

and continuous in one replicate tank 

EPA recommends temperature 22±1oC; interreplicate and inter-treatment differentials should not exceed 0.5oC.

Iodide <0.01 mg/L Twice per year 

EPA recommends aquatic iodide range 0.5 – 

10 

սg/L (supplemental iodide should not 

exceed 2 

սg/L).

Page 20 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 19 of 67

DER Template Version: 22 September 2011 

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Ammonia <0.01 mg/L Twice 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 

hardness be measured at least at the 

beginning and end of the test.

Fluoride <0.01 mg/L Twice per year Perchlorate <0.2 սg/L Twice per year Chlorate <10 սg/L Twice per year Alkalinity (mg/L as CaCO3) 

20 40 33a

Measured on Days 0 and 21 in the 

control and highest treatment group. 

Conductivity (սmhos/cm)

152 206 174a

a calculated by the reviewer from data reported in the Tables and Appendices of the study report. 

Page 21 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 20 of 67

DER Template Version: 22 September 2011 

 D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or 

Remarks 

Guideline Recommendations 

Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration 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 laevis Life stage: NF stage 49-51 Test duration: 96 hours

Page 22 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 21 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or 

Remarks 

Guideline Recommendations 

Additional details: A non-GLP range-finding study was 

conducted for 96 hours under static-renewal 

conditions with tadpoles at NF stages 49-51. 

The NOAEC and LC50 values were 200 and 

>200 սg a.i./L, respectively. Since the limit of water solubility (100 սg a.i./L) was lower than the limit dose of 100 mg a.i./L and the level of acute signs of toxicity, the limit of solubility was used as the highest test level. 

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

Page 23 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 22 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Range-finder and limit of solubility

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

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

concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations:

0.2 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 concentrations adjusted for purity? 

Yes 

Page 24 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 23 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Time-weighted average 

Limit of quantification (LOQ): 0.2 սg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

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

measured on Days 0, 1, 3, 7, 10, 14, 17, and 21 

It is recommended that test item 

concentration be measured in one tank at 

each treatment level at test initiation and 

every week thereafter.

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (if applicable): 

4 EPA and OECD recommend the use of a 

concurrent solvent control when a 

solubilizing agent is used. EPA 

recommends 4 replicates.

Page 25 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 24 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 20 larvae/rep 

Was a solvent used? Yes Solvent type (if applicable): DMF Maximum 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? No 

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

Aquatic Toxicology, 76, pp.69–92. 

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

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

Page 26 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 25 of 67

DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 12:12 

(light:dark).

Light intensity at water’s surface: 

0.623-0.890 Klux EPA recommends light intensity 0.6 – 2 

Klux (at water’s surface).

Additional details: Specific details on the appearance of the 

test solution did was observed in the study report 

Page 27 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 26 of 67

DER Template Version: 22 September 2011 

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

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration-TWA 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ NA 

Maximum allowable 

%CV (i.e., 20%) was 

exceeded for all 

treated levels. 

EPA and OECD recommend that test item 

concentrations be maintained at a 

coefficient of variation (CV) 

20%.

Solvent Control 0 <LOQ NA 

Treatment 1 0.003 0.0027 31.1 

Treatment 2 0.020 0.0141 27.3 Treatment 3 0.100 0.0744 27.4 

Abbreviations: CV Coefficient of variation. 

LOQ = 0.00020 mg a.i./L 

Page 28 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 27 of 67

DER Template Version: 22 September 2011 

E. Observations

Biological Endpoints: Days 7 and 21: whole body wet weight, SVL, HLL, normalized HLL, NF stage, and asynchronous development 

 Day 21: thyroid gland histopathology  Daily: 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 (Days 7 and 

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

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

pathology observations.

Page 29 of 300
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 71 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. 

 

Page 30 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 29 of 67

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 10th and 90th 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. 

 

Page 31 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 30 of 67

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 սg a.i./L 

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

ranged from 0.45 in the TWA 2.70 սg 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 

n1 Mean

(mm) 

±SD 

HLL: 

SVL3 n1 Mean(mm) 

±SD

HLL: 

SVL2

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). 

Page 32 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 31 of 67

DER Template Version: 22 September 2011 

Day 7 SVL ranged from 16.18 mm in the TWA 0.0744 սg a.i./L treatment group to 17.30 mm in solvent control. Day 21 SVL ranged form 28.30 mm in the TWA 0.0744 սg 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 0.0027 սg a.i./L 

treatment group to 0.48 g in the TWA 0.0414 սg a.i./L treatment group. Day 21 body weight ranged from 1.96 g in the negative control to 2.16 g in the TWA 0.0027 սg a.i./L treatment group. Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWA- measured]

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 21 Day 7 Day 21 

n2

Mean

(mm) 

±SD n2

Mean

(mm) 

±SD n2

Mean

(g) 

±SD n2

Mean

(g) 

±SD

Negative Control 4 16.58 0.59 4 28.23 0.30 4 0.43 0.03 4 1.96 0.03 

Solvent Control 4 17.30 0.71 4 29.23 0.80 4 0.44 0.08 4 2.10 0.12 

0.0027 4 16.43 0.13 4 29.05 0.37 4 0.41 0.02 4 2.16 0.04 

0.0141 4 17.58 0.74 4 28.60 0.82 4 0.48 0.05 4 2.03 0.11 

0.0744 4 16.18 0.81 4 28.30 0.42 4 0.42 0.05 4 2.04 0.12 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 1 Also referred to as “wet weight” in the test guideline. 2 Sample size (n) represents the number independent replicates

Page 33 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 32 of 67

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. 

Page 34 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 33 of 67

DER Template Version: 22 September 2011 

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

Treatment 

(mg a.i./L)

[measured]

Diagnostic Observations1

Severity

Thyroid Gland 

Hypertrophy 

Thyroid Gland 

Atrophy 

Follicular Cell 

Hypertrophy 

Follicular Cell 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

0 20 20 20 20 20 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 graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 

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

Page 35 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 34 of 67

DER Template Version: 22 September 2011 

Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. Not Assessed 

Treatment 

(mg a.i./L) 

[TWA-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 

0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

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

Solvent Control 

0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

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

0.0027 

0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

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

Page 36 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 35 of 67

DER Template Version: 22 September 2011 

Treatment 

(mg a.i./L) 

[TWA-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.0141 

0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

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

0.0744 

0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

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

Abbreviations: NA Not applicable.

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

Page 37 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 36 of 67

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 

Page 38 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 37 of 67

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 20th to the 80th 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. 

Page 39 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 38 of 67

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. 

Page 40 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 39 of 67

DER Template Version: 22 September 2011 

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

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental Stage Hind Limb Length3 Thyroid Gross and 

Histopathology 

Day 7 Day 21 Day 7 Day 21 Day 21 

Median

p Median

p % Diff. p % Diff. 

p

Treatment-Related Effects? 

(Yes/No) 

Negative Control 54 NA 57 NA 0 NA 0 NA No 

Solvent Control 54 NA 57 NA -2.10 0.39 3.20 0.44 No 

0.0027 54 >0.99 57 >0.99 -2.08 0.85 -3.21 0.75 No 

0.0141 54 >0.99 57 0.48 0.00 >0.99 1.60 0.95 No 

0.0744 54 0.62 57 1.0 6.25 0.171 1.60 0.95 No 

Statistical test Mann-Whitney U Mann-Whitney U Dunnett’s Dunnett’s NA 

Abbreviations: Diff. Difference. NA Not applicable.

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

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

2 Unless otherwise specified, effects are considered statistically significant at 

p<0.05. 

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

Page 41 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 40 of 67

DER Template Version: 22 September 2011 

Table 17: Growth Endpoints1,2 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. 

Page 42 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 41 of 67

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 

(KOC= 9840 -22,660 L/kg) and lipophilic (log POW= 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.” 

Page 43 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 42 of 67

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. 

Page 44 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 43 of 67

DER Template Version: 22 September 2011 

APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:TABLE OF STATISTICAL INTERPRETATION: EDSP VS EFED METHODS 

Endpoint Monotonic? Parametric? EDSP EFED Comments 7-day wet weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

7-stage median No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

7-day SN-vent length 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

7-day hind-limb length 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

7-day normalized HLL 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

21-day stage median 

No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

21-day wet weight No Yes Dunnett’s: 

Dose 1 p=0.018 

Dunnett’s: Dose 1 p=0.018 

EDSP and EFED same conclusions. The lowest dose was significantly (p<0.05) increased 10% relative to control. 

21-day SN-vent length 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Page 45 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 44 of 67

DER Template Version: 22 September 2011 

Endpoint Monotonic? Parametric? EDSP EFED Comments 21-day HLL No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

21-day normalized HLL 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Page 46 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 45 of 67

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 

Page 47 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 46 of 67

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 

Page 48 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 47 of 67

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 

Page 49 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 48 of 67

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 

Page 50 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 49 of 67

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 . . . . . 

Page 51 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 50 of 67

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 

Page 52 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 51 of 67

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 

Page 53 of 300
Data Evaluation Record on the Toxicity of Benfluralin to Amphibians, Metamorphosis Assay 

EPA MRID Number 48614901 

Page 52 of 67

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 . . . 

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

Page 54 of 300
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 

Page 55 of 300
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 . . 

Page 56 of 300
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) 

 

Page 57 of 300
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 

Page 58 of 300
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) 

 

Page 59 of 300
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 

Page 60 of 300
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) 

 

Page 61 of 300
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 

Page 62 of 300
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) 

 

Page 63 of 300
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 

Page 64 of 300
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) 

Page 65 of 300
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 

 

Page 66 of 300
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 

 

Page 67 of 300
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 

 

Page 68 of 300
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 

Page 69 of 300
DATA EVALUATION RECORD

BENFLURALIN

Study Type: OCSPP 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. 

Page 70 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 71 of 300
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), 

ventral 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 10-10 to 10-3 M). Ethanol was used as a vehicle at a final assay concentration of 

<3%. The assay 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 (Bmax) 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 entire 

Page 72 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ 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 IC50s 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 [3H]-R1881 binding at all concentrations of benfluralin was 77%, 78%, 

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 IC50 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 Non-Binder in the 

Androgen Receptor Binding Assay. 

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

assay (OCSPP 890.1150). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were

provided. 

Page 73 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test facility: 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: Benfluralin

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 mCi/mL

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 very low solubility in water (>0.001 g/L)

Final concentration: <3%

F

F

N F

O2N

O2N

3CH

3CH

Page 74 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

B. METHODS

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

purchased from Charles River (Wilmington, MA). Male 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 stored at approximately -80°C until 

use, and were processed as a batch and used for multiple studies. 

The cytosol was prepared by adding low-salt TEDG buffer [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 supernatant 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 Research Lab, Rockford, IL). 

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

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

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

R1881 ligand. The conditions for the saturation binding experiment are summarized in 

Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat 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 specific activity of the stock solution [3H]-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 binding. 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 [3H]-R1881 

was measured. For each batch of cytosol, the optimal protein concentration was determined 

Page 75 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ 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 Tubes 1-24 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [

3

H]-R1881. Samples were dried, and

300 μl of prostate cytosol were added.

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

H]-R1881. R1881 was added in a 

100-fold molar excess of [3

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

cytosol were added.

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

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

[

3

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

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

dried, dissolved in diluted prostate cytosol (300 μL), and incubated 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, and 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. 

Page 76 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ 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 concentration 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 pages 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 [3H]-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. 

Page 77 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

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

Solvent Control Reference standard Weak positive control Test Chemical None

Ethanol R1881 Dexamethasone Benfluralin

Tubes 11-13 and 80-82 Tubes 14-31 and 83-85 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 pages 18-20 and 38-43 of the study report.

b Each concentration of each chemical was run in triplicate for a total of 85 tubes per run. Tubes 11-85 contained 

50 μL of triamcinolone acetonide and 30 μL [3

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

were added. Tubes 11-85 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tubes 14-16 and 83-85 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 14-16 and 83-85 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) 

R1881. 

4. Data Analysis: The maximal binding capacity (Bmax) and dissociation constant (Kd) 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 (IC50) was

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. 

