## UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



## MEMORANDUM

Date: 6/29/2015

SUBJECT: 2,4-D: Data Evaluation Records (DERs) for EDSP Tier 1 Assays

PC Code: 030001


## Decision No.: NA
Petition No.: NA DP Barcode: D398637

, D398638, D398640 

## Registration No.: NA

Risk Assessment Type: NA

TXR 

## No.: 0052104


## MRID No.: See Table 

## Regulatory Action: NA
Case No.: NA
CAS No.: 94-75-7


## 40CFR: NA

FROM: Greg Akerman, Ph.D.

# ~?) 

# .,A-------

- Immediate Office
Health Effects Division (7509P)



## THROUGH: Jess Rowland

## c\c-a-~~ Deputy Director
Health Effects Division



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



## I. ACTION REQUESTED Ver .A pr. 2010



## The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects
Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted in response to the agency ' s Test Order for 2

, 4-D: Test Order# CON-030001-1.

Page 1 of2

### Page 2 of 2 

## II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for 2,4-D.

III

. MRID Table

Chemical: 2,4

- D PC Code: 030001
Guideline Assay MRID
890.1100 Amphibian Metamorphosis Assay (Frog) 48317002
890.1150 Androgen Receptor Binding (Rat Prostate) 48614301
890.1200 Aromatase Assay (Human Recombinant) 48614302
890.1250 Estrogen Receptor Binding 48614303
890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa

- 9903) 48614304

890.1350 Fish Short

- Term Reproduction 48317001
890.1400 Hershberger (Rat) NA
890.1450 Female Pubertal (Rat) NA
890.1500 Male Pubertal (Rat) NA
890.1550 Steroidogenesis (Human Cell Line

– H295R) 48614305
890.1600 Uterotrophic (Rat) NA NA= Not Applicable (Requirement satisfied by other scientifically relevant information (OSRI)).

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 1 of 61

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 388579

OECD Data Point 231

EPA MRID 48317002

EPA Guideline 890.1100

Amphibian Metamorphosis Assay (Frog)

Test Material: 2,4-Dichlorophenoxyacetic acid Purity (%): 98.6

Common Name 2,4-D

Chemical Name IUPAC

CAS Name

CAS No. 94-75-7

Synonyms 2,4-D

2,4-D acid

EPA PC Code 030001

Primary Reviewer: Catherine Aubee Signature:

Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 07/30/2012

Additional Reviewer: Alicia Korol Signature: No longer with EPA

Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 06/24/2011

Additional Reviewer: Anita Ullagaddi Signature: No longer with EPA

Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 0 2/24/2012

Final Additional Reviewer: Robin Sternberg Signature:

Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 05/27/2015

Date Evaluation Completed: 05/27/2015 Digitally signed by ROBIN

STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126

Date: 2015.06.01 13:09:51 -04'00' 

### 2015.06.01

13:15:13
-04'00'

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 2 of 61

DER Template Version: 22 September 2011 CITATION: Coady, K.K., T.A. Marino, and J. Thomas. November 29, 2010. 2,4-Dichlorophenoxyacetic Acid:

The Amphibian Metamorphosis Assay Using the South African Clawed Frog, Xenopus laevis. The Dow

Chemical Company. Midland, Michigan. Laboratory Project Study ID 101025. Sponsor: Industry Task Force

II on 2,4-D Research Data.

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 Inform ation (OSRI). In order to determine if a chemical has

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

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

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

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

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 3 of 61

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of 2,4-D (purity 98.6%) on amphib ian metamorphosis of South 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 a negative control and test chemical

nominal concentrations of 0.4, 4.0, 40.0, and 100.0 mg a.i./L. Mean measured concentrations were <0.120

(<LOQ, negative control), 0.273, 3.24, 38.0, and 113 mg a.i./L. The test system was maintained at 21.9 to

22.7 °C and a pH of 7.0 to 7.8.

Only one incidence of tadpole mortality occurred in the mid-low treatment group; the cause of death was

unknown. No clinical signs of toxicity were noted.

2,4-D did not affect Day 7 normalized (for snout-vent length) hind-limb length (HLL). However, there was a

statistically significant (p<0.05) decrease of 15% in Day 21 normalized HLL at the highest treatment level

compared with the negative control. There was no significant effect on median NF developmental stage, snoutvent length (SVL), or body weight at Day 7 or Da y 21. Asynchronous development was not observed. There

were no effects on thyroid gland histopathology. Late stage (>NF stage 60) tadpoles were observed in the

negative control and in all treatment levels; consistent with the guideline recommendations, these tadpoles

were excluded from analyses of growth and normalized HLL.

The study met all performance and validity criteria with the exception that the coefficients of variation (CVs)

for measured concentrations of the low and low-mid treatment groups were 56 and 22%, respectively,

exceeding the guideline performance criterion of ≤ 20%. This was likely due to biodegradation of the test

material in the test vessels.

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

Guideline 890.1100).

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 4 of 61

DER Template Version: 22 September 2011 Results Synopsis:

Test organism NF stage at test initiation: 51

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

Test type: Flow-through

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

### in the Amphibian

Metamorphosis Assay (AMA) with 2,4-D.

Treatment

(mg a.i./L)

[mean- measured] NF Developmental

Stage Hind Limb

Length 3 

### Asynchronous

Development Thyroid Gross

and

Histopathology

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

0.273 No No No No No No No

3.24 No No No No No No No

38.0 No No No No No No No

113 No No No Yes No No No

1 

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

unless otherwise specified.

2 

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

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

3 

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 5 of 61

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

Guideline Followed: This study was conducted following guidelines outlined in United States

Environmental Protection Agency (USEPA) 2009, Endocrine Disrupter

Screening Program Test Guidelines OPPTS 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. OECD231 Adopted 7 September 2009. The following deviations was

noted:

1. The CVs for measured concentrations of the low and low-mid treatment groups were 56 and 22%,

respectively, exceeding the guideline performance criterion of ≤ 20%. This was likely due to

biodegradation of the test material in the test vessels.

This deviation did not impact the interpretation of the study.

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

statements were provided. This study was conducted in compliance with the

following: European Community (EC) — European Parliament and Council

Directive 2004/1 0/EC (O.J. No. L 50/44, 20/02/2004); Organisation

for Economic Co-Operation and Development (OECD) – OECD Series on

Principles of Good Laboratory Practice and Compliance Monitoring, Number

1. OECD Principles on Good Laboratory Practice (as revised in 1 997)

ENV/MC/CHEM(98)17; and US Environmental Protection Agency -- FIFRA

GLPs Title 40 CFR, Part 160 - Federal Insecticide, Fungicide and

Rodenticide Act (FIFRA); Good Laboratory Practice Standards, Final Rule.

A. Test Material 2,4-Dichlorophenoxyacetic acid

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 6 of 61

DER Template Version: 22 September 2011 CAS No 94-75-7

Description:

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.: 2006 2433 8006-USA

Purity: 98.6%

Impurities: None reported

Stability of Compound: The CVs for mean-measured concentrations were 55, 22, 16, and 17% for

the low, low-mid, high-mid, and high concentrations, respectively. This was

likely due to biodegradation of the test material in the test vessels. Overall

recoveries were 68, 81, 95, and 113% of nominal for the low, low-mid, highmid, and high concentrations, respectively.

Storage Conditions of

Test Chemicals: Not reported. Aqueous stock solutions held in amber glassware or covered

during storage to prevent photodegradation.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page7of61 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:South African ClawedFrog 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

recommend ed 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):1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the EndocrineDisruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention(OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page8of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained as inhouse stock?YesBreeding pairs were ordered fromXenopus Express (Brooksville, Florida).Adult male and female frogs wereinjected with human chorionicgonadotropin at least twelve hoursbefore the desired breeding events. EPA recommends that larvae used in the assay be derived from in -house adults.Were parental acclimationconditions same as definitivetest?NoNot reportedAcclimation period for parentalfrogs (if applicable):daysDetails on parental feeding:Not reportedDetails on parental health:Not reportedData Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page9of61 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?NoNot reported EPA and OECD recommend that the best 2 - 3 individual spawns, with a minimum of

1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn (i.e.,the spawns are not comixed)Number of spawns evaluated (ifapplicable):Not reportedNumber of eggs sampled perspawn:Not reportedNF stage at test initiation51 EPA recommends that the definitive study be initiated with larvae at Nieuwkoop - Faber (NF) developmental stage 51 (17 days post-fertilization).Age at test initiation:18 days postfertilization (dpf)Test initiation began when tadpoleswere 16 days old (discrepancy)Mean total length at testinitiation (if reported):mmNot reportedRange of total length at testinitiation (if reported):mm : mmNot reportedData Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page10of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the optional size selectionmethod used?NoNot reportedDetails on larval selection:Not reportedLoading rate (rearing density):Not reported for larvae; 10 tadpoles/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow - through systems or 4 tadpoles/L in static - renewal exposure systems.Type of food:Sera Micron® 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 AmericaIodide concentration in diet (ifknown):53.7 ug/gData Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page11of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of feeding:2 times/day EPA recommends that feeding occur at least twice per day.Details on feeding regime:The feeding regimenwasthe same asguidelinerecommendations(Table 2). 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

increa se 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).Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page12of61 DER Template Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:flow-throughEPA recommends the use of a flowthrough system.Type of flow-through dilutionsystem (if applicable):continuous-flow dilutersystem Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):32 mL/min± 3 (complete volume turnoverapproximately every 2.7 hours for eachreplicate test vessel) Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).Details on toxicant mixing forflow-through systems (ifapplicable):Stainless steel mixingchamber was used.The dilution water flow and resulting testsolution volume in the mixing chamber wasmaintained using a stainless steel floatvalve. The test solution was gravity fedRecommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing;2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page13of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsfrom the mixing chamber and split equallyto replicate test vessels (contained in atemperature controlled water trough) via aTeflon manifold with Teflon delivery tubing.The diluter system was calibrated prior totest initiation, and diluter operation wasmonitored at least twice daily throughoutthe test. If there was an indication that thediluter calibration had changed (e.g.,diluter malfunction, change in water qualityor measured concentrations), calibration ofnecessary diluter components was checkedand adjusted as appropriate. 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy

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

hours.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page14of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration?No EPA recommends maintaining dissolved oxygen concentrations >40% air

saturation ( >3.5 mg/L) . Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on the tadpoles.Source of dilution water:natural waterWater was obtained from the upperSaginaw Bay of Lake Huron off WhitestonePoint by the City of Midland WaterTreatment Plant and supplied to The DowChemical Company prior to municipaltreatment for human consumption. Waterwas limed and flocculated with ferricchloride, sand-filtered, pH-adjusted withgaseous CO2, carbon-filtered, and UVirradiated. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants, including known

substrates of the iodine transporter of the thyroid gland (e.g., fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page15of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesLaboratory dilution water is routinelyanalyzed for pesticides, organics, metals,and other inorganics twice per year. Inparticular, chlorate, perchlorate, and iodidelevels are also determined.Iodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 μ g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g. in Sera Micron).Test vessel type/materials:Glass sealed togetherwith clear siliconeadhesiveEPA 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).Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page16of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel size:30 x 14.5 x 20 cm deepFill volume:5.2 LAdditional details on exposuresystem:Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3)64 76 67 7days EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH7.07.87daysEPA recommends pH 7.5 ± 1, interreplicate and inter - treatment differentials should not exceed 0.5.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page17of61 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsDissolved oxygen (mg/L)5.3 7.9 7days EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration

>3.5 mg/L (>40% air saturation).Temperature21.922.5 7days EPA recommends temperature 22±1 o C;

inter -replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide<LOQ<LOQ EPA recommends aquatic iodide range

0.5 - 10 μ g/L (supplemental iodide should not exceed 2 μ g/L).Ammonia<0.10.244 months 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 qualityFluoride0.10.1Perchlorate<0.2<0.2Chlorate<10<10Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page18of61 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline Recommendations 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.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page19of61 DER Template Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?NoIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?NA EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever concentration is lowest.Species:NALife stage:NATest duration:NAAdditional details:NAData Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page20of61 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive test:The solubility limit andother reference studieswere used to select thehighest testconcentration. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):Morgan et al., 1996;Palmer and Krueger,1997a; Palmer andKreuger, 1997b;Alexander et al., 1985Separation of testconcentrations:0.1-0.4 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page21of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of test concentrations:4 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?No"Standards were not corrected forpurity." (Appendix B)Indicate the type of valuespresented for measuredconcentrations:Average measuredLimit of quantification (LOQ):0.120 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement:7 days It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week thereafter.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page22of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable): EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of larvae per treatmentat test initiation:80Was a solvent used?NoSolvent type (if applicable):Maximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends3Hutchinson 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.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page23of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12

(light:dark).Light intensity at water'ssurface:0.618-0.882 Klux EPA recommends light intensity 0.6 - 2 Klux (at water's surface).4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications.Series on Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page24of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:Information on test solution appearancedid not appearto be reported in thereport.Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with 2,4-D.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQ EPA and OECD recommend that test item concentrations be maintained at a coefficient
of variation (CV)≤ 20%.Treatment 10.40.27355.67Treatment 243.2421.60Treatment 34038.016.29Treatment 410011316.90Abbreviations:CVCoefficient of variation.LOQ = 0.120 mg a.i./LData Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page25of61 DER Template Version: 22 September 2011E. ObservationsBiological Endpoints:Mortality (throughout study); NF stage and asynchronous development; wet weight, snout-vent length, hind limb length (Day7 and 21); thyroid histopathology (Day 21)Were raw (individual) data provided?Yes EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (D ays 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-base d larval selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also incl udes thyroid gross

pathology observations.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 26 of 61

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

A. Results

Only one larvae in the 3.24 mg a.i./L treatment level died during the 21-day study.

Table 9: Larval Mortality in South African Clawed Frog.