Page 78 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ 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

Bmax: maximal binding capacity (Not provided)

Kd: dissociation constants (Not provided)

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

by the competitor

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

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 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 Cytosola

Parameter Run 1b Run 2 b Run 3 b Mean Runs 1-2 c

R2 (unweighted) 0.9937 0.9871 ND 0.9871-0.9937

Bmax (nM) 0.06194 0.06590 ND 0.06392

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

Kd (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 R2 is reported and the mean is reported for the other parameters. 

R2 Goodness of fit for curve calculated for specific binding,

ND not determined

Page 79 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

Non-specific, specific, and total binding curves for [3H]-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 [3H]-R1881 to the Androgen Receptor during the Saturation 

Binding Experiment.

Figure 2 is a Scatchard plot that illustrates the binding of [3H]-R1881 to the androgen receptor. 

The data fit results in a linear plot. 

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

COMPETITIVE BINDING EXPERIMENT: The results from the 3 competitive binding 

experiments are summarized in Table 7 and shown graphically in Figures 3-6. The estimated 

Page 80 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

log IC50s 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%, 78%, and 75% resulting in run classifications of non-binder, nonbinder and equivocal in runs 1, 2, and 3, respectively. An estimated mean log IC50 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 AR 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 0.9997

Test substance 0.9912 0.9888 0.9861 NA

Log IC50 (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

IC50 (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 % IC50) c Positive control 0.0036 0.0028 0.0037 0.0034

Test substance NA NA NA NA

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

NA Not applicable. 

r

2 Goodness of fit (r2 is more appropriately expressed as a range, as opposed to a mean).

c Calculated by reviewer: [IC50 (in M) positive control or benfluralin / IC50 (in M) R1881] x 100% 

RBA (%) relative binding affinity

FIGURE 3. Relative binding activity in Assay 1. 

Page 81 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ 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. 

Page 82 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

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

functioning properly, each run was evaluated using the following criteria (Table 8):

TABLE 8. Criterion a Tolerance 

Limit(s) b Value Yes No

Ligand depletion is minimal. The recommended ratio of total 

binding in the absence of competitor to total amount of [

3

H]-

R1881 added per assay tube.

≤15% 4.8 to 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 (dexamethasone) fitted curve parameters

Top (% binding) 87 to 106 99.9 to 102.7 X

Bottom (% binding) -12 to 12 -1.5 to 0.3 X

Hill Slope -1.4 to -0.6 -1.0 X

Saturation Binding Experiment Kd (nM) 0.685-1.57 0.4641 X

Non-specific binding (%)c <20% 6.5-7.4% X

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

b These values represent ranges 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 increase 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 binding sites 

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

variability. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: 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. Benfluralin 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, the estimated mean 

log IC50 for the test material could not be determined as it did not cross 50% binding. The 

Page 83 of 300
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1150/ OECD None

estimated log IC50s 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 [3H]-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). This minor deviation did not impact the study results 

as evidenced by expected IC50 and Hill Slope values for R1881. 

Page 84 of 300
DATA EVALUATION RECORD

BENFLURALIN

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. 

Page 85 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 86 of 300
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, 104 , 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. 

Page 87 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / 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 runs.

The standard curves for 4-OH ASDN 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 aromatase activity curves after exposure to benfluralin either did not fit the model 

(Run 1) or were not lower than 75% of full activity (Runs 2 and 3). The log IC50 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 satisfies the EDSP Tier 1 Test Order requirements for an Aromatase assay (OCSPP 

890.1200). 

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

Assurance statements were provided. 

Page 88 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Benefin TGAI

Description: Yellow Solid

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

Lot/Batch #: Lot No. ACD13683, TSN100037

Purity: 97.5%

Storage conditions: 5oC - Ambient

Stability: Not reported

Solvent: DMSO

Solubility (in Test Solvent): 0.1 M

Highest Concentration Tested: 1x10-4.5 M

Stock Solution Preparation: Serial Dilution

Molecular Weight: 335.3 g/mol

CAS #: 1861-40-1

Structure:

2. Non-Labeled Substrate: 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 Substrate: 1-β [

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

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

Lot/Batch #: Lots: 3615304 and 619344

Radiochemical Purity (Supplier): Lot 619344: >97%

Lot 3615304: Radiochemical 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 Control: 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 (Vehicle Control): DMSO

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

Lot 037K0766

Justification for Choice of Solvent Greater solubility compared to ethanol

Concentration (% of Total Volume in Assays): 1%

N

O2N NO2

CF3

H3CH2C (CH2)3CH3

Page 89 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / OECD None

6. Test Microsomes: Human Recombinant Aromatase (CYP19) Microsomes

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

Lot/Batch #: Lot 74101

Protein Concentration: 0.004 mg protein per mL 

Cytochrome C Reductase Activity: 560 nmole cytochrome c reduced/mg protein/min

Aromatase Activity: 4.4 pmol product/pmol P450/min

B. METHODS

1. Assay Components and Preparations: A mixture of non-labeled and radiolabeled 

[

3

H]ASDN was prepared such that the final concentration of ASDN in the assay was 

approximately 2 μM, 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 solvent(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 formulated in DMSO. 

Fresh dilutions of the stock solution were prepared in the same solvent as the stock solution 

on the day of use. Dilutions were prepared such that the target concentrations of the positive 

control substance (0.1–10,000 nM; Table 4) were achieved by the addition of 20 μL of the 

dilution for a final assay volume of 2 mL.

Human recombinant microsomes were purchased from 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

Sodium phosphate buffer (pH 7.4) 0.1M

Microsomal Protein 0.004 mg/mL a

NADPH 0.3 mM

[

3H]ASDN 100 nM

Propylene Glycol 5%

Temperature 37°C

Incubation Time 15 min

a The concentration of microsomal protein was optimized for microsomes that produced approximately 4.4 pmol 

product/pmol P450/min.

2. Suitability Assessments: The protein concentration was reported to have been determined 

on each day the aromatase assay was run. Protein concentration was determined in 

Page 90 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / 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 was greater than the minimum acceptable aromatase activity of 

0.1 nmol/mg-protein/min.

3. Aromatase Assay: 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 3H2O in the aqueous fraction was quantified for each assay tube by liquid 

scintillation counting (LSC), and aromatase activity was reported in units of 

nmol∙mg-protein-1∙min-1. 

4. Demonstration of Proficiency: There was no 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 Proficiency: The positive control data 

(new/historical data for laboratory) were not reported in regards to the following criteria: 

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

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

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

concentration of 4-OH ASDN was <15%.

Performance criteria were reported for the test runs only (Table 2), and served as guidance 

in identifying runs that provided parameters in the preferred ranges. 

(2) Demonstration of Proficiency of New Technician for Conducting Assay (when 

applicable): Demonstration of proficiency by a new technician, if applicable, was not 

reported. 

Page 91 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / 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 IC50 (M) −7.3 −7.0 –7.4 –7.3

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

b. Proficiency Chemicals: 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. 

TABLE 3. Proficiency Chemicals

Compound CAS# Class Concentrations

Econazole 24169-02-6 Inhibitor Not reported

Fenarimol 60168-88-9 Inhibitor Not reported

Nitrofen 1836-75-5 Inhibitor Not reported

Atrazine 1912-24-9 Non-inhibitor Not reported

5. Determination of Aromatase Activity with Test Chemical(s): The response of aromatase 

activity to the presence of eight concentrations of a test chemical per run, in triplicate, was 

tested during three independent runs. 

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions (Tubes) Description Reference or Chemical (M)

Full Activity Control 4 All test componentsa plus solvent vehicle N/A

Bkgd Activity Control 4 Same as above without NADPH N/A

4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10-5

4-OH ASDN Conc 2 2 All test components plus 4-OH ASDN 1×10-6

4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10-6.5

4-OH ASDN Conc 4 2 All test components plus 4-OH ASDN 1×10-7

4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10-7.5

4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10-8

4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10-9

4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10-10

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, [3H]ASDN, and NADPH. 

Page 92 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / OECD None

C. DATA ANALYSIS

1. Raw Data: 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/min 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. Statistical Methods: Statistical analyses and displays such as summary tables, graphical 

displays, analysis of variance, and multiple comparisons 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 symbols for each run) were 

included on the same graph with their respective fitted response curves. In addition, the 

average percent of control values for each run versus the logarithm of test chemical 

concentration were plotted on a separate graph along with the average concentration 

response curve across runs were superimposed on the same plot. 

In order to determine the consistency among runs, the slope and log IC50 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

Average 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 runs is greater 

than 75% activity

Non-inhibitor

Data do not fit model ---

Page 93 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / 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 compared to the previous two assay runs. 

A different (and older) lot of [3

H]ASDN was used for the third assay run. 

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase 

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 (10-5M). The standard curves for 4-OH ASDN 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 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 overall standard deviation and % CV. The 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 SEMa Overall %CVa

4-OH ASDN 

(Positive Control)

-5 3 -1.28a 3.10a 1.26 -242

-6 3 4.68a 2.36a 0.96 50.4

-6.5 3 13.3a 2.69a 1.10 20.2

-7 3 32.3a 4.41a 1.80 13.6

-7.5 3 62.2a 4.92a 2.01 7.92

-8 3 84.3a 5.19a 2.12 6.16

-9 3 105a 8.71a 3.55 8.26

-10 3 110a 7.31a 3.66 6.63

Benfluralin -4.5 3 102b 4.15b 2.45 7.18

-5 3 106b 3.34b 2.40 6.77

-5.5 3 102b 12.9b 5.11 15.0

-6 3 102b 3.85b 2.28 6.71

-7 3 104b 6.88b 3.04 8.77

-8 3 101b 11.1b 4.60 13.6

-9 3 99.8b 4.24b 1.93 5.81

-10 3 100b 3.71b 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 Variance

Page 94 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / OECD None

FIGURE 1. Inhibition Response Curves for 4-OH ASDN. 

Page 95 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / 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 Response Curves for Benfluralin From Each Test Run.

Page 96 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 11 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / OECD None

FIGURE 3. Mean Inhibition Response Curve for Benfluralin.

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

estimated log IC50 ranged from -9.0 to -5.9 M for the test material (average not determined) 

compared to the positive control 4-OH ASDN, 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 ASDN. Confidence in the 4-OH ASDN data is high due to the small variation, but 

confidence is low for benfluralin due to the high variation in the IC50 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 Benfluralin on Aromatase Activity (as Percent of Control) From Independent Runs

Chemical Run 1 Run 2 Run 3 Mean SD %CV

Log IC50 (M)

Benfluralin ND -9.0 -5.9 -8.0b ND ND

4-OH ASDN -7.4a -7.3 -7.3 -7.33b NR NR

Slope

Benfluralin ND 7.5 0.54 18b ND ND

4-OH ASDN -0.93 -0.91 -0.94 -0.93b NR NR

a The log IC50 was outside the guideline recommended performance criteria range of -7.3 to -7.0 

b Mean log IC50 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, 

Page 97 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / 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 standard 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 [3H]ASDN may have contributed to this 

difference. 

 

 The investigators stated that if 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 (R2 > 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 ASDN 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 Run 3. These 

deviations from the guideline recommendations for performance criteria were considered 

minor by the investigators because acceptable Hillslope values and goodness of fit values 

were obtained for all three assay runs. The log IC50 value in assay 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 

were reported as potential reasons for the issues with the background count. As described 

previously, a different (and older) lot of [3H]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.

 Aromatase 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 IC50 from the mean benfluralin response curve was -8.0 M compared to 

the log IC50 from the mean positive control 4-OH ASDN curve, which averaged -7.3 M; the

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

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

lower for benfluralin due to the considerably higher variation reported for the IC50 and 

Hillslope data (95% confidence intervals of 10-7 to 107 and 10-10 to 1010, respectively). 

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 non-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: 

Page 98 of 300
Aromatase (Human Recombinant) Assay (2011) / Page 13 of 13

BENFLURALIN (BENEFIN) / 084301 OCSPP 890.1200 / OECD None

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

chemical). 

• Two different lots of test substrate [3H]ASDN were used separately for different assay 

runs; initial radiochemical purity of the older lot (No. 36153054, 770 days) was not 

reported. 

• The stabilities of the 4-OH ASDN positive control and benfluralin stock solutions were 

not verified analytically. 

• The maximum storage 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 ASDN percent aromatase 

activity from the individual assay runs (or the average curve), and SEM and %CV values 

were not reported for 4-OH ASDN or benfluralin percent aromatase activity. These 

values were calculated by the Reviewer and reported in Table 7 of the DER. 

Page 99 of 300
DATA EVALUATION RECORD

BENFLURALIN

Study Type: OCSPP 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: 11/14/2011

Secondary Reviewer Signature:

David A. McEwen, B.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. 

Page 100 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 101 of 300
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 expected 

Page 102 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 2 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ 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 experiment was conducted on the batch of cytosol preparation used for the competitive 

binding experiment.

For the competitive binding experiment, all performance criteria were met for appropriate 

responses by 17β-estradiol and 19-norethindrone. The log IC50 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 from the three runs, benfluralin is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

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

Assay (OCSPP 890.1250).

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were

provided. 

Page 103 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 3 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ 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: 17β-estradiol

Supplier: Sigma, St. Louis, MO

Catalog # Not Reported

Batch #: 098K1372

Purity: 100%

CAS #: 50-28-2

4. Radioactive ligand: [

3

H]-17β-estradiol

Supplier: Perkin-Elmer, Boston, MA

Catalog #: NET517001MC

Batch #: 639068

Radiochemical purity: >97%

Specific activity: Not Reported

Concentration of stock: 162.9 Ci/mmol

*Information on adjusted specific activity was not available. 

5. Positive control: 19-Norethindrone

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 # : 2943-75-1

Page 104 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 4 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ 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 Rat Uterine 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 concentration 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. Saturation (radioligand) Binding Experiment: A saturation binding experiment was 

conducted to measure total and non-specific binding of [3H]-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 experiment 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 nMb

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 Data were not included in the study report, but are reported in the study profile, submitted separately (MRID 

48675404).

b This value was slightly higher than the suggested range in the guideline; however, all other values, including 

minimal ligand depletion, indicated 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 concentrations 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-

Page 105 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 5 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

fold greater than each respective radiolabeled concentration to result in final concentrations 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. 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

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 Data were not included in the study report, but are reported in the study profile, submitted separately (MRID 

48675404).

b Total binding = [3H]-17β-estradiol bound to ER.

c Non-specific binding = [3H]-17β-estradiol and 100-fold greater non-labeled bound to ER.

d Total [3H]-17β-estradiol alone for dpm determination at each concentration.

e Final concentrations of [3H]-17β-estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f Final concentrations of non-labeled 17β-estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.

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

Competitive Binding Experiment is presented in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat Uterine Cytosol 

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 pages 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 [3H]-17βestradiol was not reported, but the study authors stated that it was adjusted for decay over 

Page 106 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 6 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

time. [3H]-17β-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 

achieve the concentrations shown in Table 4. Each assay consisted of three runs, and each 

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

TABLE 4. Molar (M) concentrations in Competitive 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, 73-96, 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 x10-9

10-4 3.16 x 10-5 10-4 10-8

10-3 10-4 10-3 10-7

a

 Data were obtained from pages 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 nonlabeled 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]-17β-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. DATA ANALYSIS: For the competitive binding assay, 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 

IC50 as a parameter to be estimated. For the competitive binding experiment (log IC50 and 

RBA), mean and standard deviation were calculated for each run and mean and standard error 

were calculated for the composite three runs using [software not reported].

Page 107 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 7 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

1. Definitions

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

three runs. Each run was first individually classified as follows: 

Interactive = lowest point on the fitted curve within the range of the data is less than 50% 

(i.e., >50% of the radiolabeled estradiol has been displaced from the ER). 

Not interactive = there are usable data points at or above 10-6M 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 10-6M and either a binding curve can be fit but 

≤50% of the radiolabeled estradiol has been displaced from the ER or a binding 

curve cannot be fit and the lowest average percent binding among 

concentration groups in the data is >50%. 

Equivocal = A run is classified as equivocal if it does not fall into any of the categories 

above. 

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

Run Classification Numerical Value

Interactive 2

Equivocal 1

Not interactive 0

Equivocal up to the limit of concentrations tested “missing”

The values for each run were then averaged across runs and the chemical classified using the 

following ranges: 

Test Material Classification Numerical Range

Interactive average ≥1.5

Equivocal 0.5≥ average <1.5

Not interactive average <0.5

Equivocal up to the limit of concentrations tested “missing”

b. Descriptors for receptor binding: 

Bmax: maximum specific binding number (not provided)

Kd: dissociation constant (not provided)

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

the receptor

Relative Binding Affinity (RBA %): IC50 of 17β-estradiol ÷ IC50 of test substance × 100 

Page 108 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 8 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The Kd for [3H]-17β-estradiol was 0.1032 nM and the Bmax (nM) 

was 0.07097 for the prepared rat uterine cytosol used in these experiments. The Kd for the 

run was within the expected range of 0.03 to 1.5 nM. The Bmax 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 17β-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

Bmax (nM) 0.07097 N/A N/A N/A

Bmax (fmol/100 μg protein) 59.28 N/A N/A N/A

Kd (nM) 0.1032 N/A N/A N/A

a Data were not included in the study report, but are reported in the study profile, submitted separately (MRID 

48675404).

b Only a single run of the saturation binding experiment was conducted on the batch of cytosol used for this 

competitive binding experiment.

r

2 Goodness of fit for curve calculated for specific binding 

Specific, non-specific, and total binding curves for [3H]-17β-estradiol to the estrogen receptor 

are presented in Figure 1 below. The specific binding reached a plateau, and non-specific

binding was less than 20% of total binding at all concentrations (range 1.7%-8.6%). Figure 2 is 

a Scatchard plot that illustrates the binding of [3H]-17β-estradiol to the estrogen receptor. The 

data fit results in a linear plot. 

FIGURE 1. Binding of [3H]-17β-estradiol to the ER during the Saturation Binding

Experiment. 

Page 109 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 9 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

FIGURE 2. Scatchard Plot of the Binding of [3H]-17β-estradiol to the 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 was 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 competitive binding experiments are summarized in Table 6 and 

shown graphically in Figures 3-6. The estimated mean log IC50 for the test material could 

not be determined as it did result in 50% displacement of radiolabeled estradiol at any 

concentration. The mean log IC50 for 17β-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).

Page 110 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 10 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

TABLE 6. Competitive Binding Assay of Benfluralin with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1b Run 2b Run 3b Meanc

r

2 (unweighted), 17β-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 IC50 (M), 17β-estradiol -8.913 -8.948 -8.934 -8.934

Positive control -5.431 -5.462 -5.435 -5.442

Benfluralin NA NA NA NA

IC50 (M), 17β-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 %IC50)

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 R2 is reported and the mean is reported for the other parameters. 

d Calculated by reviewer: [IC50 (in M) positive control or benfluralin / IC50 (in M) R1881] x 100% 

RBA (%) Relative binding affinity

NA Not applicable

TABLE 7. Binding Classification of Benfluralin with Estrogen Receptor a

Run 1 2 3 Mean Binding Classificationc

Classification category valueb 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 <0.5

Page 111 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 11 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test 

Compound. Assay #1.

FIGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test 

Compound. Assay #2.

FIGURE 5. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test 

Compound. Assay #3.

Page 112 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 12 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ OECD None

FIGURE 6. Mean and Standard Error of the Mean for 3 Independent Assays - Relative 

ER Binding Activity for Benfluralin. 

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

properly, each run was evaluated using the following criteria in Table 8: 

TABLE 8. Criterion a Tolerance 

Limit(s)

Value Yes No

17β-estradiol fitted curve parameters

Loge 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 (log10(M)-1) -1.1 to -0.7 -0.99 X

Weak Positive control (norethynodrel) fitted curve parameters

Loge 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 (log10(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]-17β-estradiol from the ER on average across all concentrations

≤25% 13.5c 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 [3H]-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 the curve. The 

percent binding at this 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 runs 

Page 113 of 300
Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (2011) / Page 13 of 13

BENFLURALIN (BENEFIN)/084301 OCSPP 890.1250/ 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 guideline. 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, the investigators concluded that 

benfluralin had no appreciable effect on the binding of 17β-estradiol 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 study 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, non-specific, and total binding curves for [3H]-17β-estradiol to the estrogen receptor 

were reported in a study profile (MRID 48675401). The maximum binding capacity (Bmax) 

was 59.28 fmol/100 μg protein and the dissociation constant (Kd) was 0.1032 nM. The Bmax

fell within the expected range of 10 to 150 fmol/100 μg protein and the Kd for the run was 

within the expected range of 0.03 to 1.5 nM. Only one saturation binding run was 

performed instead of the recommended three runs. For the competitive binding experiment, 

the log IC50 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 preparations. 

Page 114 of 300
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, 

Building 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: 11/11/11

Program Manager: Signature:

Jack D. Early, M.S. Date: 11/17/11

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 11/17/11

Disclaimer

This Data Evaluation Record may have been altered by the Endocrine Disruptor Review Team 

subsequent to signing by CSS-Dynamac Corporation personnel. 

Page 115 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 116 of 300
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 Signaturt:'-:?~: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 PCMax was the limit of solubility and cytotoxicity, 10-

4 M. 

Page 117 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 2 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ OECD 455

Because the RPCMax > PC10 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. 

Page 118 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 3 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ 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 # 079KI0131

Purity: 100% 

CAS # : 50-28-2

17α-estradiol (weak estrogen)

Supplier: Sigma, St. Louis, Missouri

Catalogue and Batch #: Cat# E-8750 Lot# 029K4116

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 concentration): 0.1%

Page 119 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 4 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ OECD 455

B. METHODS

1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese 

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% CO2 at 37oC. Cells 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 Assays: 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 × 104 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). Cells containing luciferase 

were lysed and luciferase substrate was added. The resulting chemiluminescence was 

measured immediately after adding luciferase reagents in a Packard TopCount NXT 

luminescence counter. In a typical in vitro cell culture assay, TopCount has a detection limit 

of ~5 cells in 1000-μL portion of medium.

a. Preliminary Test: 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 insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: The proficiency chemicals test was not provided.

c. Reference Chemicals: To ensure the stability of the response from the cell line, six 

concentrations of each of the following reference chemicals were included in each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class

17β-estradiol 50-28-2 10−14 to 10−8 Strong estrogen

17α-estradiol 57-91-0 10−12 to 10−6 Weak estrogen

Corticosterone 50-22-6 10−10 to 10−4 Negative compound

17α-methyltestosterone 58-18-4 10−11 to 10−5 Very weak agonist

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control 

(PC), the luminescence signals from the concurrent plate were analyzed by subtracting the 

mean value of the vehicle control from each well value to normalize the data; each 

Page 120 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 5 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ OECD 455

normalized value was then divided by the mean value of the normalized PC. The resulting 

value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. Graph Pad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA) was 

used to calculate the EC50, PC10, PC50, RPCMax, and PCMax for benfluralin when applicable. 

The test material was defined as positive for inducing estrogen receptor transcriptional 

activation if the RPCMax ≥ PC10 in at least 2 of 2 (or 2 of 3) runs. LogEC50 and Hill slope 

values are calculated only if a positive response is observed. Coefficients of variation (CV) 

were calculated for the luminescence data triplicates. Concentrations showing >20% 

cytotoxicity or evidence of insolubility were excluded from analyses.

4. Definitions

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response induced 

by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response induced 

by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS

A. PRELIMINARY TEST: 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 

assay (Table 1). 

TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Benfluralina

Concentration (M) % Viability b Commentsc

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 NRe

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 Reported

Page 121 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 6 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ 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 Chemicals: Values derived from the concentration response curve (e.g., log

PC50, log PC10, log EC50, and Hill 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 17α-methyltestosterone which had a log PC10 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 acceptable value for 17αestradiol in one run. 