Treatment (mg a.i./L)

[mean-measured] Larval Mortality

Day 7 1 

### Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 0 0 60 0 0

0.273 80 0 0 60 0 0

3.24 80 0 0 60 1 1.7

38.0 80 0 0 60 0 0

113 80 0 0 60 0 0

1 

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 27 of 61

DER Template Version: 22 September 2011 Day 7 median NF stage was 54 for the negative control and all treatment levels. Day 21 median NF

stage was 58 for the highest treatment level and 59 for the negative control and all other treatment levels.

There were no asynchronous tadpoles.

Table 10: Larval Development in South African Clawed Frog- Developmental Stage and Asynchronous

Development.

Treatment (mg a.i./L)

[mean-measured] Developmental Stage

Day 7 Day 21

n Median

Stage 1 

### # Asynchronous n Median

Stage 1 

### # Asynchronous

Negative Control 4 54 0 4 59 0

0.273 4 54 0 4 59 0

3.24 4 54 0 4 59 0

38.0 4 54 0 4 59 0

113 4 54 0 4 58 0

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 28 of 61

DER Template Version: 22 September 2011 Day 7 HLL ranged from 2.2 to 2.4 mm across the control and all treatment levels. Day 21 HLL ranged from

16.2 mm in the high treatment group to 18.0 mm in the low treatment group.

Table 11: Larval Development in South African Clawed Frog - Hind Limb Length.

Treatment

(mg a.i./L)

[mean-measured] Hind Limb Length (HLL)

Day 7 Day 21

n Mean

(mm) ±SD HLL:

SVL 1 n Mean

(mm) ±SD HLL:

SVL 1



### Negative Control 4 2.4 0.2 0.1 4 17.2 0.8 0.7

0.273 4 2.2 0.1 0.1 4 18.0 1.0 0.7

3.24 4 2.3 0.3 0.1 4 17.1 0.9 0.7

38.0 4 2.2 0.1 0.1 4 17.5 1.0 0.7

113 4 2.2 0.1 0.1 4 16.2 1.0 0.6

Abbreviations: SD 

### Standard deviation.

In this table, “n” represents the number of independent replicates per treatment level.

1 

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 29 of 61

DER Template Version: 22 September 2011 Day 7 SVL ranged from 18.00 to 18.5 mm and Day 21 SVL ranged from 26.9 to 28.1 mmm across the

negative control and all treatment levels. Day 7 body weight ranged from 0.48 g to 0.53 g and Day 21 body

weight ranged from 1.63 g to 1.91 g acorss the negative control and all treatment levels.

Table 12: Larval Growth in South African Clawed Frog.

Treatment

(mg a.i./L)

[measured] Snout-Vent Length (SVL) Body Weight 1



### Day 7 Day 21 Day 7 Day 21

n Mean

(mm) ±SD n Mean

(mm) ±SD n Mean

(g) ±SD n Mean

(g) ±SD



### Negative

Control 4 18.5 0.5 4 26.8 1.4 4 0.53 0.04 4 1.63 0.17

0.273 4 18.0 0.5 4 27.4 0.6 4 0.48 0.04 4 1.73 0.12

3.24 4 18.3 0.6 4 26.9 1.6 4 0.49 0.05 4 1.63 0.24

38.0 4 18.1 0.2 4 27.7 0.31 4 0.49 0.02 4 1.75 0.06

113 4 18.4 0.3 4 28.1 0.40 4 0.50 0.04 4 1.91 0.22

Abbreviations: SD 

### Standard deviation.

In this table, “n” represents the number of independent replicates per treatment level.

1 

### Also referred to as "wet weight" in the test guideline.

There were no treatment-related histopathologic changes in the thyroid gland in any of the treatment

groups. There was no evidence of glandular atrophy or hypertrophy or follicular cell hyperplasia in any of

the thyroid glands examined across all treatment groups. The incidence of tall columnar cells lining the

follicles (follicular cell hypertrophy) did not show any treatment-related differences and was interpreted to

be within normal limits at all concentrations of 2,4-D.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 30 of 61

DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in South African Clawed Frog.

Treatment

(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity 

### Thyroid Gland

Hypertrophy Thyroid Gland

Atrophy Follicular Cell

Hypertrophy Follicular Cell

Hyperplasia

n Incidence n Incidence n Incidence n Incidence



### Negative

Control 0 20 20 20 20 20 5 20 20

1 20 0 20 0 20 15 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.273 0 20 20 20 20 20 3 20 20

1 20 0 20 0 20 17 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

3.24 0 20 20 20 20 20 6 20 20

1 20 0 20 0 20 14 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

38.0 0 20 20 20 20 20 5 20 20

1 20 0 20 0 20 15 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

113 0 20 20 20 20 20 4 20 20

1 20 0 20 0 20 16 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.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page31of61 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in South African Clawed Frog.Treatment(mg a.i./L)[time-weighted,mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA0.2730NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA3.240NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANAData Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page32of61 DER Template Version: 22 September 2011Treatment(mg a.i./L)[time-weighted,mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence38.00NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA1130NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANAAbbreviations:NANot applicable.1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 33 of 61

DER Template Version: 22 September 2011 No abnormal behaviors or clinical signs of toxicity were noted among control or 2,4-D exposed tadpoles.

Table 15: Clinical Signs in Xenopus laevis

.

Treatment

(mg a.i./L)

[TWA- measured] Clinical Signs 1



### Type n Incidence

Negative Control None NA NA

Solvent Control None NA NA

0.00264 None NA NA

0.0262 None NA NA

0.273 None NA NA

Abbreviations: NA 

### Not applicable.

1 Note that asynchronous development (unable to stage) is reported previously in Table 10 and not here.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 34 of 61

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

There were no signs of overt toxicity among exposed tadpoles in the present study. Throughout the

entire exposure period, there was only one incidence of tadpole mortality. The study author identified

no indications of developmental delay or abnormal behavior and therefore concluded that concentrations

of 2,4-D used in the present study (up to 113 mg/L 2,4-D) were not overtly toxic to developing X.

laevis . There were also no signs of advanced development (as measured by developmental stage and

hind limb length) or asynchronous development among 2,4-D exposed tadpoles relative to control

tadpoles on either Day 7 or Day 21 of exposure. Finally, compared to thyroid glands from controls,

there were no significant histopathological effects observed among thyroid glands from 2,4-D exposed

tadpoles.

C. Reviewer's Analysis and Conclusions

Statistical Methods: Day 21 values for NF developmental stage and normalized HLL were consistent

with a monotonic response and were analyzed using the Jonckheere-Terpstra test. Values for Day

7 SVL and Days 7 and 21 body weight showed no apparent monotonic response and met the

assumptions of normality and homogeneity of variance; therefore, these endpoints were analyzed using

the parametric Dunnett’s test. Day 7 NF stage and Day 21 SVL were inconsistent with a monotonic

response and were analyzed with the non-parametric Mann-Whitney test. All analyses were performed

using the FROG program with Statistical Analysis Software (SAS v. 9.3).

Late stage (> NF 60) tadpoles were excluded from analyses of SVL, body weight, and normalized

HLL. Late stage tadpoles were excluded from every treatment level: control-6 tadpoles, 0.273 mg

a.i./L-10 tadpoles, 3.25 mg a.i./L-6 tadpoles, 38.0 mg a.i./L-7 tadpoles, 113 mg a.i./L-6 tadpoles.

Histopathology results were evaluated visually based on severity and incidence data, within the context

of the narrative pathology report.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 35 of 61

DER Template Version: 22 September 2011 Conclusions:

The only statistically-significant effect was for Da y 21 HLL which was decreased by 15% at the high

treatment level compared to the negative control after excluding "late stage" tadpoles from the analysis.Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis AssayEPA MRID Number 48317002Page36of61 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with 2,4-D.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21MedianpMedianp%Diff.p%Diff.p%Diff.p%Diff.pTreatment-RelatedEffects? (Yes/No)Negative Control54NA59NANANANANANANANANANA0.273540.48590.750NA-3.70.250NA0NANo3.2454>0.99590.940NA-3.70.250NA0NANo38.054>0.99590.590NA-3.70.260NA0NANo11354>0.99580.170NA-14.80.020NA0NANoStatistical TestMann-WhitneyJonckheereTerpstraNAJonckheere-TerpstraNA NA NAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Negative values indicate reductions or decreases relativeto the negative control. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Hind-limb length is normalized to snout-vent length (SVL).

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 37 of 61

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

### in the AMA with 2,4-D.

Treatment

(mg a.i./L)

[mean-measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

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

Diff. p

Negative Control NA NA NA NA NA NA NA NA

0.273 -2.8 0.31 2.3 0.78 -9.0 0.32 6.3 0.82

3.24 -1.1 0.92 0.3 >0.99 -6.3 0.61 -0.8 >0.99

38.0 -2.2 0.53 3.4 0.78 -7.2 0.50 7.9 0.68

113 -0.7 0.98 4.9 0.24 -5.8 0.68 17.5 0.10

Statistical Test Dunnett’s Mann-Whitney 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

The coefficients of variation for the measured concentrations of the 0.273 and 3.24 mg a.i./L treatment

groups exceeded the guideline performance criterion of 20%. This was likely due to biodegradation of

the test material in the test vessels. All other validity and performance criteria were met.

F. Reviewer's Comments

Unlike the study author, the reviewer detected a statistically significant decrease in Day 21 normalized

HLL (p<0.05) at the highest treatment level after excluding "late stage" tadpoles from the analysis.

No other signs of toxicity were observed at this or any other treatment level. Late stage tadpoles were

excluded from every treatment level: control-6 tadpoles, 0.273 mg a.i./L-10 tadpoles, 3.25 mg a.i./L6 tadpoles, 38.0 mg a.i./L-7 tadpoles, 113 mg a.i./L-6 tadpoles. Late stage tadpoles were included

in the analysis of potential effects on developmental stage.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 38 of 61

DER Template Version: 22 September 2011 The individuals selected for histopathology were not stage-matched to the median stage in the controls.

However, there were no noteworthy differences between histopathology observations in control and

treatment specimens; therefore, this guideline deviation does not appear to have substantively affected

the interpretation of results.

III. REFERENCES

Alexander, H. C., Gersich, F. M., and Mayes, M. A. (1985). Acute Toxicity of Four Phenoxy Herbicides

to Aquatic Organisms. Bulletin of Environmental Contamination and Toxicology 35, 314-321.

ASTM. (2004). Standard Guide for Conducting the Frog Embryo Teratogenesis Assay - Xenopus (FETAX).

E 1439-98

ASTM. (2007). Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes,

Macroinvertebrates, and Amphibians. American Society for Testing and Materials, ASTM £729-

96(2002), Philadelphia, Pennsylvania.

Cooke, A. S. (1972). The effects of DDT, Dieldrin , and 2,4-D on amphibian spawn and tadpoles. Environ.

Pollut. 3, 51-68.

ECOTOX database. 2009. Search of the United States Environmental Protection Agency's ECOTOX

database on CAS# 94757 and Pimephales promelas conducted on 12/17/09;

http://cfpub.epa.gov/ecotox/.

Hansch C, Leo A, and Hoekman D. (1995). Exploring QSAR Hydrophobic, Electronic and Steric Constants,

ACS, Washington, D.C.

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 39 of 61

DER Template Version: 22 September 2011 Hopkins, D. L. (1987). 2,4-Dichlorophenoxyacetic Acid: Determination of the water solubility. Unpublished

report of The Dow Chemical Company. Laboratory Report No: ES-994. November 25, 1987. MRID

No. 41332002.

Lezotte F., Van Hoven R., and Nixon, W. (2002). Determination of Water Solubility of 2,4- D Acid by the

Shake Flask Method: Lab Project Number: 467C-103: 467/121801/7840/SUB467. Unpublished

study prepared by Wildlife International, Ltd. MRID No. 45692501.

Morgan, M. K., Scheuerman, P. R., Bishop, C. R., and Pyles, R. A. (1996). Teratogenic potential of

atrazine and 2,4-D using FETAX. Journal of Toxicology and Environmental Health 48, 151-168.

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

New York

Organization for Economic Cooperation and Development (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.

Organization for Economic Cooperation and Development OECD. (2007). Guidance Document on

Amphibian Thyroid Histology. Environmental Health and Safety Publications. Series on Testing and

Assessment. No. 82. Paris

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.

Palmer, S. J. and Krueger, H. O. (1997a). 2,4-D Dimethylamine salt: A 96-hour static acute toxicity test

with the leopard frog tadpoles (Ranapipiens). Study ID 467-103. Wildlife International Ltd., Easton,

Maryland. MRID No. 44517306.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 40 of 61

DER Template Version: 22 September 2011 Palmer, S. J. and Krueger, H. O. (1997b). 2,4-D (2,4-Dichlorophenoxyacetic acid): A 96-hour static

acute toxicity test with the leopard frog tadpoles (Ranapipiens). Study ID 467-102. Wildlife International

Ltd., Easton, Maryland. MRID No. 44517307.

Sinton, G. L., Fan, L. T., Erickson, L. E., and Lee, S. M. (1986). Biodegradation of 2,4-D and related

xenobiotic compounds. Enzyme Microb. Technol. 8, 395-403.

United States Environmental Protection Agency (USEPA) (2005). Reregistration Eligibility Decision for

2,4-D. EPA 738-R-05-002, June 2005.

United States Environmental Protection Agency (USEPA) (2009). Endocrine Disrupter Screening Program

Test Guidelines OPPTS 890.1100: Amphibian Me tamorphosis (Frog). EPA 740-C-09-002, October

2009.

Wo C. (2008). Certificate of Analysis for 2,4-D Acid, Lot# 2006 2433 8006-USA. PSL Reference No.

081210-2H. Eurofins [Product Safety Laboratories, Dayton, New Jersey.

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 41 of 61

DER Template Version: 22 September 2011 Appendix I: Output of Reviewer’s Statistical Analysis

test for amphib metamorph screen study - 2,4-d 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
0.909 0.061 0.655 0.632 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.53 0.04 0.02 8.03 0.46, 0.59 Dose1 4 0.48 0.04 0.02 8.86 0.41, 0.55 Dose2 4 0.49 0.05 0.02 9.97 0.41, 0.57 Dose3 4 0.49 0.02 0.01 4.57 0.45, 0.52 Dose4 4 0.50 0.04 0.02 8.41 0.43, 0.56

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

Ctrl 0.52 0.48 0.58 . .