TABLE 2. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter

Acceptable Range Values Acceptable

Run 1 Run 2 Run 4 Yes No

17β-estradiol

Log PC50 −11.4 to −10.1 −10.6 −10.6 -11.0 X

Log PC10 <−11 −11.9 −12.4 -12.9 X

Log EC50 −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 PC50 −9.6 to −8.1 −8.7 −9.0 −9.4 X

Log PC10 −10.7 to −9.3 −10.0 −10.0 −10.6 X

Log EC50 −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 PC50 −6.0 to −5.1 −5.4 −6.1 −8.0 X

Log PC10 −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. 

Page 122 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 7 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ 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

4d 2323.4 261.9 14443.2 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, therefore 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. Concentration-response curves 

depicting fold induction of relative transcriptional activation are presented in Figure 1 below. 

The RPCMax was 13.3-19.1%, and the associated PCMax was the limit of solubility and 

cytotoxicity, 10−4 M for all three runs. Because the RPCMax ≥ PC10 in all three runs, 

benfluralin was considered positive for estrogen receptor transcriptional activation. 

The PC50 could not be determined. 

TABLE 4. Relative Transcriptional Activation (RTA) of Benfluralina 

Parameter RTA (mean ± SD); % of Positive Control (PC)

Run 1 Run 2 Run 4c 

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 EC50b −4.7 −5.6 −6.0

Hill Slopeb 1.1 1.1 1.3

RPCMax 19.1% 18.8% 13.3%

PCMax 10−4 10−4 10−4

PC50 NA NA NA

PC10 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, therefore the assay was repeated.

NR = Not Reported

NA= Not Applicable

Page 123 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 8 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ 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 Criteria: 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 experiment 

Page 124 of 300
Estrogen Receptor Transcriptional Activation (2009) / Page 9 of 9 

BENFLURALIN/PC Code 084301 OCSPP 890.1300/ OECD 455

fell within the acceptable range (Table 3). However, the PC50 and PC10 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 luciferase activity of the 

positive control was greater than 4-fold that of the mean vehicle control on each plate. PC10

values of concurrent PC were 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 benfluralin 

(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 RPCMax 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 RPCMax for benfluralin was greater than 10% of the response of the positive control 

(13.3-19.1%) indicating a positive result. The PC10 was 10−5.3 to 10−4.8 M, but the activity of 

benfluralin was not strong enough to allow the calculation of a PC50. It is pertinent to note 

that increased sensitivity to a known weak agonist of the estrogen receptor, 17αmethyltestosterone, may result in a false positive response. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

• The proficiency test results were not provided. 

Page 125 of 300
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'

Page 126 of 300
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.

Page 127 of 300
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 25.5o

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 

Page 128 of 300
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. 

Page 129 of 300
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-through 

Page 130 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 6 of 91

Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Benfluralin. 

Treatment 

(mg a.i./L) 

[TWA 

measured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2 

M F M F M F M F M F M F

0.000302 No No No No No No No No No No NA NA NA NA 

0.00314 No No No No Yes No No No No No NA NA NA NA 

0.03653 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 

Abbreviations: E2 17ȕ-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin. NR Not reported. 1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified. 2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavily 

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

3 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.

Page 131 of 300
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. 

Page 132 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 8 of 91

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, 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. 

Page 133 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 9 of 91

B. Test organism: Table 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Pimephales promelas

Species strain (if stated): Not reported Were fish obtained from a single laboratory stock? 

Yes New England Bioassay, Inc. (Manchester, 

Connecticut)

EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test?

Yes EPA recommends that fish be acclimated under 

water quality and illumination conditions that are 

similar to the definitive test. 

Acclimation period: 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. 

Page 134 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 10 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Mortalities 0.7% during 20 day acclimation 

period; 0.5% in 7 days prior to pre-exposure 

period.

Fish did not receive any treatment for disease during acclimation, pre-exposure or 

exposure periods. 

Moribund fish or fish with clinical signs of disease were not used in the 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/day Details on feeding: Equal measured portions of shrimp were fed 

to each tank. Food was withheld for at least 12 hours prior to treatment termination to aid in histological processing. 

Page 135 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 11 of 91

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 6 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months old.

Mean weight of males at test initiation (if determined): 

3.8 g Based 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 test initiation (if determined): 

Not reported Individual weights within ±20% of the 

estimated mean. Average male weight was 

3.8 ± 0.6 g (CV=15.0%; n=60) 

Mean weight of females at test initiation (if determined): 

1.8 g Based on all female fish selected for the test 

(pre-exposure). 

Range of individual weights (females) at test initiation (if determined): 

Not reported Individual weights within ±20% of the 

estimated mean. Average female weight was 1.8 ± 0.3 g (CV=17.1%; n=120) 

Mean length of males at test initiation (if determined): 

Not reported 

Mean length of females at test initiation (if determined): 

Not reported 

Duration of pre-exposure phase: 16 days EPA recommends a minimum of 14 days. 

Page 136 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 12 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical to the definitive test? 

Yes EPA recommends that pre-exposure conditions, 

including temperature, photoperiod, feeding, etc., 

be identical to definitive test conditions. 

Number of pre-exposure tanks: 30 tanks 10 extra replicates were established and 

included in the total number of pre-exposure 

tanks (30 tanks).

EPA recommends that additional tanks set up at 

the beginning of pre-exposure will ensure that 

sufficient replicates with the correct sex ratio are 

available for the definitive test. 

Number of males per tank: 2 males/tank Number of females per tank: 4 females/tank Pre-exposure fecundity: >15 eggs/female/ 

reproductive day/replicate

EPA recommends that pre-exposure fecundity in 

each replicate (tank) selected for use in the 

definitive test be at least 15 

eggs/female/reproductive day/replicate during 

the 7 days prior to the definitive test. 

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. 

Page 137 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 13 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on pre-exposure: 2 pre-exposure replicates were excluded 

due to mortality and 4 pre-exposure replicates were excluded due to poor reproductive performance. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flow-through 

system. As noted in the Corrections and 

Clarifications document1 , the use of a static 

renewal system is not recommended for this 

assay.

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

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

Page 138 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 14 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution system:

Continuous flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: 90 mL/min The flow rate is greater than the USEPA 

guideline (45 ml/min) and was chosen to help maintain test concentrations during the exposure due to the adsorption properties of the test substance. 

Recommended flow-through rate is 45 

mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

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

Page 139 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 15 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

A continuous-flow syringe pump delivery using gas-tight syringes continuously delivered stock 

solution at designated rate to designated stainless steel mixing chamber and mixed with dilution water; no test substance or solvent stock were added to the negative control. 

Concentrations of the solvent in the treatment and solvent control were identical. Test solution was split equally among four replicate test vessels (maintained in temperature controlled water trough) via Teflon manifold and stopcock. Diluter operation was monitored at least two times daily during the test and flow rates assessed. The flow rates of the dose stock solutions were measured 6 times/week and the diluent measured daily during exposure and targeted not to vary more than 20% during exposure. 

Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration is 

not recommended for mixing; 3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is within 

10%.

Aeration? Not reported Dissolved oxygen levels were maintained at 

>60% saturation. 

EPA recommends aeration if dissolved oxygen 

reaches <4.9 mg/L (<60% saturation).

Page 140 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 16 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Natural water Laboratory dilution water was Lake Huron water 

supplied by the City of Midland Water Treatment Plant and limed and flocculated with ferric chloride. The water was pumped to the lab prior to municipal treatment for human consumption. 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 for pesticides, heavy metals, and other contaminants?

Yes Periodically, the water is analyzed for total 

organic carbon (TOC), total suspended solids (TSS), pesticides, organics, metals and other inorganics. 

Test vessel type/materials: Glass aquaria sealed with 

clear 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 covered overflow drain.

EPA recommends the use of 18 L test 

chambers (e.g., 40 x 20 x 20 cm).

Page 141 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 17 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Fill volume: 10 L EPA recommends 10 L solution per tank. Spawning substrate material: Aged PVC Spawning tiles were constructed of ca. 9 cm 

long x 10 cm diameter PVC pipe cut and 

sectioned lengthwise and inverted to form a semicircle arch. Each tile positioned on a lipped tray of stainless steel (ca. 13 cm long) 

to contain eggs that do not adhere to tile and a stainless steel screen to prevent fish from access 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 cm 

diameter PVC pipe 

Page 142 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 18 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system:

The test substance was absorptive (Koc = 9840-11,660 L/kg) and lipophilic (log Pow = 

5.29). Therefore, the exposure system was 

equilibrated at least 8 days prior to initiating exposure to achieve equilibrium. Nominal ‘dosing’ concentrations were increased to 2.5X of target exposure concentrations (i.e. 0.083, 

0.0083, and 0.00083 mg/L) in an effort to 

achieve and maintain the target exposure concentrations (0.033, 0.0033, and 0.00033 

mg/L) in the water column of the test vessels. Water samples from all replicate vessels and 

concentrated stock solutions were sampled on Days 0, 1, 2, 4, 7, 11, 14, 18 and 21 to better characterize benfluralin exposure. Finally, test 

solution flow rate was doubled from 45 mL/min 

to 90 mL/min (6 to 12 turnovers/day). 

Page 143 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 19 of 91

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 24.3 25.5 24.9 

Weekly in all replicate 

tanks and continuously 

in one replicate tank. 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials should not exceed 1oC.

pH 7.5 7.8 7.6 Weekly 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 47 

Weekly from the 

negative control and 

highest treatment level 

with surviving fish. 

EPA recommends total alkalinity >20 mg/L 

as CaCO3.

Hardness (mg/L as CaCO3) 60 92 74 

Weekly from the 

negative control and 

highest treatment level 

with surviving fish. 

Total organic carbon (mg/L) Not reported Measured periodically 

from source water. 

EPA recommends that total organic carbon in 

dilution water be <2 mg/L. 

Page 144 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 20 of 91

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Unionized ammonia (Pg/L) Ammonia (as Nitrogen); <0.1 mg/L (<LOD) 

Measured periodically 

from source water; 

results from most 

recent analysis. 

EPA recommends that unionized ammonia in 

the dilution water be <1 Pg/L. 

Residual chlorine (Pg/L) <0.02 mg/L (<LOD) 

Measured periodically 

from source water; 

results from most 

recent analysis. 

EPA recommends that residual chlorine in 

dilution water be <10 Pg/L. 

Other (pesticides, organics, inorganics, metals)

Measured concentrations of pesticides, organics, 

metals, and other inorganics were below levels of 

concern. 

Measured periodically 

from source water; 

results from most 

recent 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. 

Page 145 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 21 of 91

D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? No EPA recommends conducting a range-finder if 

96-hour LC50 data for the fathead minnow are 

unavailable. 

If yes, what was the method for determining the highest test concentration in the 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 applicable Life 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. 

Page 146 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 22 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional 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 concentration-response curve. 

Based upon the results, the highest concentration 

that does not result in increased mortality or 

signs of overt morbidity compared to controls, or 

1/3 the derived 96-hr LC50, may be selected as 

the highest exposure concentration in the 21-day 

test.

Page 147 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 23 of 91

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

test be 21 days. 

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

Other reference study (1/3rd the 96-hour LC50)

The acute toxicity 96-hour LC50 value for benfluralin with fathead minnows was reported to be 0.100 mg/L (Mayer and Ellersieck, 1986). From the acute toxicity study, the high test concentration for the present study was selected as 1/3 of 0.100 

mg/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 LC50), whichever concentration is lowest.

Page 148 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 24 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Reference study citation (if applicable):

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. 

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 acceptable1 . 

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

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Page 149 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 25 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Time-weighted average 

Limit 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 reported Frequency of measurement: Test concentrations 

measured on 0, 2, 4, 7, 11, 14, 18 and 21 from all replicate tanks. 

Additional analysis of test concentrations was necessary to characterize benfluralin exposure concentrations due to the adsorptive and lipophilic nature of the test chemical. 

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 complete block 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. 

Page 150 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 26 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (if applicable): 

4 EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is used. 

EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

Page 151 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 27 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a solvent used? Yes Dose selection determined from the limit 

concentration (100 ug/L) reported in published literature and the solubility limit of the test chemical. The study report recognizes use of a solvent (DMF) to prepare test chemical solutions is a last resort. However, test chemical concentrations in an aqueous solution were likely to be unstable and the solubility limits of test chemical made saturator columns impractical for the selected test concentrations. 

Solvent type (if applicable): DMF N’, N-dimethylformamide. Maximum solvent concentration (if applicable):

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 other 

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

Toxicology, 76, pp.69–92. 

Page 152 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 28 of 91

Parameter Value(s) Details or Remarks Guideline Recommendations 

adverse effects and that concentration not be 

greater than 0.1 ml/L4.

Was a positive control used? No Positive control (if applicable): Not applicable Positive control concentration(s) (if applicable): 

Not applicable 

Photoperiod: 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 was 

not located in study report. 

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

Assessment. No. 23. Paris, France. 

Page 153 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 29 of 91

Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Benfluralin. 

Treatment ID 

Nominal 

Concentration

(mg a.i./L) 

TWA Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative control 0 <LOQ NA The coefficient of variation for measured 

concentrations of 0.000302 and 0.00314 mg a.i./L exceeded 20%, ranging from 21.9 

to 52.5%, reflecting the loss of test substance in the water column during the first week of the study (the 0.033 mg a.i./L 

was terminated at 11 days). Several measures were taken to compensate for the uncontrollable loss of test substance in the water column to provide exposure concentrations near the target (see additional details on exposure system). 

EPA recommends that test 

item concentrations be 

maintained at a coefficient of 

variation (CV) 20%.

Solvent control 0 <LOQ NA 

Treatment 1 0.00033 0.000302 43.4 

Treatment 2 0.0033 0.00314 27.1 Treatment 3 0.033 0.0365 12.5 

Abbreviations: CV Coefficient of variation. NA Not applicable. 

LOQ=0.0000505 mg a.i./L

Page 154 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 30 of 91

E. Observations

Biological Endpoints: Daily, observations were conducted for mortality, external abnormalities (e.g. hemorrhage, discoloration) and any abnormal 

behavior relative to the controls (e.g. hyperventilation, loss of equilibrium, uncoordinated swimming, atypical quiescence and feeding abstinence), fecundity and fertilization. At test termination (Day 21) fish were observed for behavioral and secondary sex 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 on surviving fish at test termination.

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least 

daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, 

plasma vitellogenin, and plasma sex steroids (testosterone and 17ȕ-estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio 

of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. 

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal behavior. 

Page 155 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 31 of 91

II. RESULTS AND DISCUSSION 

A. Results 

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 terminated and removed from the study on exposure Day 11 and excluded from any subsequent statistical analysis. The toxicity in the high treatment group may have been due to the necessity of dosing the test solution at 2.5X the target exposure concentrations to achieve target water concentrations during the exposure. The nominal dosing concentration for the high treatment group was 0.083 mg a.i./L, which was near the reported 96-hour LC50 value 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, the survival rate in the control groups satisfied the minimum acceptable control value criteria of 90% according to the USEPA OCSPP 890.1350 

guideline. Excluding the highest treatment level, minimal signs of abnormal behavior and appearance were observed in the other treatment groups during the exposure and were not treatment related. 

Page 156 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 32 of 91

Table 9: Adult Fish Survival in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured] 

Males Females 

n # Surviving % Survival n # Surviving % Survival 

Negative control (<LOQ) 8 8 100 16 15 94 

Solvent control (<LOQ) 8 8 100 16 15 94 

0.000302 8 8 100 16 15 94 

0.00314 8 8 100 16 16 100 

0.03651 NA NA NA NA NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.0000505 mg a.i./L 

1 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. 

Page 157 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 33 of 91

Table 10: Size at Test Termination in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured]

Body Weight Length 

Males Females Males Females 

n1

Mean

(g) 

±SD n1

Mean

(g) 

±SD n1

Mean

(mm) 

±SD n1

Mean

(mm) 

±SD

Negative control 

(<LOQ) 

4 4.73 0.742 4 1.91 0.200 4 57.6 3.35 4 44.4 1.49

Solvent control 

(<LOQ) 

4 4.03 0.386 4 1.96 0.208 4 53.5 1.07 4 44.4 2.01 

0.000302 4 4.26 0.269 4 1.82 0.119 4 55.2 1.32 4 43.9 0.76 

0.00314 4 4.64 0.405 4 1.89 0.182 4 56.1 1.53 4 43.7 1.66 

0.03652 NA NA NA NA NA NA NA NA NA NA NA NA 

Abbreviations: NA Not applicable. SD Standard deviation. 

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. 

Page 158 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 34 of 91

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] 

Fecundity1 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 98.84 0.52 

0.00314 24.2 8.39 98.7 0.83 

0.03653 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. 

 

Page 159 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 35 of 91

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 

n1 Median Tubercle 

Score n1 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 

0.03652 NA NA NA NA 

Page 160 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 36 of 91

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 

n2 Mean GSI1 (%) ±SD n2 Mean GSI1 (%) ±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 

0.03653 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. 

 

Page 161 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 37 of 91

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 Stage1 n Median Stage2

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 

0.03653 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. 

 

Page 162 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 38 of 91

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 Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of TestisOva 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hypertrophy/ 

Hyperplasia 

n Incidence2 n Incidence2 n Incidence2 n Incidence2

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 

Page 163 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 39 of 91

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of TestisOva 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hypertrophy/ 

Hyperplasia 

n Incidence2 n Incidence2 n Incidence2 n Incidence2

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 

0.03653 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, 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 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. 

Page 164 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 40 of 91

Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured] 

Additional Diagnostic Observations1

Severity

Decreased Proportion 

of Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence2 n Incidence2 n Incidence2 n Incidence2 n Incidence2

Negative control (<LOQ) 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

Solvent control (<LOQ) 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.000302 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

Page 165 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 41 of 91

Treatment 

(mg a.i./L) 

[TWA-measured] 

Additional Diagnostic Observations1

Severity

Decreased Proportion 

of Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence2 n Incidence2 n Incidence2 n Incidence2 n Incidence2

0.00314 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.03653 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

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, 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 no 

Page 166 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 42 of 91

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. 

Page 167 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 43 of 91

Table 17: Gonadal Histopathology in Female Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

Severity 

Increased Oocyte Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

n Incidence2 n Incidence2 n Incidence2

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 

0.03653 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. 

Page 168 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 44 of 91

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, 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 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 Observations1

Severity 

Interstitial Fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence2 n Incidence2 n Incidence2 n Incidence2

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 

Page 169 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 45 of 91

Treatment 

(mg a.i./L)

[TWA-measured] 

Additional Diagnostic Observations1

Severity 

Interstitial Fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence2 n Incidence2 n Incidence2 n Incidence2

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 

0.03653 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, 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 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. 

 

Page 170 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 46 of 91

Table 18b: Additional Gonadal Histopathology Observations in Female Fathead Minnows (Pimephales 

promelas). 

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observation1

Severity

Inflammation; 

Subacute to Chronic; 

multifocal

Edema; 

Interstitium; 

Bilateral

n Incidence2 n Incidence2

Negative control 

(<LOQ) 

0 15 8 15 14 

1 15 6 15 14 

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 

Page 171 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 47 of 91

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observation1

Severity

Inflammation; 

Subacute to Chronic; 

multifocal

Edema; 

Interstitium; 

Bilateral

n Incidence2 n Incidence2

0.03653

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, 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 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. 

 

Page 172 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 48 of 91

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 

n1 Mean

(mg/mL plasma) 

±SD n1 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 

0.03652 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./L 

Page 173 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 49 of 91

Plasma 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) 

Males Females Males Females 

n

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD

Negative control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

Solvent control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

0.000302 NA NA NA NA NA NA NA NA NA NA NA NA 

0.00314 NA NA NA NA NA NA NA NA NA NA NA NA 

0.0365 NA NA NA NA NA NA NA NA NA NA NA NA 

Abbreviations: NA Not applicable. SD Standard deviation. 

LOQ=0.0000505 mg a.i./L 

Page 174 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 50 of 91

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 terminated and removed from the study on exposure Day 11 and excluded 

from any subsequent statistical analysis. Excluding the highest treatment level, minimal signs of abnormal behavior and appea rance were observed during the exposure and were not treatment related. Overall, no concentration-dependent overt signs of toxicity were observed during the study except for the high treatment 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 notable effects were not mentioned, except for the one fish in the negative control which was reported as not having a very pronounced fatpad. 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 Termination 

Males Females 

Type n2 Incidence Type n2 Incidence 

Negative control 

(<LOQ) 

Fatpad not very pronounced 8 1 Injured pectoral fin 15 1 

Very thin 8 1 No notable effects 15 14 

No notable effects 8 6 

Page 175 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 51 of 91

Treatment 

(mg a.i./L) 

[TWA-measured] 

Secondary Sex Characteristics and Clinical Signs in Surviving Fish at Test Termination 

Males Females 

Type n2 Incidence Type n2 Incidence 

Solvent control (<LOQ) No notable effects 8 8 Slight darkening of body 15 1 

Bloated 15 1 

No notable effects 15 13 

0.000302 No notable effects 8 8 No notable effects 15 15 

0.00314 Loss of territorial aggressiveness 8 1 Slightly bloated 16 1 

No notable effects 8 7 Slight vertical banding present 16 1 

Aggressive behavior 16 1 

No notable effects 16 13 

0.03651 NA NA NA NA NA NA 

LOQ=0.0000505 mg a.i./L 1 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. 

2 n is the number of fish present at test termination, not replicates 

Page 176 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 52 of 91

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 

Page 177 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 53 of 91

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 

Page 178 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 54 of 91

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.

Page 179 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 55 of 91

Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with Benfluralin. 

Treatment 

(mg a.i./L) 

[TWA-measured] 

Tubercle Score GSI 

Gonadal Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p Effect? (Yes/No) % Diff. p % Diff. p % Diff. p 

Negative control 

(<LOQ) 

30 NA 0 NA No 0 NA NA NA NA NA

Solvent control 

(<LOQ) 

26 0.494 4.35 0.657 No 70.94 0.362 NA NA NA NA

0.000302 25 0.529 25.0 0.678 No -29.9 0.325 NA NA NA NA 

0.00314 30 0.976 27.2 0.029 No -24.8 0.443 NA NA NA NA 

0.03653 NA NA NA NA NA NA NA NA NA NA NA 

Statistical test Dunnett’s4 Mann-Whitney NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin.LOQ=0.0000505 mg a.i./L 1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathology 

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 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 Mean 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. 

Page 180 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 56 of 91

Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with Benfluralin. 

Treatment 

(mg a.i./L) 

[TWA-measured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2

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

Effect? 

(Yes/No) 

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

Negative control (<LOQ) 0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA 

Solvent control (<LOQ) -38.8 0.231 -0.10 0.748 0 NA -7.09 0.354 No4 -9.56 0.634 NA NA NA NA 

0.000302 -29.2 0.602 -0.35 0.662 0 NA -9.49 0.546 No4 -22.4 0.764 NA NA NA NA 

0.00314 -21.0 0.759 -0.45 0.476 0 NA 1.30 0.987 No 26.5 0.691 NA NA NA NA 

0.03653 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

Statistical test Dunnett’s Dunnett’s NA Dunnett’s NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG

Vitellogenin.

LOQ=0.0000505 mg a.i./L

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathology 

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 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 incidences of grade 4 oocyte atresia 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. The reason for this is not known, and the study authors did not think it was treatment-related. The replicates where this atresia was observed also had reduced fecundity.

Page 181 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 57 of 91

Table 24: Growth Endpoints1,2 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 

0.03653 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 

Page 182 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 58 of 91

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. 

Page 183 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 59 of 91

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 

Page 184 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 60 of 91

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.