Dose1 0.47 0.44 0.54 90.97 9.03

Dose2 0.47 0.46 0.57 93.68 6.32

Dose3 0.49 0.46 0.51 92.78 7.22

Dose4 0.49 0.46 0.54 94.25 5.75

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

4 15 0.78 0.556

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

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

Ctrl 0.53 . 0.53 . . . . . .

Dose1 0.48 0.320 0.49 0.127 . . . . .

Dose2 0.49 0.611 0.49 0.135 0.986 . . . .

Dose3 0.49 0.504 0.49 0.139 0.997 1.000 . . .

Dose4 0.50 0.680 0.49 0.142 0.973 1.000 0.999 . .

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 42 of 61

DER Template Version: 22 September 2011 Degrees of Freedom TestStat P-value
4 3.62 0.460

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 0.52 . .

Dose1 0.47 0.235 0.074

Dose2 0.47 0.233 0.120

Dose3 0.49 0.230 0.200

Dose4 0.49 0.494 0.275

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

4 15 0.78 0.556

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

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

Ctrl -0.53 . -0.50 . . . . . .

Dose1 -0.48 0.320 -0.50 0.906 . . . . .
Dose2 -0.49 0.611 -0.50 0.927 0.986 . . . .

Dose3 -0.49 0.504 -0.50 0.937 0.997 1.000 . . .

Dose4 -0.50 0.680 -0.50 0.943 0.973 1.000 0.999 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 3.62 0.460

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 -0.52 . .

Dose1 -0.47 0.235 0.926

Dose2 -0.47 0.233 0.880

Dose3 -0.49 0.230 0.800

Dose4 -0.49 0.494 0.725

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 43 of 61

DER Template Version: 22 September 2011 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for amphib metamorph screen study - 2,4-d 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

0.509 <.001 9.000 <.001 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 54.00 0.00 0.00 0.00 . , . Dose1 4 53.75 0.50 0.25 0.93 52.95, 54.55 Dose2 4 54.00 0.00 0.00 0.00 . , . Dose3 4 54.00 0.00 0.00 0.00 . , . Dose4 4 54.00 0.00 0.00 0.00 . , .

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

Ctrl 54.00 54.00 54.00 . .

Dose1 54.00 53.00 54.00 99.54 0.46

Dose2 54.00 54.00 54.00 100.00 0.00

Dose3 54.00 54.00 54.00 100.00 0.00

Dose4 54.00 54.00 54.00 100.00 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

4 15 1.00 0.438

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 54.00 . 54.00 . . . . . .

Dose1 53.75 0.357 53.94 0.415 . . . . .

Dose2 54.00 1.000 53.94 0.443 0.530 . . . .

Dose3 54.00 1.000 53.94 0.458 0.530 1.000 . . .

Dose4 54.00 1.000 53.94 0.468 0.530 1.000 1.000 . .

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 44 of 61

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
4 4.00 0.406

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 54.00 . .

Dose1 54.00 0.478 0.159

Dose2 54.00 1.000 0.500

Dose3 54.00 1.000 0.673

Dose4 54.00 1.000 0.760

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

4 15 1.00 0.438

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 -54.00 . -53.88 . . . . . .

Dose1 -53.75 0.357 -53.88 0.855 . . . . .

Dose2 -54.00 1.000 -54.00 0.618 0.530 . . . .

Dose3 -54.00 1.000 -54.00 0.636 0.530 1.000 . . .

Dose4 -54.00 1.000 -54.00 0.648 0.530 1.000 1.000 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 4.00 0.406

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 -54.00 . .

Dose1 -54.00 0.478 0.841

Dose2 -54.00 1.000 0.500

Dose3 -54.00 1.000 0.327

Dose4 -54.00 1.000 0.240

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 45 of 61

DER Template Version: 22 September 2011 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for amphib metamorph screen study - 2,4-d 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
0.977 0.890 0.425 0.788 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 18.53 0.54 0.27 2.90 17.67, 19.38 Dose1 4 18.00 0.49 0.24 2.72 17.22, 18.78 Dose2 4 18.33 0.56 0.28 3.03 17.44, 19.21 Dose3 4 18.13 0.21 0.10 1.14 17.80, 18.45 Dose4 4 18.40 0.29 0.15 1.60 17.93, 18.87

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

Ctrl 18.60 17.80 19.10 . .

Dose1 18.00 17.40 18.60 97.17 2.83

Dose2 18.20 17.80 19.10 98.92 1.08

Dose3 18.15 17.90 18.30 97.84 2.16
Dose4 18.40 18.10 18.70 99.33 0.67

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

4 15 0.92 0.476

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 18.53 . 18.53 . . . . . .

Dose1 18.00 0.305 18.21 0.198 . . . . .

Dose2 18.33 0.915 18.21 0.211 0.831 . . . .

Dose3 18.13 0.528 18.21 0.218 0.994 0.965 . . .

Dose4 18.40 0.983 18.21 0.223 0.703 0.999 0.898 . .

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 46 of 61

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
4 3.39 0.494

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 18.60 . .

Dose1 18.00 0.332 0.117

Dose2 18.20 0.673 0.328

Dose3 18.15 0.341 0.254

Dose4 18.40 0.887 0.580

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

4 15 0.92 0.476

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 -18.53 . -18.24 . . . . . .

Dose1 -18.00 0.305 -18.24 0.881 . . . . .

Dose2 -18.33 0.915 -18.24 0.905 0.831 . . . .

Dose3 -18.13 0.528 -18.24 0.917 0.994 0.965 . . .

Dose4 -18.40 0.983 -18.40 0.803 0.703 0.999 0.898 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 3.39 0.494

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 -18.60 . .

Dose1 -18.00 0.332 0.883

Dose2 -18.20 0.673 0.672

Dose3 -18.15 0.341 0.746

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 47 of 61

DER Template Version: 22 September 2011 Dose4 -18.40 0.887 0.420

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for amphib metamorph screen study - 2,4-d 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
0.949 0.355 4.063 0.020 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 2.35 0.19 0.10 8.15 2.05, 2.65 Dose1 4 2.18 0.05 0.03 2.30 2.10, 2.25 Dose2 4 2.25 0.26 0.13 11.76 1.83, 2.67 Dose3 4 2.20 0.08 0.04 3.71 2.07, 2.33 Dose4 4 2.23 0.05 0.02 2.25 2.15, 2.30

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

Ctrl 2.40 2.10 2.50 . .

Dose1 2.20 2.10 2.20 92.55 7.45

Dose2 2.20 2.00 2.60 95.74 4.26
Dose3 2.20 2.10 2.30 93.62 6.38

Dose4 2.20 2.20 2.30 94.68 5.32

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

4 15 0.77 0.561

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

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

Ctrl 2.35 . 2.35 . . . . . .

Dose1 2.18 0.343 2.21 0.134 . . . . .

Dose2 2.25 0.768 2.21 0.143 0.956 . . . .

Dose3 2.20 0.471 2.21 0.147 0.999 0.990 . . .

Dose4 2.23 0.618 2.21 0.150 0.990 0.999 0.999 . .

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 48 of 61

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
4 2.48 0.648

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 2.40 . .

Dose1 2.20 0.270 0.089

Dose2 2.20 0.673 0.185

Dose3 2.20 0.335 0.207

Dose4 2.20 0.399 0.326

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

4 15 0.77 0.561

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 -2.35 . -2.24 . . . . . .

Dose1 -2.18 0.343 -2.24 0.901 . . . . .

Dose2 -2.25 0.768 -2.24 0.923 0.956 . . . .

Dose3 -2.20 0.471 -2.24 0.933 0.999 0.990 . . .

Dose4 -2.23 0.618 -2.24 0.939 0.990 0.999 0.999 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 2.48 0.648

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -2.40 . .

Dose1 -2.20 0.270 0.911

Dose2 -2.20 0.673 0.815

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 49 of 61

DER Template Version: 22 September 2011 Dose3 -2.20 0.335 0.793

Dose4 -2.20 0.399 0.674

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for amphib metamorph screen study - 2,4-d 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

. . . . NO DATA FOR TEST

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.10 0.00 0.00 0.00 . , . Dose1 4 0.10 0.00 0.00 0.00 . , . Dose2 4 0.10 0.00 0.00 0.00 . , . Dose3 4 0.10 0.00 0.00 0.00 . , . Dose4 4 0.10 0.00 0.00 0.00 . , .

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

Ctrl 0.10 0.10 0.10 . .

Dose1 0.10 0.10 0.10 100.00 0.00
Dose2 0.10 0.10 0.10 100.00 0.00

Dose3 0.10 0.10 0.10 100.00 0.00

Dose4 0.10 0.10 0.10 100.00 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

. . . 1 . . . . . . .

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 0.10 . . . . . . . .

Dose1 0.10 . . . . . . . .

Dose2 0.10 . . . . . . . .

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 50 of 61

DER Template Version: 22 September 2011 Dose3 0.10 . . . . . . . .

Dose4 0.10 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 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 0.10 . .

Dose1 0.10 1.000 .

Dose2 0.10 1.000 .

Dose3 0.10 1.000 .

Dose4 0.10 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

. . . 1 . . . . . . .

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 -0.10 . . . . . . . .

Dose1 -0.10 . . . . . . . .

Dose2 -0.10 . . . . . . . .

Dose3 -0.10 . . . . . . . .

Dose4 -0.10 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 51 of 61

DER Template Version: 22 September 2011 Ctrl -0.10 . .

Dose1 -0.10 1.000 .

Dose2 -0.10 1.000 .

Dose3 -0.10 1.000 .

Dose4 -0.10 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL
Williams Dose1

Jonckheere Dose1

test for amphib metamorph screen study - 2,4-d 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
0.944 0.284 6.750 0.003 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 58.50 0.58 0.29 0.99 57.58, 59.42 Dose1 4 58.75 0.50 0.25 0.85 57.95, 59.55 Dose2 4 59.00 0.00 0.00 0.00 . , . Dose3 4 58.50 0.58 0.29 0.99 57.58, 59.42 Dose4 4 58.25 0.50 0.25 0.86 57.45, 59.05

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

Ctrl 58.50 58.00 59.00 . .

Dose1 59.00 58.00 59.00 100.43 -0.43

Dose2 59.00 59.00 59.00 100.85 -0.85

Dose3 58.50 58.00 59.00 100.00 0.00

Dose4 58.00 58.00 59.00 99.57 0.43

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

4 15 1.39 0.284

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 58.50 . 58.75 . . . . . .

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 52 of 61

DER Template Version: 22 September 2011 Dose1 58.75 0.874 58.75 0.841 . . . . .

Dose2 59.00 0.421 58.75 0.869 0.946 . . . .

Dose3 58.50 1.000 58.50 0.636 0.946 0.599 . . .

Dose4 58.25 0.874 58.25 0.322 0.599 0.234 0.946 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.15 0.273

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 58.50 . .

Dose1 59.00 0.624 0.753

Dose2 59.00 0.223 0.941

Dose3 58.50 1.000 0.592

Dose4 58.00 0.624 0.173

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

4 15 1.39 0.284

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 -58.50 . -58.50 . . . . . .

Dose1 -58.75 0.874 -58.63 0.427 . . . . .

Dose2 -59.00 0.421 -58.63 0.455 0.946 . . . .

Dose3 -58.50 1.000 -58.63 0.471 0.946 0.599 . . .

Dose4 -58.25 0.874 -58.63 0.481 0.599 0.234 0.946 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.15 0.273

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 53 of 61

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

Ctrl -58.50 . .

Dose1 -59.00 0.624 0.247

Dose2 -59.00 0.223 0.059

Dose3 -58.50 1.000 0.408

Dose4 -58.00 0.624 0.827

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for amphib metamorph screen study - 2,4-d 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
0.948 0.345 1.312 0.310 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.63 0.17 0.08 10.32 1.36, 1.89 Dose1 4 1.73 0.12 0.06 6.75 1.54, 1.91 Dose2 4 1.61 0.24 0.12 14.76 1.23, 1.99 Dose3 4 1.75 0.06 0.03 3.23 1.66, 1.84 Dose4 4 1.91 0.22 0.11 11.42 1.56, 2.26

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

Ctrl 1.64 1.43 1.78 . .

Dose1 1.76 1.57 1.82 106.27 -6.27

Dose2 1.69 1.26 1.80 99.17 0.83

Dose3 1.75 1.70 1.82 107.92 -7.92

Dose4 1.93 1.63 2.15 117.51 -17.51

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

4 15 1.94 0.156

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

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 54 of 61

DER Template Version: 22 September 2011 Ctrl 1.63 . 1.73 . . . . . .

Dose1 1.73 0.819 1.73 0.863 . . . . .

Dose2 1.61 1.000 1.73 0.889 0.875 . . . .

Dose3 1.75 0.681 1.73 0.902 0.999 0.770 . . .

Dose4 1.91 0.104 1.73 0.909 0.580 0.157 0.708 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.41 0.247

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 1.64 . .

Dose1 1.76 0.346 0.876

Dose2 1.69 1.000 0.500

Dose3 1.75 0.494 0.772

Dose4 1.93 0.156 0.969

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

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

4 15 1.94 0.156

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 -1.63 . -1.63 . . . . . .

Dose1 -1.73 0.819 -1.67 0.428 . . . . .

Dose2 -1.61 1.000 -1.67 0.457 0.875 . . . .

Dose3 -1.75 0.681 -1.75 0.203 0.999 0.770 . . .

Dose4 -1.91 0.104 -1.91 0.021 0.580 0.157 0.708 . .

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

4 5.41 0.247

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 55 of 61

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

Ctrl -1.64 . .

Dose1 -1.76 0.346 0.124

Dose2 -1.69 1.000 0.500

Dose3 -1.75 0.494 0.228

Dose4 -1.93 0.156 0.031

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL
Williams Dose4

Jonckheere Dose4

test for amphib metamorph screen study - 2,4-d 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
0.981 0.945 5.995 0.004 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 26.78 1.39 0.70 5.21 24.56, 28.99 Dose1 4 27.40 0.56 0.28 2.04 26.51, 28.29 Dose2 4 26.85 1.63 0.81 6.06 24.26, 29.44 Dose3 4 27.68 0.31 0.15 1.12 27.18, 28.17 Dose4 4 28.08 0.40 0.20 1.44 27.43, 28.72

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

Ctrl 26.80 25.30 28.20 . .

Dose1 27.25 26.90 28.20 102.33 -2.33

Dose2 26.90 24.90 28.70 100.28 -0.28

Dose3 27.60 27.40 28.10 103.36 -3.36

Dose4 28.10 27.60 28.50 104.86 -4.86

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

4 15 1.18 0.361

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

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 56 of 61

DER Template Version: 22 September 2011 Ctrl 26.78 . 27.36 . . . . . .

Dose1 27.40 0.798 27.36 0.859 . . . . .

Dose2 26.85 1.000 27.36 0.885 0.937 . . . .

Dose3 27.68 0.550 27.36 0.899 0.995 0.779 . . .

Dose4 28.08 0.253 27.36 0.907 0.877 0.460 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
4 4.19 0.381

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 26.80 . .

Dose1 27.25 0.780 0.668

Dose2 26.90 1.000 0.529

Dose3 27.60 0.780 0.787

Dose4 28.10 0.235 0.971

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

4 15 1.18 0.361

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

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

Ctrl -26.78 . -26.78 . . . . . .

Dose1 -27.40 0.798 -27.13 0.377 . . . . .

Dose2 -26.85 1.000 -27.13 0.403 0.937 . . . .

Dose3 -27.68 0.550 -27.68 0.150 0.995 0.779 . . .

Dose4 -28.08 0.253 -28.08 0.058 0.877 0.460 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
4 4.19 0.381

MannWhit - testing each trt median signif. different from control

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 57 of 61

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -26.80 . .

Dose1 -27.25 0.780 0.332

Dose2 -26.90 1.000 0.471

Dose3 -27.60 0.780 0.213
Dose4 -28.10 0.235 0.029

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL Williams >highest dose (no sign. differences)

Jonckheere Dose4

test for amphib metamorph screen study - 2,4-d 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
0.955 0.443 0.118 0.974 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 17.23 0.77 0.38 4.46 16.00, 18.45 Dose1 4 18.00 1.02 0.51 5.65 16.38, 19.62 Dose2 4 17.08 0.91 0.46 5.35 15.62, 18.53 Dose3 4 17.48 1.00 0.50 5.71 15.89, 19.06 Dose4 4 16.20 0.98 0.49 6.07 14.64, 17.76

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

Ctrl 17.20 16.40 18.10 . .

Dose1 17.70 17.20 19.40 104.50 -4.50

Dose2 16.90 16.20 18.30 99.13 0.87

Dose3 17.60 16.30 18.40 101.45 -1.45

Dose4 16.45 14.80 17.10 94.05 5.95

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

4 15 1.96 0.153

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 58 of 61

DER Template Version: 22 September 2011 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 17.23 . 17.61 . . . . . .

Dose1 18.00 0.607 17.61 0.799 . . . . .

Dose2 17.08 0.998 17.28 0.650 0.642 . . . .

Dose3 17.48 0.986 17.28 0.669 0.930 0.973 . . .

Dose4 16.20 0.378 16.20 0.094 0.100 0.686 0.350 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.67 0.225

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 17.20 . .

Dose1 17.70 0.413 0.845

Dose2 16.90 0.889 0.384

Dose3 17.60 0.678 0.500

Dose4 16.45 0.283 0.086

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

4 15 1.96 0.153

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 -17.23 . -17.20 . . . . . .

Dose1 -18.00 0.607 -17.20 0.602 . . . . .

Dose2 -17.08 0.998 -17.20 0.637 0.642 . . . .

Dose3 -17.48 0.986 -17.20 0.656 0.930 0.973 . . .

Dose4 -16.20 0.378 -17.20 0.668 0.100 0.686 0.350 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.67 0.225

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 59 of 61

DER Template Version: 22 September 2011 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 -17.20 . .

Dose1 -17.70 0.413 0.155

Dose2 -16.90 0.889 0.616
Dose3 -17.60 0.678 0.500

Dose4 -16.45 0.283 0.914

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for amphib metamorph screen study - 2,4-d 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
0.794 <.001 0.750 0.573 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.68 0.05 0.03 7.41 0.60, 0.75 Dose1 4 0.65 0.06 0.03 8.88 0.56, 0.74 Dose2 4 0.65 0.06 0.03 8.88 0.56, 0.74 Dose3 4 0.65 0.06 0.03 8.88 0.56, 0.74 Dose4 4 0.58 0.05 0.02 8.70 0.50, 0.65

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

Ctrl 0.70 0.60 0.70 . .

Dose1 0.65 0.60 0.70 96.30 3.70

Dose2 0.65 0.60 0.70 96.30 3.70

Dose3 0.65 0.60 0.70 96.30 3.70

Dose4 0.60 0.50 0.60 85.19 14.81

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

4 15 1.92 0.160

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

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 60 of 61

DER Template Version: 22 September 2011 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.68 . 0.68 . . . . . .

Dose1 0.65 0.914 0.65 0.315 . . . . .

Dose2 0.65 0.914 0.65 0.337 1.000 . . . .

Dose3 0.65 0.914 0.65 0.349 1.000 1.000 . . .
Dose4 0.58 0.066 0.58 0.013 0.341 0.341 0.341 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.87 0.209

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 0.70 . .

Dose1 0.65 0.624 0.247

Dose2 0.65 0.624 0.246

Dose3 0.65 0.624 0.256

Dose4 0.60 0.100 0.019

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL
Williams Dose4

Jonckheere Dose4

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

4 15 1.92 0.160

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 -0.68 . -0.64 . . . . . .

Dose1 -0.65 0.914 -0.64 0.880 . . . . .

Dose2 -0.65 0.914 -0.64 0.905 1.000 . . . .

Dose3 -0.65 0.914 -0.64 0.916 1.000 1.000 . . .

Dose4 -0.58 0.066 -0.64 0.924 0.341 0.341 0.341 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.87 0.209

### Data Evaluation Record on the Toxicity of 2,4-D to Amphibians, Metamorphosis Assay

EPA MRID Number 48317002

Page 61 of 61

DER Template Version: 22 September 2011 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 -0.70 . .

Dose1 -0.65 0.624 0.753
Dose2 -0.65 0.624 0.754

Dose3 -0.65 0.624 0.744

Dose4 -0.60 0.100 0.981

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

# DATA EVALUATION RECORD

2,4- DICHLOROPHENOXYACETIC ACID (2,4

- D)

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

EPA Contract No. EP10H001452
Task Assignment No. 2-26-2012 (MRID 48614301)

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

Arlington, VA 22202

Prepared by
CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Sandra Hastings Date: 3 / 19 /2012

Secondary Reviewer : Signature: Scott D. Studenberg

, Ph. D., D.A.B.T. Date: 3/26 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 3 / 26 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 3/26 /2012

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

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathway s. 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).2,4-D/ 030001 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page I of 15
OCSPP 890.1150/ OECD None



## Primary Reviewer: Chester Rodriguez, Ph.D.


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


## Health Effects Division

# Signa<-~;)~~~ <

## Date:_~4( Signature:
-..- ............**~----** 

## Date: 

# CO/J r- b r
Template version 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890

. 1150



## PC CODE: 030001 

## DP BARCODE: D398638



## TXR#: 0052104 

## CAS No.: 94- 75-7



## TEST MATERIAL (PURITY): 2,4-D (98.5% a.i.)



## SYNONYMS: (2,4-dichlorophenoxy) acetic acid



## CITATION: LeBaron, M

. J., Schisler, M.R., and Visconti, N.R. (2011). Evaluation of2,4- Dichlorophenoxy Acetic Acid (2,4-D) In An 

## In Vitro Androgen Receptor Binding Assay. The Dow Chemical Company, Toxicology & 

### Environmental Research

and Consulting, Midland, Ml. Laboratory Project Study ID: 111111, October 27,
2011. MRID #48614301. Unpublished.



## SPONSOR: Industry Task Force II on 2,4-D Research Data, c/o McKenna Long & 

### Aldridge LLP, 1900 K Street NW, Washington, D.C.



## TEST ORDER# : CON-030001-1



## EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48614301),
ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment to measure the binding of a single concentration of [3H]-Rl 881 (1 nM) in the presence of increasing concentrations (10- 11 

### to 10- 4 

### M) of2,4-D (98.5% purity).

The test guideline recommends testing up to 10- 3 

### M; however, the sponsor selected 10 4 

### M as the highest concentration based on 

## in vivo toxicokinetic analyses in the rat; concentrations higher

than 10- 4 

### M were not relevant for testing in this assay as they are substantially above the

inflection point for linear toxicokinetics (see Appendix B of study report). Ethanol was used as a solvent at a final concentration of <3%. A total of three independent runs were performed, and

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

Saturation binding experiments were conducted to demonstrate that the AR in the rat prostate cytosol was present at adequate levels and functioning with appropriate affinity for the

radiolabeled ligand. The saturation binding experiment resulted in a maximum binding capacity (Bmax) of 3.245 fmol/100 μg protein and the dissociation constant (Kd) was 0.4641 nM. Although these values were slightly below the range of values from the validation studies, the

results were highly reproducible and all other performance criteria and the competitive bindingAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 2 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## assays indicated acceptable performance of the assay. 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 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%).

There were no appreciable alterations in R1881 AR binding activity at 2,4-D concentrations ranging from 10

- 11 

## to 10 -4 

## M in the c ompetitive binding e xperiments , therefore, t he log IC 50 

## and relative binding affinity (RBA) for 2,4-D could not be calculated. The log IC 50 

## values for R1881 alone and the positive co ntrol, dexamethasone, were −9.0 and

− 4.4 M , respectively. Compared to R1881, the RBA for dexamethasone was 0.0027 %. In all instances, R1881 and the positive control met the QC performance criteria established in the test guideline.

Based on the results from the three runs, 2,4- D is classifie d as a Non-binder in the Androgen Receptor Binding Assay.

The study satisfies the EDSP Tier 1 Test Order requirement s for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE : Signed and dated GLP and Quality Assurance statements were pr ovided in the study report.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 3 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

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

Location: Midland, MI Study Director: Schisler, M.R.
Other Personnel: LeBaron, M.J. (Lead Scientist); Visconti, N.R. (Research Biologist); Gollapudi, B.B. (Technical reviewer) Study Period: July 11, 2011

– October 27, 2011



## 2

. Test substance : 2,4

- D

Description: Off

- white powder
Source: NuFarm Americas, Inc. (Burr Ridge, IL)
Lot/Batch #: 2006 2433 8006

- USA ( expir y date : March 3, 2013) Purity: 98.5 % a.i. Solubility : Up to 315 mg/L in water; soluble in ethanol up to 30 mM Volatility : 1.9 x 10

- 5 Pa at 25 ° C Stability: 2

- yr shelf life Storage conditions: Ambient
CAS #: 94

- 75

- 7 Molecular weight: 221.0
Structure:



## 3

. Non

- labeled ligand : R1881
Supplier : Perkin

- Elmer ( Boston, MA) Catalog # : Lot #: R0908
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: July 11 , 2011 to July 28 , 2011 Radiochemical purity: >97 % Specific activity : 85.1 Ci/mmol
Concentration of stock : 1.0 mCi/mL



## 5

. Positive control : Dexamethasone
Supplier : Sigma (St. Louis, MO) Catalog #
Lot #: D4902
BCBC9269 Purity: 98.9%
CAS # : 50

- 02

- 2Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 4 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## 6

. Solvent/vehicle control : Ethanol

Justification for choice of
solvent : None provided

Final Concentration : <3%



## B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River Laboratories (Wilmington, MA). Male Sprague Dawley rats (number not reported) were castrated at approximately 90 days of age and euthanized approximately 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, 1 mM sodium molybdate, 1.5 mM EDTA, 10% glycerol and 1 mM phenylmethylsulfonyl fluoride (PMSF) with 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 collected, pooled from all tissues, aliquoted (amounts not reported) and
stored at

− 80°C until used. Protein concentration of the cytosol prepared 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 : The summary of conditions for the saturation binding experiment is provided in Table 1 below.

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 as a separate validation report.
b As indicated in the guideline for acceptable 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 [ 3 

## H] -R1881 was not adjusted for decay over time, and serial dilutions in TEDG buffer were prepared to achieve the final concentrations in cytosol of 0.25, 0.5, 0.7, 1.0, 1.5, 2.5, 5.0, and 10.0 nM to determine total 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 [ 3 

## H] -R1881Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 5 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## was measured. For each batch of cytosol, the optimal protein concentration was determined by calculating specific binding to differing amounts of protein per tube, using 0.25 n M 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 acceptable assay performance the recept or 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. E ach run contained three concurrent replicates at eac h 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) [ 3 

### H]

- R1881 (μL)

0.25 0.25 25 10 7.5
0.50 0.50 50 10 15

0.70 0.70 70 10 21
1.00 1.00 100 10 30
1.50 1.50 150 10 45
2.50 2.50 250 100 7.5
5.00 5.00 500 100 15

10.00 10.00 1000 100 30

a Data were not included in the study report, but are reported as a separate validation report.
b Each concentration was run in triplicate for a total of 72 samples.

c Tubes 1

- 24 contained 50 μL of triam cinolone acetonide and 7.5

- 45 μL [3H]

- R1881. Sam ples were dried, and 300 μ L of prostate cytosol were added.
d Tubes 25

- 48 contained 50 μL of triamcinolone acetonide and 7.5

- 45 μL [3H]

- R1881. R1881 was added in
a 100

- fold molar excess of [3H]

- 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 [3H]

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

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



## Following addition of triamcinolone acetonide, [ 3 

## H] -R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated for approximately 16
hours at 2- 8°C. Samples were maintained at temperatures of ~4°C except during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, 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 vortexi ng. 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, which had highly similar binding profiles. Final determination of acceptable AR binding assay performance was primarily based on guideline suggested standards for the competitive binding assay, although the saturation binding parameters
were evaluated.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 6 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## 3. Competitive Binding Experiment : A summary of the assay conditions for the c ompetitive binding e xperiment is included in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a 

### Source of receptor Rat ventral prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 4.3

- 5.2 % of 1.0 nM radioligand b


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

− 11 

### to 10 −4 

### M Incubation Temperature 4

- 8 oC

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

EDTA 1.5 mM Glycerol 10% (v/v) Phenylmethylsulfonyl fluoride
with DTT 1 mM

Sodium molybdate 1 mM

Protease inhibitor 60 μM

a Data were obtained from pages 15, 18, and 19 of the study report.
b Data were obtained from pages 37, 39 and 4 1 of the study report ; p rotein concentrations (μg/tube) were not reported.