Page 185 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 61 of 91

APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION: 

TABLE OF STATISTICAL INTERPRETATION: EDSP VS EFED METHODS

Endpoint Monotonic?1 Parametric? EDSP EFED Comments Female body weight 

NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body weight NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female body length 

NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body length NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female VTG NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male VTG NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female GSI NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male GSI NA No Mann-Whitney 

mid dose p=0.029 

Mann-Whitney mid dose p=0.029 

EDSP and EFED same conclusions 

Page 186 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

EPA MRID Number 48614906 

Page 62 of 91

Endpoint Monotonic?1 Parametric? EDSP EFED Comments Female tubercle score 

NA NA NA NA No score 

Male tubercle score 

NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Fecundity NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Fertility NA Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

F testosterone NA NA NA NA NA M testosterone NA NA NA NA NA F estradiol NA NA NA NA NA M estradiol NA NA NA NA NA 

1 Given that there were only two treatment groups available at test termination, the ability to make inferences about monotonicity and trends are 

limited. Therefore, the reviewer analyzed all data using non-trend tests. 

Page 187 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

Page 63 of 91

 

Page 188 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

Page 64 of 91

 

Page 189 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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 

Page 190 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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 

Page 191 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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) ) 

Page 192 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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 

Page 193 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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 

Page 194 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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 

Page 195 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

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 

Page 196 of 300
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 . . . . 

Page 197 of 300
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 

Page 198 of 300
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 . . . . . . 

Page 199 of 300
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 

Page 200 of 300
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 

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

Page 201 of 300
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 . . . . . . . . 

Page 202 of 300
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 

Page 203 of 300
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 

Page 204 of 300
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 . . . . 

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

Page 205 of 300
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 

Page 206 of 300
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 . . . . . , . 

Page 207 of 300
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 

Page 208 of 300
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 . . . 

Page 209 of 300
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 

Page 210 of 300
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 

 

Page 211 of 300
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 

Page 212 of 300
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 

Page 213 of 300
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 

Page 214 of 300
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 0.0209 

Page 215 of 300
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Benfluralin 

 EPA MRID Number 48614906 

Page 91 of 91

Control Comparisons 

% Mort. (all) 

% Mort. (male) 

% Mort. (female) Fecundity % Fert. 

Wt (male) 

Length (male) Wt. (female) Length (female) 

Neg Control 0 0 0 49.0 99.2 3.72 53.4 1.81 43.3 

0 0 0 23.9 99.0 4.86 57.3 1.78 43.2 0 0 0 13.2 99.1 5.51 61.6 2.21 46.4 

16.7 0 25 36.6 99.3 4.86 58.2 1.85 44.8 

Average 4.2 0.0 6.250 30.7 99.2 4.74 57.6 1.91 44.4 Solvent Control 0 0 0 26.9 99.3 4.16 53.3 2.16 45.9 

0 0 0 26.0 99.2 4.26 54.5 2.02 45.4 0 0 0 11.1 99.5 3.46 52.1 2.00 44.8 

16.7 0 25 11.0 98.2 4.26 54.2 1.67 41.4 

Average 4.2 0.0 6.250 18.8 99.1 4.04 53.5 1.96 44.4 t-test p value 1.000 #DIV/0! 1.000 0.231 0.748 0.145 0.060 0.741 0.970 Control Comparisons 

GSI (male) 

GSI (female) 

Tubercle (male) 

VTG (male) 

VTG (Female) 

GSI (male) 

GSI (female) 

Tubercle (male) 

VTG (male) 

VTG (Female) 

Neg Control 1.01 11.4 22.0 0.001010 159.55 1.01 11.4 22.0 0.001010 159.55 

1.32 15.6 30.0 0.000626 69.52 1.32 15.6 30.0 0.000626 69.52 1.22 14.1 31.0 0.001090 95.74 1.22 14.1 31.0 0.001090 95.74 1.07 15.2 30.5 0.000673 101.31 1.07 15.2 30.5 0.000673 101.31 

Average 1.2 14.1 28.4 0.000850 106.53 1.2 14.1 28.4 0.000850 106.53 Solvent Control 1.1 13.9 28.5 0.002110 94.08 1.1 13.9 28.5 0.002110 94.08 

1.3 12.7 31.0 0.002720 90.52 1.3 12.7 31.0 0.002720 90.52 1.3 13.2 24.0 0.000948 117.11 1.3 13.2 24.0 0.000948 117.11 1.1 12.5 21.0 0.000033 83.69 1.1 12.5 21.0 0.000033 83.69 

Average 1.2 13.1 26.1 0.001453 96.35 1.2 13.1 26.1 0.001453 96.35 t-test p value 0.657 0.354 0.494 0.362 0.634 0.657 0.354 0.494 0.362 0.634 

Page 216 of 300
DATA EVALUATION RECORD

BENFLURALIN

Study Type: OCSPP 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, 

Building 100, Suite B

Durham, NC 27713 

Primary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 11/15/2011

Secondary Reviewer: Signature:

Rebecca L. Bryan, B.S. Date: 11/15/2011

Program Manager: Signature:

Jack D. Early, M.S. Date: 11/15/2011

Quality Assurance: Signature:

Jack D. Early, M.S Date: 11/15/2011

Disclaimer

This Data Evaluation Record may have been altered by the Endocrine Disruptor Review Team 

subsequent to signing by Dynamac Corporation personnel. 

Page 217 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 218 of 300
Page 219 of 300
In vivo Hershberger Assay (2011) / Page 2 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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 14C-testosterone clearance experiment was conducted to examine if the decrease in 

androgen-responsive 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 via 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 subcutaneous injection. 14C-Testosterone was 

administered on Days 1 and 10 to measure plasma clearance of 14C-testosterone 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 collected 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, 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. 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 

performance 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 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) 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) 

Page 220 of 300
In vivo Hershberger Assay (2011) / Page 3 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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 criteria 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 14C-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≤0.05) 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 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 nonsignificant 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 14C-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 lower (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 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 positive for anti-androgenicity 

in the Hershberger assay as statistically significant decreases 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 overt toxicity at the dose that showed 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. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

Page 221 of 300
In vivo Hershberger Assay (2011) / Page 4 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

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

Assurance statements were provided.

Page 222 of 300
In vivo Hershberger Assay (2011) / Page 5 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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. Radiolabeled androgen for 

testosterone clearance exp.:

[

14C]-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.

 

Page 223 of 300
In vivo Hershberger Assay (2011) / Page 6 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

7. Test Animals:

Species: Rat

Strain: Sprague Dawley (Crl:CD[SD])

Age/weight at dose initiation: Post-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 14C-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: Tap water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

22±1oC

40-70% 

12-15/hr

12 h light/12 h dark

Acclimation period: Prior to castration: 5-6 days (animals castrated by Supplier on PND 45)

Post-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-life Dates: 

 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 assay was conducted to screen for both 

potential anti-androgenic and androgenic activity. The 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 subcutaneous 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 days and 

sacrificed approximately 24 hours after the final dose administration. On necropsy day, 

animals were anesthetized with isofluorane/O2 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 results of the main Hershberger assay, 

a testosterone clearance experiment was conducted to determine if benfluralin could enhance 

Page 224 of 300
In vivo Hershberger Assay (2011) / Page 7 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

testosterone metabolism by inducing hepatic enzymes resulting in increased TP clearance 

and decreased accessory sex tissue weights. 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 14C-testosterone by intravenous injection (approximately 13 

μg/kg). Unlabeled TP was administered s.c. on Days 2-9. To measure plasma clearance of 

14C-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 benfluralin-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 evaluated clinical signs, body weight and target 

organ weights as described for the Hershberger Assay.

 

3. Study Schedule: Sexually mature male Sprague Dawley rats were castrated on PND 45 by 

the supplier 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 64. Rats were euthanized on PND 65 approximately 24 hours after the last dose and 

necropsied for organ weight measurements.

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1 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. 

Page 225 of 300
In vivo Hershberger Assay (2011) / Page 8 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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

Vehicle 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

FT (oral gavage) (+ TP) 0 7 14C-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 study1 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 1000 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 suspending 

appropriate amounts of the test compound in corn oil. Flutamide and TP were prepared as 

solutions in corn oil. 14C-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. 

 

Page 226 of 300
In vivo Hershberger Assay (2011) / Page 9 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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 Administration: 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. 14C-Testosterone was administered intravenously via indwelling jugular 

vein cannulae on Days 1 and 10 at target doses of approximately 0.85 μCi/rat. The actual 

radioactive dose administered was determined gravimetrically. Doses 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 performed. 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 sequential method (Grubbs, 1969), and excluded if 

justified by sound scientific reasons. Descriptive statistics were used for the 

14C-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: All animals 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 body 

Page 227 of 300
In vivo Hershberger Assay (2011) / Page 10 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

weight gains (relative to Day 1) were calculated for Days 4, 7, and 11. For the 

14C-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, food consumption (g/rat/day) was measured for 

Days 1-4, 4-7, and 7-11. Food consumption was not measured in the 14C-testosterone 

clearance experiment. 

4. Serum Hormone Measurements: Serum from animals dosed with TP alone or TP and the 

test compound 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. 14C-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 

14C-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. 

Page 228 of 300
In vivo Hershberger Assay (2011) / Page 11 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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 14C-testosterone clearance assay, 6/7 rats dosed with 750 mg/kg/day had 

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

Day 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 controls; 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) cumulative body weight gains for 

Days 1-7 (↓69%) and Days 1-11 (↓57%). There were no effects on body weights or body 

Page 229 of 300
In vivo Hershberger Assay (2011) / Page 12 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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 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.

TABLE 3. Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen 

Assaya

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 presented 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 14C-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. Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the 14C-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 presented in parentheses.

N Number of animals in the group

SD= Standard Deviation

* Significantly different from controls; p≤0.05

Page 230 of 300
In vivo Hershberger Assay (2011) / Page 13 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

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 increased (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%).

Animals 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 14C-testosterone clearance study.

Page 231 of 300
In vivo Hershberger Assay (2011) / Page 14 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

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 presented 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 Number 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 increases (p≤0.05) 

as follows: 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 values 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 were less than the maximum 

recommended values indicated in the test guideline.

Page 232 of 300
In vivo Hershberger Assay (2011) / Page 15 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

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 weights 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 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 as follows: 51% in Cowper’s gland; 23% in glans penis; 36% in LABC; 

73% in ventral prostate; 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 previously 

discussed, the %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.

Page 233 of 300
In vivo Hershberger Assay (2011) / Page 16 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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 Number of animals in the group

SD Standard Deviation

%CV Percent Coefficient of Variation

* Significantly different from controls; p≤0.05

Page 234 of 300
In vivo Hershberger Assay (2011) / Page 17 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

Accessory sex organ weights and liver weights for the 14C-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 38% in this group. 

Accessory Sex Organ Weights (mg) and Liver Weights (g) in the 14C-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-related gross pathological findings. 

G. 14C-TESTOSTERONE CLEARANCE: Pharmacokinetic parameters for 

the 14C-testosterone clearance study are presented in Table 11. Rats treated for 10 days with 

750 mg/kg/day of the test compound cleared 14C-radioactivity 3.7-fold faster (p≤0.05) than 

the TP-only controls. This was a result of the elimination t1⁄2α 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 14C-Testosterone a

Parameters

Dose (mg/kg/day)

TP Control 750

N Mean SD N Mean SD

Day 1

Elimination t1⁄2 (h) 7 2.679 1.379 7 2.420 0.581

AUC0→t (μg 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 t1⁄2α (h)b 3 3.641 1.094 4 0.290* 0.056

Elimination t1⁄2β (h)b 3 --- --- 4 3.217 1.357

AUC0→t (μg 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 t1⁄2 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≤0.05

Page 235 of 300
In vivo Hershberger Assay (2011) / Page 18 of 19

BENFLURALIN/084301 OCSPP 890.1400/ 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. Based 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, the significant increase in liver weight, decrease in serum testosterone 

levels, enhanced clearance of 14C-testosterone-derived radioactivity at this dose level, 

coupled with previous toxicity and mode-of-action data, indicate that the anti-androgenic 

effects 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 750 mg/kg/day. Additionally at this dose, 

body 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 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%, 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 weights: glans penis (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 750 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. 

Page 236 of 300
In vivo Hershberger Assay (2011) / Page 19 of 19

BENFLURALIN/084301 OCSPP 890.1400/ OECD None

In the 14C-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 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 similar decreases (NS) in accessory sex organ weights as rats in the antiandrogen 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 38% in this group. Rats treated for 10 days with 750 mg/kg/day of the test compound 

cleared 14C-radioactivity 3.7-fold faster (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 positive was antiandrogenicity in the Hershberger assay as statistically significant decreases 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 

 

Page 237 of 300
DATA EVALUATION RECORD

BENFLURALIN (BENEFIN)

Study Type: OCSPP 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. 

Page 238 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 239 of 300
Female Pubertal Assay (2012)/ Page I of Error! Bookmark not defined. 

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-ethyl-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. 

Page 240 of 300
Female Pubertal Assay (2012)/ Page 2 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

Animals 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 largely 

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 mean ages at VO were 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 levels. 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 T4 levels were 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: decreased (p<0.05) alkaline phosphatase 

(↓16%) and increased (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, decreases (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 body 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. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

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

statements were provided. 

Page 241 of 300
Female Pubertal Assay (2012)/ Page 3 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

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

Location: Midland, MI

Study Director: C. L. Zablotny

Other Personnel: M.S. Marty, Ph.D., Lead Scientist

R. R. Hukkanen, D.V.M., Pathologist

Study Period: June 20, 2011, to January 25, 2012

2. Test Substance: Benfluralin

Description: Technical, molecular weight 335.3

Source: Dow AgroSciences, LLC, Indianapolis, IN

Lot/Batch #: Lot # ACD13683

Purity: 97.5 %

Stability: Not provided

CAS #: 1861-40-1

Structure:

N

N

+

N

+

O O

O

O

CH3

F

F

F

CH3

3. Vehicle: Corn oil

4. Test Animals:

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 MI

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. STUDY DESIGN

1. In-Life Dates: 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 using 

Page 242 of 300
Female Pubertal Assay (2012)/ Page 4 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / 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 this female pubertal assay.

3. Animal Assignment: 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 Selection Rationale: Prior to the start of the current assay, a probe study1 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 

female 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, respectively. 

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 were 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.

 

Page 243 of 300
Female Pubertal Assay (2012)/ Page 5 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / 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 Administration: 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 transformed) and aspartate 

aminotransferase (log transformed). Initial body weight, body weight gain, clinical 

chemistry, 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 (ANOVA). 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 (ANCOVA, alpha 

= 0.05) using body weight on PND 22 as the covariate. When 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 Clinical Examinations: Cageside examinations and observations for 

mortality and morbidity were performed on all animals at least twice daily. Hand-held 

clinical examinations were conducted each treatment day at approximately the same time 

following dosing (no additional details included in the report). 

2. Body Weight: 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 Opening (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. 

Page 244 of 300
Female Pubertal Assay (2012)/ Page 6 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

4. Estrous Cyclicity: 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 interval between VO and first 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 T4 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. T4 and 

TSH levels were analyzed using chemiluminescent immunoassay (T4) or radioimmunoassay 

(TSH).

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus X Total cholesterol

X Potassium X Globulins

X Sodium 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 Guideline 890.1450.

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination. 

UROGENITAL OTHER

XX Ovaries (paired, without oviducts)*+ XX Thyroid*+ (after fixation)

XX Uterus*+ (imbibed, with uterus & cervix) X Liver*

XX Kidneys (paired)*+ X Adrenals (paired)*

X Pituitary*

* Weights required based on Guideline 890.1450

+ Histopathological examination required based on guideline 890.1450

Page 245 of 300
Female Pubertal Assay (2012)/ Page 7 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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 weight). 

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 staining 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 was dissected from the 

trachea, processed into paraffin blocks, sectioned, and stained 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 Signs of Toxicity: All rats treated with benfluralin at 300 mg/kg/day were 

observed to have dark yellow/orange urine. It was stated that this is a common finding 

following dinitroaniline herbicide treatment. No other clinical signs were reported. 

C. General Growth and Vaginal Opening: 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 gain (p<0.05) of 15-42% for each interval up to 

PND 22-35. 

Page 246 of 300
Female Pubertal Assay (2012)/ Page 8 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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 differences from control, presented 

in parentheses, were calculated by the reviewers. 

* 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 body 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. Age 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 significantly affected 

at doses ≤75 mg/kg/day. All animals in the study achieved VO by PND 42.

Page 247 of 300
Female Pubertal Assay (2012)/ Page 9 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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/

SEb 

CV 

(%) N Mean

SD/ 

SEb 

CV 

(%) N Mean

SD/

SEb 

CV 

(%) N Meanb 

SD/

SEb 

CV 

(%)

Initial body weight

(PND 22; 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 deviation

SE Standard error of the mean

CV Coefficient of variation

* Significantly different from controls at p<0.05.

NA Not applicable

NR Not required

Page 248 of 300
Female Pubertal Assay (2012)/ Page 10 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

D. Organ Weights: 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 decreased (p<0.05) at 

300 mg/kg/day by 12-19%; at lower doses, pituitary weights 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 blotted 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 unaffected by treatment at doses 

≤75mg/kg/day. There were no effects of treatment on weights of kidneys, adrenals, thyroid 

or wet uterine at any dose level in this study. 

Page 249 of 300
Female Pubertal Assay (2012)/ Page 11 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

TABLE 4. Organ Weights at Necropsya

Organ

Vehicle Control Benfluralin

25 mg/kg/day)

Benfluralin

75 mg/kg/day

Benfluralin

300 mg/kg/day

N Mean

SD/ 

SEb 

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%) N Mean

SD/ 

SEb

CV 

(%)

Liver

(g) U 16 7.26 1.07 14.71 16 7.92 0.85 10.75 15 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 15 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 15 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 15 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, blotted (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 15 298.5 17.16 NA 16 231.0*

(↓21)

16.61 NA

Thyroid, fixed (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 reviewers. 

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.

Page 250 of 300
Female Pubertal Assay (2012)/ Page 12 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

E. Estrous Cyclicity: Estrous 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 the 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 was 0, 1, 2 

and 4 for the control, 25, 75 and 300 mg/kg/day groups, respectively. There were no intergroup 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 Cyclinga

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 

(75)

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 reviewers. 

N Number of animals examined

* Significantly different from controls at p<0.05.

F. Clinical Chemistry and Hormone Levels: Mean hormone levels are presented in Table 6. 

There were dose-dependent decreases (p<0.05) in serum T4 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 T4 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 

increased (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. 

Page 251 of 300
Female Pubertal Assay (2012)/ Page 13 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

TABLE 6. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated

Vehicle Control Benfluralin

25 mg/kg/day

Benfluralin

75 mg/kg/day

Benfluralin

300 mg/kg/day

N Mean SD

CV 

(%) N Mean SD

CV 

(%) N Mean SD

CV 

(%) N Mean SD

CV 

(%)

Hormones

Serum T4, 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 15 278 51 18 16 250*

(↓16%)

45 18

Alanine aminotransferase (U/L)b 16 55 8 15 16 59 9 15 15 59 6 11 16 57 12 21

Aspartate aminotransferase (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 0.05 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 pages 59 and 60 of the study 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 – Number of animals examined; SD – Standard deviation; SE – Standard error; and CV – Coefficient of variation

* Significantly different from controls at p<0.05.

Page 252 of 300
Female Pubertal Assay (2012)/ Page 14 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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; these 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 

Gradeb Incidence Gradeb 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

Benfluralin

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

Benfluralin

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 observed

E Number examined

NA Not applicable

Page 253 of 300
Female Pubertal Assay (2012)/ Page 15 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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 discordance between estrus staging 

(comparison of vaginal cytology and histopathology). One control animal (#6996) showed 

estrus histologically in both the ovary and uterus while vaginal cytology demonstrated 

diestrus. One 300 mg/kg/day animal (#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; however, uterine tissue for this animal was lost in 

processing. Finally, 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 Kidneya

Findings

Dose Level (# mg/kg bw/day)

Vehicle Control Benfluralin (25) Benfluralin (75) Benfluralin (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 examinedmissing 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 applicable, tissues not examined.

Page 254 of 300
Female Pubertal Assay (2012)/ Page 16 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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 than 

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 

interlaboratory validation study did not meet the performance criteria for terminal body 

weight and liver weight CVs. 

There was no treatment-related 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 were 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. Significant 

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 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. 

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 ages at VO were 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 T4 levels were decreased in a dose-dependent manner at ≥75 

Page 255 of 300
Female Pubertal Assay (2012)/ Page 17 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1450 / OECD None

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. Decreases in pituitary (unadjusted, adjusted, and 

relative) ovaries (unadjusted and adjusted), and blotted uterus (adjusted) weights 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 body 

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 T4 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%). 

Page 256 of 300
DATA EVALUATION RECORD

BENFLURALIN (BENEFIN) 

Study Type: OCSPP 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. 

Page 257 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 258 of 300
Male Pubertal Assay (2012)/ Page 1 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 08/2011

DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 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: pubertal 

development and thyroid function in intact juvenile/peripubertal male 

Crl:CD(SD) rats. Toxicology & Environmental Research and Consulting, The 

Dow Chemical Company, Midland, MI. Laboratory Study No.: 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 (MRID 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 (T4), and thyroid stimulating hormone (TSH) levels 

were analyzed using radioimmunoassay (RIA) or chemiluminescent immunoassay. 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 coagulating 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 adrenals, kidneys, and testes, and gross and 

microscopic examinations of the testes, epididymides, and kidneys. 

Systemic findings were observed at 400 mg/kg/day. Dark 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% 

Page 259 of 300
Male Pubertal Assay (2012)/ Page 2 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 15%. Serum alkaline phosphatase levels were 

decreased (p<0.05) by 40%, and serum 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 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%. 

At 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 T4 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 decrease 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 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%. Animals treated with 400 mg/kg/day had increases 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. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500).

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

statements were provided. 

Page 260 of 300
Male Pubertal Assay (2012)/ Page 3 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 1/25/2012

2. Test Substance: Benfluralin

Description: Technical, molecular weight 335.3

Source: Dow AgroSciences, LLC, Indianapolis, IN

Lot/Batch #: Lot # ACD13683

Purity: 97.5 %

Stability: Not provided

CAS #: 1861-40-1

Structure:

N

N

+

N

+

O O

O

O

CH3

F

F

F

CH3

3. Vehicle: Corn oil

4. Test Animals:

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 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. STUDY DESIGN

1. In-Life Dates: 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 age 

Page 261 of 300
Male Pubertal Assay (2012)/ Page 4 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

relative to birth date. Litters were culled to 10 pups on PND 4 using 5 males and 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 Assignment: 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 mates 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 Selection Rationale: Prior to the start of the current assay, a probe study1 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 Day 9 through Day 15. On PND 44, body weight 

decreases 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/kg/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. Liver weights were increased at all dose levels. Some 

hepatocellular 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 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 were 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 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 Dose 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 Chemical 

Company, Midland, MI.

 

Page 262 of 300
Male Pubertal Assay (2012)/ Page 5 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 variation

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: 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. Other 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 Clinical Examinations: Cageside examinations and observations for 

mortality and morbidity were performed on all animals at least twice daily. Hand-held 

clinical examinations were conducted each treatment day at approximately the same time

post-dosing (no additional details included in the report). 

2. Body Weight: 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 puberty 

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. Analyses for testosterone, T4, 

and TSH were conducted at WIL Research Laboratories, LLC (Ashland, Ohio). The rats 

were then euthanized by exsanguination followed by decapitation. A complete necropsy 

Page 263 of 300
Male Pubertal Assay (2012)/ Page 6 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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, T4, and TSH levels were analyzed using chemiluminescent immunoassay 

(testosterone and T4) or radioimmunoassay (TSH).

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus X Total cholesterol

X Potassium X Globulins

X Sodium 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 Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination.