## The c ompetitive binding e xperiment was performed according to the protocol provided in
the EPA Test Guidelines OCSPP 890.1150. The competitive b inding e xperiment measures the binding of a single concentration of [ 3 

## H] -R1881 (specific activity of 85.1 Ci/mmol ) to the AR in the presence of increasing conce ntrations of a test substance. The amount of cytosolic protein used in the assay contained enough receptor to bind 4.3-5.2% of the [ 3 

## H]

- R1881.

Ethanol was used as the solvent vehicle, and the solubility of the test material in the vehicle and assay buffer was evaluated visually. No precipitation was noted.

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. E ach as say consisted of three independent runs on three different days. For each run, a set of duplicate blanks and triplicate tubes with 1 μ M R1881 (non- specific binding, NSB ) were run at the beginning and end of each run. Each run also included triplicate sam ples of each centration of the reference standard, the weak positive control, and 2,4-D, resulting in a total of 77 samples per run. In addition, duplicate blanks followed by six replicates [ 3 

## H] -R1881 only (for total binding calculations) were run the day before each analysis run (the day of preparation of sample tubes).Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 7 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / 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 2,4

- D

Tubes 3

- 5 and 72

- 74 Tubes 6

- 23 and 75

- 77 c 

### Tubes 24

- 47 Tubes 48

- 71 Tubes 1

- 2

1×10

− 6 

### 1×10

− 3 

### 1×10

− 4



### 1×10

− 7 

### 1×10

− 4 

### 1×10

− 5



### 1×10

− 8 

### 1×10

− 5 

### 1×10

− 6



### 1×10

− 9 

### 1×10

− 6 

### 1×10

− 7



### 1×10

− 10 

### 1×10

− 7 

### 1×10

− 8



### 1×10

− 11 

### 1×10

− 8 

### 1×10

− 9



### -- 1×10

− 9 

### 1×10

− 10



### -- 1×10

− 10 

### 1×10

− 11



### a Data were obtained from pages 3 7-42 of the study report.
b Each concentration of each chemical was run in triplicate, plus duplicate blanks for a total of 77 tubes per run.
Tubes 3

- 77 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

### H]

- R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 3

- 77 also contained 10 μL of the solvent control, reference standard (non

- radiolabeled R1881), weak positive control, or test substance, wi th the exception of Tubes 6

- 8 and 75

- 77 that contained 30 μL of non

- radiolabeled R1881 (used to evaluate non

- specific binding). Six tubes analyzed the day prior to each run analysis contained only 30 μL of [ 3 

### H]

- R1881 to determine ligand activity.

c Tubes 6- 8 and 75

- 77 were used to evaluate non

- specific binding by adding 100x of cold (non

- radiolabeled)
R1881.



## Sample tubes were stored overnight at 4-8°C in the dark to allow the reaction to reach equilibrium, bound R1881 was separated from free R1881 by washing with HAP buffer and extraction with ethanol, followed by scintillation counting of bound [ 3 

## H] -R1881.

4. Data Analysis: The top and bottom of the curve, Hill slope, inhibition concentration (IC 50 

## ), and standard deviations were assessed using GraphPad Prism v. 5, and the data were fitted

to a “one site binding” non- linear regression model (GraphPad Prism v. 5)

.



## 5. Definitions

a. Classification of test material

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

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

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

− 0.6 to

− 1.4).

Non

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

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

− 6 

## M and the binding curve does not cross 50%, the chemical is judged to be untestable.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 8 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



### Table 5. Data Interpretation Criteria Specified in OCSPP 890.1150

Criteria Classification Values

Data fit 4

- parameter nonlinear regression
model Average curve across runs crosses 50% a 

### Binder 2
Average lowest portion of curves across runs is between 50% and 75% activity b 

### Equivocal 1

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

### Non

- Binder 0

Data do not fit the
model ----

a If the curve fell outside the range for the weak positive control (see test acceptability criteria), the run as classified as equivocal.
b If the test compound was not soluble above 10 -6 

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



## b. Descriptors for receptor binding

B max 

## : maximal binding capacity
K d 

## : dissociation constant
IC 50 

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

## Relative Binding Affinity (RBA) : IC 50 

## of R1881 × 100 ÷ IC 50 

## of test substance

II. RESULTS

A. SATURATION BINDING EXPERIMENT : Saturation binding e xperiment parameters are presented in Table 6. The dissociation constant (K d 

## ) for [ 3 

## H] -R1881 was 0.4641, and the estimated B max 

## (nM) was 0.06392 for the batch of prostate cytosol that was used for this study. The K d 

## was below the recommended range reported in the test guideline (0.685-1.57 nM). Confidence in these numbers is high according to the goodness of fit (R 2 

## = 0.9871-0.9937) and the small variation among runs.

TABLE 6 . Saturation Binding Experiment of R

- 1881 with Androgen Receptor from Rat Prostate Cytosol a



### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean Runs 1

- 2 c



### R 2 

### (unweighted) 0.9937 0.9871 ND 0.9871

- 0.9937

B max 

### (nM) 0.06194 0.06590 ND 0.06392

B max 

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

K d 

### (nM) 0.4359 0.4922 ND 0.4641

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

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

c The range of R 2 

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

R 2 

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

ND N ot determined



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

## H] -R1881 to the androgen receptor. 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 the highest concentration in Run 1 (24.6%). All other valu es indicated acceptable performance ofAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 9 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



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

## H] -R1881 to the androgen receptor. The data fit results in a linear plot.

F IGURE 1. Binding of [ 3 

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

F IGURE 2. Scatchard Plot of the Binding of [3H]- R1881 to the Androgen Receptor. 0 2 4 6 8 0 1 2 3 4



## R1881 (nM)Specific Binding(fM/100μg)



## Specific binding Run 2 (fM/100 μ M)

Total Run 2
Nonspecific Run 2 Specific Binding Run 1 (fM/100 μ g)

Total Run 1 Nonspecific Run 1



# Scatchard

0 1 2 3 4 0 2 4 6 8



## run 2 run 1

Specific Bound (fM/100 μ g)Specific Bound/FreeAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 10 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## B. COMPETITIVE BINDING EXPERIMENT : The results from the three c ompetitive binding experiments are summarized in Tables 7 and 8 and shown graphically in Figures 3
and 4 . The estimated mean log IC 50 

## for 2,4- D could not be determined as it did not result in 50% displacement of the radioligand at any concentration. The estimated average log IC 50 

## s for R1881 and the weak positive con trol (dexamethasone) were

− 9.0 and

− 4.4 M, respectively . The mean RBA for the positive control was 0.0027%. Confidence in these numbers is high due to the small variation. No precipitation of the test compound was visually observed at any concentration (≤10

− 4 

## M). The solvent control responses indicated no drift in the study assay.

TABLE 7. Competitive Binding Experiment Results for Strong and Weak Positive with AR from Rat Prostate Cytosol a,b

a Data were obtained from page 30 of the study report. b The mean and standard deviations are reported for the combined runs.
NA Not applicable.

R 2 Goodness of fit
IC 50 C oncentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor RBA (%) R elative binding affinity Assay #1 Assay #2 Assay #3 Mean ± SEM Performance Criteria
R1881 Percent (%) of Total Binding

10 - 11 99.8 96.3 101.7 99. 0 ±1.6

--- 10 - 10 92.1 87.3 93.5 90.9 ±1.9

--- 10 - 9 50.0 51.1 52.5 51.2 ±0.7

--- 10 - 8 10.0 9.5 9.7 9.7 ±0. 2

--- 10 - 7 0. 2 0. 2 0 .1 0. 2 ±0.0

---

Bottom (%)

- 0.6

- 1.6

- 1.1

- 1. 1

- 2.0 to 2.0 Top (%) 101 97 103 10 1 82 to 114 Log IC 50 (M)

- 8.993

- 8.986

- 8.956

- 8.97 8

--- IC 50 (M) 1.01 7 e

- 009 1.032e

- 009 1.10 6 e

- 009 1.05 2 e

- 009 Hill slope

- 1.0

- 0.9

- 1.0

- 1.0

- 1.2 to

- 0.8

R 2 0.99 9 8 0.9999 1.000 1.000

--- RBA (%) 1 00 1 00 100 100 100

Dexamethasone Percent (%) of Total Binding

10 - 10 98.2 97.9 104.2 100.1±2.0

--- 10 - 9 98.1 97.5 104.0 99.9 ±2.1

--- 10 - 8 97.9 99.0 10 2.1 99.7 ±1. 3

--- 10 - 7 96.5 98. 0 10 4.7 99.7 ±2. 5

--- 1 0 - 6 94.7 96.3 99.3 96.8 ±1.3

--- 10 - 5 79. 6 79.1 80.5 79.7 ±0.4

--- 10 - 4 29.7 25.3 27.7 2 7 .6 ± 1.3

--- 10 - 3 3. 9 2.3 3.6 3.2 ±0.5

---

Bottom (%)

- 0. 5

- 0.5

- 0. 6

- 0. 6

- 12 to 12 Top (%) 98 98 104 100 87 to 106 Log IC 50 (M)

- 4.3 79

- 4.4 40

- 4.41 6

- 4.4 12

--- IC 50 (M) 4.1 77 e

- 005 3.63 0 e

- 005 3.8 4 0 e

- 005 3. 871 e

- 005 Hill slope

- 1.0

- 1.1

- 1.0

- 1.0

- 1.4 to

- 0.6

R 2 0.999 8 0.9999 0.999 5 1.000

--- RBA (%) 0.0024 0.0028 0.0029 0.002 7

---Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 11 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



### TABLE 8. Competitive Binding Experiment Results for 2,4

- D with AR from Rat Prostate Cytosol a,b



### a Data were obtained from page 31 of the study report.
b The mean and standard deviations are reported for the combined runs.
n/a Not applicable.

R 2 Goodness of fit
IC 50 C oncentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor RBA (%) R elative binding affinity



## Collective Responses of the Independent AR Binding Assays
The collect ive responses of the three independent AR binding assays for 2,4- D indicates no apparent alterations in radiolabeled R1881 binding at any of the concentrations tested. The mean results of the three independent assays are shown in Figure 4 (R1881 and d exam ethasone controls) and Figure 5 (2,4-D) of the study report. The final classification of 2,4- D was based on the average of the three valid assays with a mean value of “0” ( i.e. , (0+0+0)/3) as described in Text Table 5 of the study report. Based on these data, 2,4- D was classified as non -binding at concentrations up to 10 -4 

## M.

Data Interpretation

Test Validity The QC criteria for the reference chemicals in the AR binding assay indicated that the assay performed according to the specified criteria. As with any biological system, there was slight Assay #1 Assay #2 Assay #3 Mean ± SEM 2,4

- D Percent (%) of Total Binding

10 - 1 1 

### 98.79 98.24 99.89 99.0 ± 0.5

10 - 10 

### 102.44 99.72 99.55 100.6 ± 0.9
10 - 9 

### 103.53 97.07 99.95 100.2 ± 1. 9
10 - 8 

### 103.22 99.22 104.25 10 2

. 2 ± 1.5

10 - 7 

### 101.49 99.04 103.98 101.5 ± 1.4
10 - 6 

### 103.17 98.32 104.51 102.0 ± 1. 9
10 - 5 

### 102.3 3 97.65 105.58 101.9 ± 2.3
10 - 4 

### 105. 64 99.8 106.32 103.9 ± 2.1

Bottom (%)

(95% CI) 103.2 ( 101.1 to 105.4 ) n/a 99.70 (9 7.9 to 101.5 ) n/a

Top (%) (95% CI) ~ - 146.6 ( very wide ) n/a 105.1 ( 103.8 to 106.4 ) n/a

Log IC 50 

### (M)

(95% CI) n/a n/a n/a n/a

IC 50 

### (M) (95% CI) n/a n/a n/a n/a

Log E C 50 

### (M)

(95% CI) ~ - 13.15 (very wide) n/a

- 8.363 (

- 9.431 to

- 7.294 ) n/a

E C 50 

### (M) (95% CI) ~ 7.139e

- 014 (very wide) n/a 4.339e

- 009 (3.707e

- 010 to

5.078 e

- 00 8 ) n/a

Hill slope (95% CI) ~ - 0.8 113 (very wide ) n/a 1.939 (

- 3.018 to 6.895 ) n/a

R 2 

### 0. 63 18 n/a 0. 9371 n/a

Data Interpretation negative negative negative n/a RBA (%) n/a n/a n/a n/aAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 12 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## variability between assays, but the overall robustness of the responses for R1881 (strong positive control/standard curve) and d examethasone (weak positive control) indicated that each assay included in this assessment performed as expected. Thus, assay #1, #2, and #3 of the AR binding assay with 2,4- D were considered v alid.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 13 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## FIGURE 3. Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and 2,4-D (Assays 1 – 3).

Assay #1

Assay #2

Assay #3Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 14 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## FIGURE 4. Mean of Percentage R1881 Bound to the Androgen Receptor in the Presence of 2,4- D from Three Assays.