Page 264 of 300
Male Pubertal Assay (2012)/ Page 7 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

UROGENITAL OTHER

XX Testes (left and right separately)*+ XX Thyroid*+

XX Epididymides (left and right separately)*+ X Liver*

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)*

X Ventral prostate* X Pituitary*

X Dorsolateral prostate*

X Levator ani-bulbocavernosus (LABC) muscle complex*

XX Kidneys (paired)*+

* Weights required based on Guideline 890.1500

+ Histopathological examination required based on Guideline 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 testes and epididymides were fixed in Bouin’s solution overnight 

for 24-72 hours; the 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 histopathological evaluation. The other tissues were fixed in 

10% buffered formalin for at least 24 hrs. Following fixation, the thyroid was 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 Signs 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 Growth and Preputial 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 days for the 0, 25, 100 and 400 mg/kg/day

groups respectively. There were no significant differences in either age or body weight at 

PPS in any of the treated groups. 

Page 265 of 300
Male Pubertal Assay (2012)/ Page 8 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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/

SEb 

CV 

(%) N Mean

SD/ 

SEb 

CV 

(%) N Mean

SD/

SEb 

CV 

(%) N Mean

SD/

SEb 

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 differences 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 23 

A Adjusted for body weight on PND 23 

N Number of animals examined

SD Standard deviation

CV Coefficient of variation

* Significantly different from controls at p<0.05.

NA Not applicable

-- The report states this is not required as part of the test guideline.

Page 266 of 300
Male Pubertal Assay (2012)/ Page 9 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 Weights: 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. At 25 mg/kg/day, 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 15% 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) 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%. 

Kidney, adrenals and testis weights were unaffected by treatment with benfluralin at any of 

the dose levels tested. 

Page 267 of 300
Male Pubertal Assay (2012)/ Page 10 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsya

Organ

Vehicle Control Benfluralin

25 mg/kg/day

Benfluralin

100 mg/kg/day

Benfluralin

400 mg/kg/day

N Mean SD/ SEb CV (%) N Meanc SD/ SEb CV (%) N Mean

SD/ 

SEb CV (%) N Mean SD/ SEb CV (%)

Liver

(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 0.04 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 27.20 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 NA

Page 268 of 300
Male Pubertal Assay (2012)/ Page 11 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsya

Organ

Vehicle Control Benfluralin

25 mg/kg/day

Benfluralin

100 mg/kg/day

Benfluralin

400 mg/kg/day

N Mean SD/ SEb CV (%) N Meanc SD/ SEb CV (%) N Mean

SD/ 

SEb CV (%) N Mean SD/ SEb 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

LABC

(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

Thyroid, 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 pages 59-61 of the study report. Percent differences from controls, 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 23 

A Adjusted for body weight on PND 23 

R Organ-to-body weight ratio (relative to body weight)

Page 269 of 300
Male Pubertal Assay (2012)/ Page 12 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

N Number of animals examined

SD Standard deviation

CV Coefficient of variation

NA Not applicable

* Significantly different from controls at p<0.05.

Page 270 of 300
Male Pubertal Assay (2012)/ Page 13 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / 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 total serum T4

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 serum 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. 

Page 271 of 300
Male Pubertal Assay (2012)/ Page 14 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 T4, 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

(↓52) 

[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 16 338*

(↓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 pages 57-58 of the study report. Percent differences 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 deviation

CV Coefficient of variation

* Significantly different from controls at p<0.05.

F. Gross and Histopathology: The incidences of histopathological findings of the thyroid 

Page 272 of 300
Male Pubertal Assay (2012)/ Page 15 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

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 increases in follicular cell height 

(increased in Grades 3 and 4) with a related decrease 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

Gradeb Incidence Gradeb 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 Number observed

E Number examined

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 treatmentrelated histopathological effects observed in the kidneys or testis of the control or treated 

animals.

Page 273 of 300
Male Pubertal Assay (2012)/ Page 16 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / OECD None

TABLE 7. Incidence of Histopathological Lesions of the Testes, Epididymides and Kidneya

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 applicable, tissues not examined.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: All mean values met the performance criteria 

except the thyroid and kidney mean weights. Mean kidney and thyroid control weights 

were consistent with the historical control data; 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. 

These 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). These variables were likely to have little or no impact on the outcome of 

the study. Mortality, body 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. 

GGT, 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 statistically different 

Page 274 of 300
Male Pubertal Assay (2012)/ Page 17 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / 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 epididymal 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, 

benfluralin 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 findings were observed at 400 mg/kg/day. Dark 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 15%. Serum alkaline phosphatase levels 

were decreased (p<0.05) by 40%, and serum 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%. 

At 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 T4 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 

Page 275 of 300
Male Pubertal Assay (2012)/ Page 18 of Error! Bookmark not defined.

BENFLURALIN/084301 OCSPP 890.1500 / 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, animals 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 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. 

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). 

Page 276 of 300
DATA EVALUATION RECORD

BENFLURALIN

Study Type: OCSPP 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. 

Page 277 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 278 of 300
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. Dimethyl 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 

:S30% between replicate wells within a plate and between 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. 

Page 279 of 300
Steroidogenesis Assay (2011) / Page 2 of 13

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

Based on the hormone responses in three independent runs, benfluralin treatment did not result in 

statistically significant and reproducible alterations in testosterone or estradiol production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay

(OCSPP 890.1550). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

Page 280 of 300
Steroidogenesis Assay (2011) / Page 3 of 13

BENFLURALIN/084301 OCSPP 890.1550/ 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

376.67

Page 281 of 300
Steroidogenesis Assay (2011) / Page 4 of 13

BENFLURALIN/084301 OCSPP 890.1550/ 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 D3-testosterone and 13C3-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 

chloride solution (2.0 mg/mL in acetone, freshly prepared). The vials containing the 

Page 282 of 300
Steroidogenesis Assay (2011) / Page 5 of 13

BENFLURALIN/084301 OCSPP 890.1550/ 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-APPIMS/MS) was used for 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 recovery 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 

proficiency. EC50 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% CO2

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 demonstrate the assay’s response to forskolin (an inducer 

Page 283 of 300
Steroidogenesis Assay (2011) / Page 6 of 13

BENFLURALIN/084301 OCSPP 890.1550/ 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 according to the schematic presented in Table 2. 

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

1 2 3 4 5 6

A Blankb Blank Blank Blank + MeOHc 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 

proliferation 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 Measurement 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, Ontario, 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 μM) 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 tritiated-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 production of the respective hormone)

Page 284 of 300
Steroidogenesis Assay (2011) / Page 7 of 13

BENFLURALIN/084301 OCSPP 890.1550/ 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 

follows:

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 distributed, 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 Wallis). 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 this 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%.

Page 285 of 300
Steroidogenesis Assay (2011) / Page 8 of 13

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations 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 

Significanceb 

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

FIGURE 1. Changes in Testosterone Production Relative to Benfluralin Concentration, 

Assay 1.

 * Significantly different from the solvent control at p ≤ 0.05.

Page 286 of 300
Steroidogenesis Assay (2011) / Page 9 of 13

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

FIGURE 2. Changes in Testosterone Production Relative to Benfluralin Concentration, 

Assay 2.

* Significantly different from the solvent control at p ≤ 0.05.

FIGURE 3. Changes in Testosterone Production Relative to Benfluralin Concentration, 

Assay 3.

* Significantly different from the solvent control at p ≤ 0.05.

Page 287 of 300
Steroidogenesis Assay (2011) / Page 10 of 13

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

FIGURE 4. Change in Estradiol Production Relative to Benfluralin Concentration, 

Assay 1. 

* Significantly different from the solvent control at p ≤ 0.05.

FIGURE 5. Change in Estradiol Production Relative to Benfluralin Concentration, 

Assay 2.

* Significantly different from the solvent control at p ≤ 0.05.

Page 288 of 300
Steroidogenesis Assay (2011) / Page 11 of 13

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

FIGURE 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 

acceptable. 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 the 

Page 289 of 300
Steroidogenesis Assay (2011) / Page 12 of 13

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

recommended 10 passages described in the guideline; however in Assay No. 3, the QC 

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 on 

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 μM 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 guideline 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 demonstrated 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 a 

Page 290 of 300
Steroidogenesis Assay (2011) / Page 13 of 13 

BENFLURALIN/084301 OCSPP 890.1550/ OECD None

1.5-fold cut-off criterion established in the validation program for this assay, and was not 

interpreted 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 μM with no 

evidence of precipitation 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-fold in two 

of three independent assays at 10 μM and 1.3-fold in one assay at 100 μM, but no doseresponse 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 pertinent staff 

involved in the study were not reported. 

• EC50 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. 

Page 291 of 300
DATA EVALUATION RECORD

BENFLURALIN (BENEFIN)

Study Type: OCSPP 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: 12/19/2011

Disclaimer

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

signing by Dynamac Corporation personnel. 

Page 292 of 300
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 293 of 300
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 

negative in the uterotrophic assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 

890.1600). 

Page 294 of 300
In vivo Uterotrophic Assay (2010/ Page 2 of 7 

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440

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

Quality Assurance statements were provided. 

Page 295 of 300
In vivo Uterotrophic Assay (2010/ Page 3 of 7 

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: 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 Estrogen: 17α-ethynyl estradiol (EE)

Supplier: Sigma-Aldrich (St. Louis, Missouri)

Lot/Batch #: 028K1411

Purity: Not provided

CAS # : 57-63-6

3. Solvent/Vehicle Control: 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 immature 

female rats.

Final concentration: Not applicable

4. Test Animals:

Species: Rat (females only)

Strain: Sprague-Dawley (Crl:CD[SD])

Age/weight at dose initiation: PND 19; 39.1-49.1 g

Source: Charles River Laboratories, Inc., Portage, Michigan

Housing: 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, ad libitum; low phytoestrogen with total genistein equivalents <325 μg/g 

diet.

Water: Tap water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

22±3oC

40-70% 

12-15/hr (average)

12 hrs light/12 hrs dark

Acclimation period:

7 days, immature females housed with their dam. 

Page 296 of 300
In vivo Uterotrophic Assay (2010/ Page 4 of 7 

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440

B. METHODS

1. In Life Dates: Start: 9/17/10 End: 9/20/10 

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 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. 

TABLE 1. Study Designa

Test Group 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

17α-ethynyl estradiol (EE), Reference Estrogen 10 μg/kg/day 6

a Data were obtained from Text Table 1 on page 14 of the study report.

4. Dose Selection Rationale: The dose levels in the present study were selected based on the 

results from a probe study1 conducted by the Performing Laboratory in which four female 

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). Female rats given 

250 mg/kg/day had 42% lower body weight gains during days 1 through 3; body weights 

were more affected 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 Preparation and Analysis: Dose formulations were prepared once prior to initiation 

of treatment by initially dissolving the appropriate 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 noted

 

Page 297 of 300
In 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 Dose 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 Administration: 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. Statistics: 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 (ANCOVA) was performed with terminal body weight as 

the covariate. If the ANCOVA 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 Weight: 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 

CO2 inhalation, euthanized by cervical dislocation, and subjected to a gross necropsy. 

Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the 

vagina was removed just below the cervix in order to retain the luminal fluid in the uterus. 

The “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 formalin for potential 

future examination. 

Page 298 of 300
In vivo Uterotrophic Assay (2010/ Page 6 of 7 

BENFLURALIN/084301 OCSPP 890.1600/ OECD 440

5. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: No clinical signs of toxicity were observed in 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, body 

weight gains (TD 1-4) were decreased by 30% compared to the controls. 

Table 2. Selected Group Body Weights and 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 μg/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 pages 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: Uterine 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. 

Page 299 of 300
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 Ratsa

Parameter

Dose (mg/kg/day)

Untreated 

Control

Vehicle 

Control Low (25) Mid (75) High (200)

Reference

Estrogen

EE 

(10 μg/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 Body weight

N No. of animals in the group; SD Standard Deviation

* Significantly different from controls at p<0.05.

2. Microscopic Examination (Optional): Microscopic 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 study. 

Benfluralin was negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None 

Page 300 of 300