C. PERFORMANCE CRITERIA : To ensure that the c ompetitive binding a ssay was functioning properly, each run was evaluated using the criteria in Table 9. 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 H ill slope of

− 1.0 to

− 0.9. Examination across the runs indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus.

TABLE 9 . 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% 4.3

- 5.2% X

2,4

- D Top (% binding) 80 to 115 100 to 106 X

R1881 fitted curve parameters Top (% binding) 82 to 114 97 to 103 X Bottom (% binding)

− 2.0 to 2.0

− 1.6 to

− 0.6 X Hill Slope

− 1.2 to

− 0.8

− 1.0 to

− 0.9 X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 98 to 104 X Bottom (% binding)

− 12 to 12

− 0.6 to

− 0.5 X Hill Slope

− 1.4 to

− 0.6

− 1.1 to −1. 0 X Saturation Binding Experiment K d 

### (nM) (0.685

- 1.57 nM) 0.4641 X

Non

- specific binding (%) ≤ 10.0 7.30 to 8 .23 c 

### X

a Data were obtained from pages 3 0, 31 and 37 -42 of the study report.
b These values represent ranges from the validation study.

c Values reported for the three NSB tubes at the beginning of each run; does not include the three NSB tubes at
the end of the run.
NR Not reportedAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 15 of 15 2,4 -D/ 030001 O CSPP 89 0.1150 / OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Based on the combined responses in each of three independent androgen receptor binding assays, it was determined that 2,4-D had no appreciable effect in the binding of the reference androgen ([3H]-R1881) at any concentration (up to 10

− 4 

## M). The results of the in vitro AR binding assay using rat prostate cytosol indicate that, under the conditions of this study, 2,4-D was negative for AR binding

at conc entrations up to 10

− 4 

## M.

B. AGENCY C OMMENTS : The saturation binding e xperiment resulted in a maximum binding capacity ( B max 

## ) of 3.245 fmol/100 μg protein and the dissociation constant (K d 

## ) was 0.4641 nM. Although these values were slightly below the range of values from the validation studies, the results were highly reproducible and all other performance criteria and the competitive binding assays indicated acceptable performance of the assay

The test guideline recommends testing up to 10 -3 

## M; however, the sponsor selected 10 -4 

## M as the highest concentration , based on in vivo toxicokinetic analyses in the rat. S pecific binding was >75% at all concentrations tested (10 −11 

## to 10

− 4 

## M) . An IC 50 

## and RBA could not be calculated for 2,4-D as it did not result in 50% displacement at any concentration

.

The estimated average l og IC 50 

## s for R1881 and the weak positive control (dexamethasone)
were −9.0 a nd −4.4 M , respectively . The mean RBA for the positive control was 0.0027%. Confidence in these numbers is high due to the small variation. No precipitation of the test compound was visually observed at any concentration (≤10

− 4 

## M). The solvent control responses indicated no drift in the study assay , and all performance criteria were met in all
three runs.

Based on the results of the three runs, 2,4-D is classified as a non -binder for the androgen receptor at concentrations up to 10 -4 

## M.

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

• Only two saturation binding runs were conducted rather than the three runs recommended in the test guideline.

# DATA EVALUATION RECORD

2,4- DICHLOROPHENOXYACETIC ACID (2,4

- D)

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-26-2012 (MRID 48614302)

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

Arlington, VA 22202

Prepared by
CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: David A. McEwen, B.S. Date: 3 / 1 7 /2012

Secondary Reviewer : Signature: Scott D. Studenberg

, Ph.D., D.A.B.T. Date: 3 / 23 / 2012

Program Manager: Signature: Jack D. Early, M.S. Date: 3/2 6 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 3/2 6 /2012

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

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery 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).2,4-D I 030001 Aromatase (Human Recombinant) Assay (2011) I Page I of 12
OCSPP 890.1200 I OECD None



## Primary Reviewer: Patience Browne, Ph

. D.


## Office of Science Coordination and Policy Secondary Reviewer: Greg Akerman, Ph

. D.


## Health Effects Division, Office of Pesticide Programs

# Signature:**--** Date: ?12-L 

### /15 

## Signature:

# 4?-;~,,.**~~~ ==** **'** 

# ===- - Date: 7 

## @/;91 1<
Template version 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



## PC CODE: 030001 

## DP BARCODE: D398640



## TXR#: 0052104 

## CAS No.: 94- 75- 7



## TEST MATERIAL (PURITY): 2

, 4-D (98

. 5% a.i

. )



## SYNONYMS: 2,4-Dichlorophenoxyacetic acid



## CIT A TI ON: Coady

, K.K. and Sosinski, L.K. 

## (2011) 2

, 4-Dichlorophenoxyacetic acid:
Evaluation of 2

, 4-dichlorophenoxyacetic acid in the human recombinant

aromatase assay. Toxicology & 

## Environmental Research and Consulting

, The

Dow Chemical Company

, Midland

, Ml. Laboratory Project Study ID : 111036

, September 27

, 2011. MRID 48614302. Unpublished.



## SPONSOR: Industry Task Force 

## II on 2

, 4-D Research Data

, c / o McKenna Long & 

## Aldridge

LLP

, 1900 K Street NW

, Washington

, D.C.



## TEST ORDER#: CON-030001-01



## EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48614302)

, 2

, 4-D (98.5 % a.i

., lot# : 2006 2433 8006-USA) in ethanol (1 %) was incubated with human recombinant

aromatase and tritiated androstenedione**(1-~** 

## [3H(N)]-androst-4-ene-3

, 17-dione ; [3H]ASDN) for
15 minutes at 37 ° C to assess the potential of 2

, 4-D to inhibit aromata s e activity

. 2

, 4-D was

t e sted at logarithmic concentrations from 10

- 10 

## M to 10

- 4 

## M in three independ e nt runs. The te s t

g uideline recommends testing up to 10- 3 

## M; however

, the sponsor selected 10- 4 

## M as the highest
concentration based on in vi v o toxicokinetic analyses in the rat. The sponsor considered
concentrations higher than 10

- 4 

## M to be not relevant for testing in this assa y as they are substanti a lly above the inflection point for linear toxicokinetics (See Appendix B of the AR
bindin g assay stud y report

, MRID 48614301)

.

Aromatase activity was determined by measuring the amount of tritiated water produced at the

e nd of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC)

. Three independent runs were conducted and each run included a full activity control

, a background activity control

, a positive control series (10

- 1 0 

## to 10

- 5 

## M) using a known inhibitor ( 4-hydro xy androstenedione ; 4-0H ASDN)

, and the test chemical series (10

- 1 0 

## to 10

- 4 

## M) with 3 repetitions per concentration

.Aromatase (Human Recombinant) Assay (2011 ) / Page 2 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## Aromatase activity in the full activity controls ranged from 0.131 to 0.244 nmol

∙ mg

protein

− 1



## ∙ min

− 1 

## for the three test runs, with a mean and standard deviation of 0.186±0.036
nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## . Activity in the background controls ranged from 7.31 to 11.51% of the full activity controls. The responses of full activity controls were outside of the 90 to 110% range in the 2 nd 

## and 3 rd 

## replicates in the Run 1 (86.6 and 113.8%, respectively ).

Results for the positive control were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 

## , and coefficient of variation for replicates of each concentration within runs, with the exception that the bottom of the curve in Runs 2 and 3 (-8.2
and - 7.2, respectively) exceeded the acceptable range ( -5 to +6). Also, the coefficients of variations (%CVs) for replicates of each concentration of 2,4-D within a run were generally within the 15% guideline, with t he exception that the overall %CV for the highest two concentrations (-45.2 and 415.9%) exceeded the acceptable limit. For 4

- OH ASDN , the
estimated log IC 50 

## averaged −7.23 M and the slope was −0.92.

For 2,4-D, aromatase activity averaged 0.195±0.045 nmol

∙ mg -protein −1



## ∙ min −1 

## at the lowest tested concentration (10 −10 

## M) and 0.168±0.025 nmol

∙ mg -protein −1



## ∙ min −1 

## at the highest tested concentration (10 −4 

## M). The data for 2,4-D were modeled for Run 1, and the goodness of fit (R 2 

## ) value was 0.83; however, the 2,4-D data from Runs 2 and 3 could not be modeled. The average
dose- response curve indicated that the aromatase activity of the test material at concentrations ranging from 10

− 10 

## M to 10

− 4 

## M was essentially equivalent to the activity observed in the full

act ivity controls. At 10 –4 

## M, aromatase activity was approximately 91 %. Since the average lowest portion of the activity response curve was greater than 75% activity, 2,4- D is classified as a non-inhibitor of aromatase activity up to the highest concentration tested (10 -4 

## M). High CVs
were observed for 4- OH ASDN at the two highest concentrations and at a single concentration for 2,4- D. Individual values were occasionally outside of the performance criteria ranges (with the mean value within rang e).

B ased on the data from the average response curve, 2,4- D is classified as a Non-inhibitor of aromatase activity in this a ssay.

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

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality
Assurance statements were provided.Aromatase (Human Recombinant) Assay (2011 ) / Page 3 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : 2,4

- D
Description: Off

- white powder
Source: Nufarm Americas, Inc. (Burr Ridge, IL)
Lot/Batch #: 2006 2433 8006

- USA Purity: 9 8.5 % a.i. Volatility: Not reported Storage conditions: Ambient Stability: Not reported
Solvent : Ethanol Solubility (in test solvent): Soluble up to 0. 0 1 M Highest Concentration Tested: 10

− 4 M in all runs Stock Solution Preparation Methodology: Dissolved the test material in ethanol

. Molecular weight: 2 21.0
CAS #: 94

- 75

- 7
Structure:



## 2. Non

- L abeled S ubstrate : Androstenedione (ASDN)

CAS # : 63

- 05

- 8
Source: Steraloids, Inc. (Cat. # A6030100)
Lot/Batch #: L1627 Purity: 9 8.4 %



## 3

. Radiolabeled S ubstrate : 1

- β [ 3 

## H(N)]

- Androst

- 4

- ene

- 3,17

- dione ; ( [ 3 

## H] ASDN)

Source: Perkin Elmer Life and Analytical Sciences (Cat. # NET 926 )
Lot/Batch #: 619344 Radiochemical Purity (Supplier): >97% Specific activity: 2 6.3 Ci/mmol Radiochemical Purity (In

- lab determination): Not reported



## 4. Positive C ontrol : 4

- hydroxyandrostenedione (4

- OH ASDN)

CAS # : 566

- 48

- 3
Source: Sigma

- Aldrich (Cat. # F2552)
Lot/Batch #: 081K2133 Purity: 99.6%



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

Source:
Lot/Batch #: Sigma Aldrich (Cat. # E7023 )
72596PMV
Justification for choice of solvent : Not provided. Ethanol is a Guideline preferre d solvent

. Concentration (% of total volume in assays) : 1% v/vAromatase (Human Recombinant) Assay (2011 ) / Page 4 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



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

Source : Gentest (Woburn, MA; Cat. # 456260)

Lot/Batch #: 03897

Protein concentration : 7.4 mg/mL Cytochrome C reductase activity: 290 nmol/min/mg Aromatase activity: 6.0 pmol/min/pmol P450



## B. METHODS

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

## H] -ASDN was prepared to result in a 2 μM ASDN solution with a predicted radioactive
content of 1.0 μCi/mL.

Test chemical stock solutions were prepared such that the total volume of each test chemical formulation used per assay was no more than 1% v/v of the total assay volume. The report specified that ethanol was chosen because it was mentioned in the guideline as a preferred
solvent.

A stock solution of the positive control substance, 4- OH ASDN, was formulated in ethanol. Fresh serial dilutions of the stock solution were prepared each time the aromatase in hibition assay was conducted. Dilutions were prepared such that the target concentrations of the positive control substance (10

− 10 

## to 10

− 5 

## M; Table 4) were achieved by the addition of 20 μL of the dilution for a final assay volume of 2 mL.

Human recombin ant microsomes were purchased from Gentest , and aliquoted into individual vials based on protein content. Microsomes were stored at approximately

− 80o C
until use.

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

T ABLE 1. Assay Components and Conditions Assay Factor Values 0.1M sodium phosphate buffer (pH 7.4)

Microsomal Protein 0.004 mg/mL

NADPH 6 mM
[ 3 H]ASDN 2 μ M Propylene Glycol 100 μL Temperature 37°C

Incubation Time 15 min



## 2. Suitability Ass es s ments : The protein concentration in an aliquot of the microsomes was determined each day of use, and microsomes were diluted with phosphate buffer such that approximately 0.004 mg/mL protein was present in the final reaction solution. A romatase activity of the microsomes was provided by the vendor as 6.0 pmol/min/ pmol P450. The minimum aromatase activity in the full activity control samples was determined to be 0. 131
nmol/ mg

- protein/min , which was greater than the minimum acceptable aromatase activity of 0.10 nmol/mg- protein/min.
3. Aromatase A ssay : Each assay run contained four tubes for the full enzyme activity and four tubes for the background activity controls. Two tubes of each control were run at theAromatase (Human Recombinant) Assay (2011 ) / Page 5 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## beginning of the assay, and two of each control were run at the end of the assay. A full concentration curve in duplicate for the positive control, and a full concentration curve in triplicate for the test substance were established. The aromatase assay was conducted according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

## H 2 

## O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol∙mg

- protein −1 

## ∙min −1



## .

4. Demonstration of P roficiency : It was stated that all assays were performed by personnel
with demonstrated proficiency performing the assay as outlined in test guideline 890.1200. Proficiency records are archived in training records at the test facility and a copy of these
data were archived with the 2,4- D specific report.

a. Positive Control

(1) Initial Demonstration of Laboratory P roficiency : Raw data were not provided, however, summary data of proficiency exercised were equivocal. Re lative aromatase activity in th e presence of prochloraz, a known aromatase inhibitor indicated dose-dependent inhibition.
A romatase activity appeared to be un affected by ronidazole, a non- inhibitor at concentrations ranging from 10

- 10 

## to 10

- 2.5 

## M. Dose -dependent inhibition of aromatase was demonstrated in the presence of fenarimol and nitrofen, two recognized inhibitors of

aromatase ; however, the data were highly variable

.

(2) Demonstration o f P roficiency of N ew T echnician for C onducting Assay (when applicable) : Demonstration of prof iciency by a new technician, if applicable, was not reported. The positive control data for slope, top and bottom percent, and log IC 50 

## met the criteria as listed in section (i) of OCSPP 890.1200, with the exception of the bottom of the curve, which was below the recommended value for Run 2 (–8.2) and Run 3 (–7.2).

TABLE 2. Performance Criteria for the Positive Control

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

− 1.2

− 0.8

– 1.1

– 0.78 Top (%) 90 110 91 100 Bottom (%)

− 5 +6

– 8.2

– 2.6 Log IC 50 (M)

− 7.3

− 7.0

– 7.3

– 7.2

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



## b. Proficiency C hemicals : Standard curves were provided for the reference chemicals prochloraz, ronidazole, fenarimol, and nitrofen (Appendix A of the protocol on pages 84-86 of the study report).

T ABLE 3. Proficiency Chemicals
Compound CAS# Class Concentrations a

Prochloraz Not provided Inhibitor 10

- 10 to 10

- 3 M

Fenarimol 60168

- 88

- 9 Inhibitor 10

- 10 to 10

- 3 M

Nitrofen 1836

- 75

- 5 Inhibitor 10

- 10 to 10

- 3 M

Ronidazole Not provided Non

- inhibitor 10

- 10 to 10

- 3 M

a Concentration ranges taken from figures

.Aromatase (Human Recombinant) Assay (2011 ) / Page 6 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## 5. Determination of Aromatase A ctivity with T est C hemical(s) : The response of aromatase activity to the presence of eight concentrations of 2,4-D per run, in triplicate, was tested during three independent runs (Table 4) . Solubility was visually assessed (presence of cloudiness or a precipitate). No precipitation was observed at any concentration in any run. The response of each full activity control within a run was between 91 to 108 % of the average full activity, with the exception of two full activity control resp onses (87 and 114 %) from the beginning and end, respectively, of Run 1 that were outside of the guideline recommended range of 90 to 110%.

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

Sample Type Repe titions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components a plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A

4

- OH ASDN Conc 1 2 All test components plus 4

- OH ASDN 1×10

− 5

4

- OH ASDN Conc 2 2 All test components plus 4

- OH ASDN 1×10

− 6

4

- OH ASDN Conc 3 2 All test components plus 4

- OH ASDN 1×10

− 6.5

4

- OH ASDN Conc 4 2 All test components plus 4

- OH ASDN 1×10

− 7

4

- OH ASDN Conc 5 2 All test components plus 4

- OH ASDN 1×10

− 7.5

4

- OH ASDN Conc 6 2 All test components plus 4

- OH ASDN 1×10

− 8

4

- OH ASDN Conc 7 2 All test components plus 4

- OH ASDN 1×10

− 9

4

- OH ASDN Conc 8 2 All test components plus 4

- OH ASDN 1×10

− 10
2,4

- D Conc 1 3 All test components plus 2,4

- D 

### 1×10

– 4

2,4

- D Conc 2 3 All test components plus 2,4

- D 

### 1×10

– 4.5

2,4

- D Conc 3 3 All test components plus 2,4

- D 

### 1×10

– 5

2,4

- D Conc 4 3 All test components plus 2,4

- D 

### 1×10

– 6

2,4

- D Conc 5 3 All test components plus 2,4

- D 

### 1×10

– 7

2,4

- D Conc 6 3 All test components plus 2,4

- D 

### 1×10

– 8

2,4

- D Conc 7 3 All test components plus 2,4

- D 

### 1×10

– 9

2,4

- D Conc 8 3 All test components plus 2,4

- D 

### 1×10

– 10

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

.



## C. D ATA ANALYSIS

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

T ABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) DPM/mL for each portion of extracted aqueous incubation mixture 

### X Average DPM/mL for each aqueous portion (after extraction) 

### X Total DPM for each aqueous portion (after extraction) 

### X The total DPM present in the assay tube at initiation 

### X The percentage of substrate converted to product 

### X Total DPM after extraction corrected for background 

### X Aromatase activity expressed in nmol/mg protein/min 

### X Average aromatase activity in the full activity control tubes 

### X Percentage of control activity remaining in the presence of various inhibitor concentrations 

### X

DPM Disintegrations per minuteAromatase (Human Recombinant) Assay (2011 ) / Page 7 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## 2. S tatistical M ethods : Statistical analyses and graphical displays were conducted using Graph Pad Prism (Version 4.0, La Jolla, CA). Basic statistical analyses were performed on the data, which included means of replicates, standard deviation of the mean, relative standard deviation, and coefficient of variation. The Hill slope and log IC 50 

## values across three independent runs were compared based on a one- way random effects analysis of variance, treating runs as random effects.

The response curve was fitted by nonlinear regres sion analysis. Model fits were carried out using a 4-parameter regression model. For each run, percent of full activity control were plotted versus logarithm (base 10) of the test chemical concentration or 4- OH ASDN concentration. Each run was plotted with the data’s best fit curve. Additionally, the average inhibition response curve across all runs was also plotted.

3. Interpretation of Results : Interpretation of the assay results was based on the average of three runs, using 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% a Inhibitor Average lowest portion of curves across runs is
between 50% and 75% activity b Equivocal

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

- inhibitor

Data do not fit model

---

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

− 1. Unusually steep curves may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than

− 2.0, the result is classified as equivocal.
b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cross 50%, the chemical is typically
determined to be un

- testable in the aromatase assay

.



## II

. RESULTS

A. CONTROL ACTIVITY : Aromatase activity in the full activity controls ranged from
0.131 to 0.244 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## for the three test runs, with a mean and standard
deviation of 0.186±0.036 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## . Activity in the background controls ranged from 7.31 to 11.51 % of the full activity controls. The response of each full activity control was generally between 90 to 11 0% of the average full activity , with the exception of
the 2 nd 

## and 3 rd 

## replicates in the Run 1 (86.6 and 113.8%, respectively). The response of the full activity controls and background controls were acceptable.

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

- OH ASDN ), a romatase activity averaged 0.180±0.027 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## at the lowest tested concentration (10

− 10 

## M) and -0.008±0.004 nmol

∙ mg -protein -1



## ∙ min

− 1 

## at the highest tested concentration (10

− 5 

## M) . The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across all three runs is presented in Table 7, along
with the o verall standard deviation and % CV. Inhibition response curves for the positive control from each run and the average of all runs are shown in Figure 1. These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope

, log IC 50 

## , and coefficient of variation for replicates of each concentration, with the followingAromatase (Human Recombinant) Assay (2011 ) / Page 8 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## exceptions: t he overall %CV for the highest two concentration s (-45.2 and 415.9% )

excee ded the acceptable limit of 15%, and the bottom of the curve in Runs 2 and 3 (-8.2 and

- 7.2, respectively) exceeded the acceptable ran ge ( -5 to +6).

TABLE 7 . Effect of 4

- OH ASDN and 2,4

- D on Aromatase Activity (as percent of control) from Independent
Runs a

Chemical Concen. ( Log M ) # Runs Overall Mean Overall SD Overall SEM Overall %CV

4

- OH ASDN (positive control) −5 3

- 4.49 2.03 1.17

- 45.2

−6 3 1.10 4.59 2.65 415.9

−6.5 3 16.86 2.40 1.39 14.2

−7 3 34.80 3.96 2.28 11.4

−7.5 3 61.98 5.07 2.93 8.2

−8 3 81.46 3.35 1.93 4.1

−9 3 92.09 1.65 0.95 1.8

−10 3 97.17 4.21 2.43 4.3
2,4

- D −4 1 91.15 7.05 4.07 7.7

−4.5 3 92.20 7.34 4.24 8.0

−5 3 88.05 5.65 3.26 6.4

−6 3 91.77 4.17 2.41 4.5

−7 3 98.02 13.05 7.54 13.3

−8 3 105.55 9.09 5.25 8.6

−9 3 100.33 22.05 12.73 22.0

−10 3 104.10 11.19 6.46 10.7

a Values were calculated by the reviewers based on data provided on pages 66

- 68

. SD Standard Deviation
SEM Standard Error of the Mean
CV Coefficient of VarianceAromatase (Human Recombinant) Assay (2011 ) / Page 9 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



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

- OH ASDN

.

C. TEST SUBSTANCE : For 2,4-D , aromatase activity averaged 0.195±0.045 nmol

∙ mg

protein

− 1



## ∙ min

− 1 

## at the lowest tested concentration (10

− 10 

## M) and 0.168±0.025 nmol

∙ mg

protein

− 1



## ∙ min

− 1 

## at the highest tested concentration (10

− 4 

## ) . The mean aromatase activity of 2,4-D (expressed as %full control activity) for each concentration tested across all three runs is presented in Table 7 (above), along with the overall standard deviation and % CV. Inhibition response curves for 2,4-D from each run are shown in Figure 2, and the average inhibition response curve across all runs is shown in Figure 3. The overall %CV for the
10

− 9 

## M concentration (22 %) exceeded the acceptable limit of 15%.Aromatase (Human Recombinant) Assay (2011 ) / Page 10 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## FIGURE 2. Inhibition R esponse C urves for 2,4-D F rom E ach T est R un.

FIGURE 3. Mean I nhibition R esponse C urve for 2,4-D.

The data for 2,4-D were modeled for Run 1, and the goodness of fit (R 2 

## ) value was 0.83 ;however, data from Runs 2 and 3 were not amenable to modeling. The average doseAromatase (Human Recombinant) Assay (2011 ) / Page 11 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## response curve indicated that the aromatase activity of the test material at concentrations ranging from 10

− 10 

## M to 10

− 4 

## M was essentially equivalent to the activity observed in the full activity controls.

For positive control curves, the best fit Hill slope and log IC 50 

## values across three independent runs were similar

.

The effect of the positive control on inhibition of aromatase activity is presented in Table 8.
For 4

- OH ASDN , t he estimated log IC 50 

## averaged

− 7.23 M and the slope was

− 0.92. Confidence in the mean log IC 50 

## for the positive control is high due to the small variation (< 1% CV ). Confidence in the mean slope is low due to the large variation (17.8% CV).

TABLE 8 . Effect of 2,4

- D on Aromatase Activity (as P ercent of C ontrol) F rom Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean SD %CV 

### Log IC 50 (M) 

### 2,4

- D NA NA NA NA NA NA

4

- OH ASDN

− 7. 2

− 7. 3

− 7. 2

− 7. 2 3 0.06

- 0.80 Slope 

### 2,4

- D NA NA NA NA NA NA

4

- OH ASDN

− 1. 1

− 0. 88

− 0. 78

− 0.92 0.16

- 17.79

a Data were provided on page 3 2 of the study report. Mean, SD and %CV were calculated by the reviewers based on these
data.
SD Standard Deviation
CV Coefficient of Variance
NA Not applicable. Values for 2,4 -D were not suitable for modeling.



## Based on the data from the average response curve and the criteria listed above in Table 6, the results support the conclusion that 2,4-D is a non-inhibitor of aromatase activity

.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS : The average response from the three independent runs with 2,4-D did not fit the four parameter regression model. Additionally, average aromatase activity for 2,4

- D was similar to full activity controls at all concentrations tested

. Th erefore , 2,4- D is classi fied as a non- inhibitor of aromatase activity

.

B. AGENCY COMMENTS : Aromatase activity in the full activity controls ranged from
0.131 to 0.244 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## for the three test runs, with a mean and standard
deviation of 0.186±0.036 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## . Activity in the background controls ranged from 7.31 to 11.51% of the full activity controls. The responses of the full activity controls were outside of the 90 to 110% range in the 2 nd 

## and 3 rd 

## replicates in the Run 1 (86.6
and 113.8% , respectively).

For the positive control substance (4- OH ASDN ), aromatase activity averaged 0.180±0.027
nmol

∙ mg -protein −1



## ∙ min −1 

## at the lowest tested concentration (10 −10 

## M) and -0.008±0.004
nmol

∙ mg -protein -1



## ∙ min −1 

## at the highest tested concentration (1 0 −5 

## M). These results were within the recommended ranges for the top of the curve, bottom curve, Hill slope , log IC 50 

## , and coefficient of variation for replicates of each concentration, with the followingAromatase (Human Recombinant) Assay (2011 ) / Page 12 of 12 2,4 -D / 030001 O CSPP 89 0. 1200 / OECD None



## exceptions: the overall %CV for the highest two concentrations (-45.2 and 415.9%) exceeded the acceptable limit of 15%, and the bottom of the curve in Runs 2 and 3 (-8.2 and

- 7.2, respectively) exceeded the acceptable range ( -5 to +6).

For 2,4-D, aromatase activity averaged 0.195±0.045 nmol

∙ mg -protein

− 1



## ∙ min −1 

## at the lowest tested concentration (10 −10 

## M) and 0.168±0.025 nmol

∙ mg -protein −1



## ∙ min −1 

## at the highest tested concentration (10 −4 

## ). The overall %CV for the 10 -9 

## M concentration (22%) exceeded the acceptable limit of 15%. The data for 2,4- D were mod eled for Run 1, and the goodness of fit (R 2 

## ) value was 0.83. The average dose- response curve indicated that the aromatase activity of the test material at concentrations ranging from 10

− 10 

## M to 10 −4 

## M was essentially equivalent to the activity observed in the full activity controls.

For 4

- OH ASDN , the estimated log IC 50 

## averaged −7.23 M and the slope was −0.92. Confidence in the mean log IC 50 

## for the positive control is high due to the small variation (< 1 % CV) , but confidence in the mean Hill slope is low due to the large variation (17.8 % CV).

At 10 –4 

## M aromatase activity was 91% compared to the full activity controls. Since the average lowest portion of the activity response curve was greater than 75% activity, 2,4- D is
classified as a non-inhibitor of aromatase activity up to the highest concentration tested (10 -4 

## M).

C. STUDY DEFICIENCIES : None

# DATA EVALUATION RECORD

2,4- DICHLOROPHENOXY ACETIC ACID (2,4- D)

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-26-2012 (MRID 48614303)

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

Arlington, VA 22202

Prepared by
CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Michelle Sharpe

- Kass, M.S. Date: 3/15 /2012

Secondary Reviewer : Signature: Scott D. Studenberg, Ph. D.,
D.A.B.T. Date: 3/22/2012

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

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

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

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The rob ustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each
indiv idual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).2,4-D/ 030001



## Primary Reviewer: Patience Browne, Ph

. D.


## Office of Science Coordination and Policy Secondary Reviewer: Greg Akerman, Ph.D.


## Health Effects Division

# Signature:**~** Date: 

# ?/U/t?
Signature:

..**~** 

## A}

Date: 

## G ftqft a= Template version 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC);
OCSPP 890.1250



## PC CODE: 030001 

## DP BARCODE: D398640



## TXR#: 0052104 

## CAS No.: 94-75-7



## TEST MATERIAL (PURITY): 2,4-D (98.5% a.i.)



## SYNONYMS: 2,4-dichlorophenoxy acetic acid



## CITATION:- LeBaron, M.J., Shisler, M.R., Visconti, N.R. (2011). Evaluation of2,4- Dichlorophenoxy Acetic Acid**,4-D)** 

## in an in vitro 

## Estrogen Receptor Binding Assay. Toxicology & 

## Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory Study

. ID.: 111121, October 27, 2011.
MRID 48614303. Unpublished.



## SPONSOR: Industry Task Force 

## II on 2,4-D Research Data, c / o McKenna Long & 

## Aldridge

LLP

, 1900 K Street NW, Washington, DC.



## TEST ORDER#: CON-030001-1



## EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48614303) for

2

, 4-D (98.5%, Lot# 2006 2433 8006-USA)

, uterine cytosol from Sprague Dawley rats was used

as the source of ER to conduct saturation binding and competitive binding experiments in this

assay. The competitive binding experiment was conducted to measure the binding of a single
concentration of**[3H]-17~-estradiol** 

## ( 1 nM) 

## in the presence of logarithmic increasing
concentrations of 2,4-D from 10

- 11 

## to 10

- 4 

## M, rather than 10

- 10 

## to 10

- 3 

## recommended in the test guideline. The justification for lowering the top test concentration was based on toxicokinetic
data in the rat; concentrations higher than 10- 4 

## M were not relevant for testing in this assay as they are substantially above the inflection point for linear toxicokinetics

. Ethanol was used as a solvent 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, and three independent runs were performed on separate days.

Summary data pertaining to the saturation binding experiment were reported separately in the study profile submitted by the test order recipient. The Kd for**[3H]-17~-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, and the Bmax was within the expected range of 10-150 fmol/100 μg protein. The data produced a linear Scatchard plot.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 2 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## In the c ompetitive binding experiment, n o precipitation was observed at any concentration tested. The mean specific binding in the presence of 2,4- D was >95% at 2,4- D concentrations of ≤ 10

− 4



## M in all three runs. The estimated mean l og IC 50 

## and RBA was not calculated for 2,4- D as the percent binding inhibition did not reach 50% for any run

.

The estimated mean log IC 50 

## for the natural ligand, 17 β -estradiol, and the weak positive control (19-norethindrone ) was −9.0 and

− 5.5 M, respectively. The mean RBA was 0.034% for 19- norethindrone. All performance criteria were met for 17 β -estradiol, 19-norethindrone and octyltriethoxysilane.

2,4-D was tested over a concentration range that fully defined the top of the curve. The mean specific radioligand binding in the presence of 2,4-D was >95% at 2,4-D concentrations of

≤ 10

− 4 

## M. B as ed on the results from the three runs, 2,4- D is classified as Not I nteractive in the Estrogen R eceptor Binding Assay.

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

COMPLIANCE : Signed and dated GLP, D ata C onfidentiality , and Quality Assurance

statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 3 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Toxicology & Environmental Research and Consulting ; Dow Chemical

Location: Midland, MI Study Director: M.R. Schisler
Other Personnel: M.J. LeBaron (Lead Scientist), N.R. Visconti (Research Biologist ), B.B. Gollapudi, (Technical Reviewer) Study Period: September 14, 2011

- October 2 7 , 2011



## 2

. Test substance : 2,4

- D
Description: Technical, Off

- white Powder
Source: NuFarm Americas, Inc.
Lot/Batch #: 2006 2433 8006

- USA Purity: 98.5% Solubility : 315 mg/L in water, 30 mM in ethanol Volatility : 1.9 x 10

- 5 Pa at 25 ° C Stability: 2 year shelf life Storage conditions: Ambient
CAS #: 94

- 75

- 7 Molecular weight: 221.0
Structure:



## 3

. Non

- labeled ligand : 17 β

- estradiol
Supplier : Sigma, St. Louis MO Catalog # : E8875
Batch #: 098K1372 Purity: 100 %
CAS #: 50

- 28

- 2



## 4

. Radioactive ligand : [ 3 

## H]

- 17 β

- estradiol
Supplier : Perkin

- Elmer, Boston MA Catalog #: NET517001MC
Batch#: 639068 Radiochemical p urity: > 97% Specific activity : 162.9 Ci/mmol
Concentration of stock : 1.0 mCI/mL



## 5

. Positive control : 19

- norethindrone
Supplier: Sigma, St Louis, MO Catalog #: N4128
Batch #: 030M1359 Purity: 99%
CAS #: 68

- 22

- 4Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 4 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## 6

. Negative control : Octyltriethoxysilane
Supplier : Sigma, St Louis, MO Catalog # : 440213
Batch #: 72596AMV Purity: 98.58%
CAS #: 2943

- 75

- 1



## 7

. Solvent/vehicle control : Ethanol

Justification for choice of
solvent : None reported; 2,4

- D is more soluble in water than ethanol

Final Concentration : <3%



## B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): Trimmed uterine tissue from 85 -100 day old female (Crl:CD(SD)) rats that were ovariectomized approximately 7 -10 days prior to tissue harvest was purchased from Charles River (Wilmington, MA). Tissues were stored
at −10 ° C until use (up to 6 months). The uteri were weighed, placed in ice

- cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer and homogenized, followed by centrifugation for 10 min at 2500 × g at 4o C. 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 with the Pierce BCA method (Thermo Scientific Research Lab, Rockford, IL) using a protein kit compatible with DTT in the TEDG buffer. Cytosol was divided into aliquots (volume not reported) for immediate use o r storage at −80o C for up to 90 days. The cytosol preparation was identified as Batch 2, prepared on 8/10/11.

2. Saturation (radioligand) B inding E xperiment : A saturation binding e xperiment was conducted to demonstrate that the ER was present in adequate concentrations and had the appropriate affinity for the native ligand. A summary of the conditions for the saturation binding experiment are provided in Table 1.

TABLE 1 . Summar y of Conditions for Saturation Binding Experiment a 

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

- 3.0 nM

Concentration of non

- labeled ligand (100X [radioligand]) 3.0

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


### Temperature ~2

- 8 oC

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

EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM

a Data were not included in the study report, but were reported in the study profile submitted separately.
b T his 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.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 5 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## The specific activity of the stock [ 3 

## H]

- 17β -estradiol was not adjusted for decay over time on the day of the assay. Serial dilutions of radiolabeled estradiol in TEDG + PMSF buffer were prepared to achieve a final concentration of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1 and 3 nM.
Solutions of non- labeled 17β

- estradiol were prepared in a similar manner to achieve
concentrati ons that were 100- fold greater than each respective radiolabeled concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100 and 300 nM. For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 0.03 nM radiolabeled estradiol. 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 th e suggested 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

35 0 μL 30 0 μL

- -- T E DG + P M SF b u f f er
5 0 μL 5 0 μL 5 0 μL [ 3 

### H ]

- 17 β

- e s t r a d i o l ( 8 s e r ial d i l u ti on s ) e



### - -- 5 0 μL

- -- N o n

- la b eled 17 β

- e s t r a d i o l ( 8 s e r ial d il u ti on s , 100 x each r e s p ecti v e la b eled c o n ce n t r ati o n ) f



### 10 0 μL 10 0 μL

- -- Ute r i n e c y t o s o l (d il u ted to a p p ropr iate c o n c

. )

50 0 μL 50 0 μL 5 0 μL T o tal v o l u m e in ea c h a s s a y t ube

a Data were not included in the study report, but were reported in the study profile submitted separately.
b Total binding = [ 3 

### H]

- 17β

- estradiol bound to ER.

c Non

- specific binding = [ 3 

### H]

- 17β

- estradiol and 100

- fold greater non

- labeled bound to ER.
d Total [ 3 

### H]

- 17β

- estradiol alone for dpm determination at each concentration.

e Final concentrations of [ 3 

### H]

- 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 30 0 nM

.



## Tubes were incubated with for ~16 hours at ~4°C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed (4 times with 5

- minute intervals). Subsequently, the contents of each tube were washed three tim es as follows: TEDG +PMSF buffer was added, vortexed, centrifuged for 10 min at 1000 x g, and the supernatant decanted and discarded. After washing, e thanol was added to the HAP pellet remaining in each tube to extract the [3H]

- 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 approximately 4oC throughout the assay prior to extraction with ethanol. A total of two saturatio n binding runs on two batches of cytosol were performed with similar results. For the batch of cytosol used for the competitive binding assay, a single saturation run was performed.

3. Competitive B inding E xperiment : A summary of the experimental conditions for the

c ompetitive bind ing e xperiment is presented in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 6 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



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

### Source of receptor Rat U terine C ytosol Concentration of radioligand 1 nM

Concentration of receptor Sufficient to bind 6.75

- 7.15 % of radioligand Concentration of test substance (as serial dilutions) 10

- 1 1 

### to 10

- 4 

### mM Temperature 4

- 8 ° C

Incubation time 16

- 2 0 hours Composition of assay buffer Tris 10 mM (pH not reported )

EDTA 1.5 mM Glycerol 10% (v/v) Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM

a Data were obtained from page s 15 , 21, 40, 42 and 44 of the study report.



## The s olubility of 2,4-D in ethanol was evaluated visually. On the day of the assay, the specific activity of the stock solution [ 3 

## H] -17 β

- estradiol (not adjusted for decay over time )

was diluted in TEDG + PMSF buffer to achieve a final concentration of 1 nM

. For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 1.0 nM radiolabeled estradiol, until a concentration was reached that bound 6.75-7.15% of the total radioactivity added. Serial dilutions of the test substance,
weak positive control (19-norethindrone), negative control (octyltriethoxysilane), and reference material ( non- labeled 17β

- estradiol) were prepared to ach ieve the concentrations
shown in Table 4 . E ach assay co nsisted of three runs performed on separate days. E ach run included three replicates of each test substance at each co ncentration , plus four blank and six samples of the master mix to determine full radioactivity, resulting in a total of 112 samples.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 7 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



### TABLE 4. Molar (M) concentrations in Co mpetitive Binding Assay Run a , b



### 2,4 -D Positive control Negative control Reference Chemical

19

- norethindrone Octyltriethoxysilane Non

- labeled 17β

- estradiol

Tubes 81

- 104 c 

### Tubes 33

- 56 c 

### Tubes 57

- 80 c 

### Tubes 11

- 32 and 105

- 112 c



### 10

− 10 

### 10

− 8.5 

### 10

− 10 

### Solvent control d



### 10

− 9 

### 10

− 7.5 

### 10

− 9 

### 10

− 11



### 10

− 8 

### 10

− 7 

### 10

− 8 

### 10

− 10



### 10

− 7 

### 10

− 6.5 

### 10

− 7 

### 10

− 9.5



### 10

− 6 

### 10

− 6 

### 10

− 6 

### 10

− 9



### 10

− 5 

### 10

− 5.5 

### 10

− 5 

### 10

− 8.5



### 10

− 4 

### 10

− 4.5 

### 10

− 4 

### 10

− 8



### 10

− 4 

### 10

− 3 

### 10

− 7



### a Data were obtained from page s 40

- 41 of the study report.
b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or

non

- labeled 17β

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

### H]

- 17β

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

c Each concentration of each chemical was run in triplicate, for a total of 96 tubes per run.
d Solvent is ethanol



## Tubes were incubated with gentle vortexing for 16-20 hours at 4±2 ° C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and the tubes were vortexed (4 times with 5 -minute intervals). Subsequently, the contents of each tube were
washed three times as follows: TEDG+ PM SF buffer was added, vortexed, centrifuged for
10 min at 1000 × g , and the supernatant decanted and discarded. Ethanol was then added to the HAP pellet remaining in each tube to extract the [ 3 

## H] -17 β

- estradiol , allowed to sit at room temperature for 15-20 min with vortexing (4 times with 5

- minute intervals) , and centrifugation for 10 min at 1000 × g . A portion of supernatant was radioassaye d by scintillation counting. The temperature was maintained at 4±2 °C throughout the assay prior
to extraction with ethanol.

C. D ATA ANALYSIS : For the c ompetitive binding e xperiment , total binding and nonspecific binding data were modeled with a non- linear regression program [Graph Pad Prism v. 5.0 (GraphPad Software, Inc., La Jolla, CA) ]. Nonlinear regression methods were used to fit a curve for 17β -estradiol, 19-norethindrone, octyltriethoxysilane, and 2,4-D data to the Hill equation with l og IC 50 

## as a parameter to be estimated. Estimates of model parameters [e.g., l og IC 50 

### , IC 50 

### , Hillslope, R 2 

### , and relative binding affinity (RBA)] were determined with Graph Pad Prism.



## 1. Definitions

a. Classification of test material : Classification of the test material is based on the average of
three runs. Each run wa s first individually classified as follows:

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

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

− 6 

## M and either the lowest point on the fitted response curve within the range of the data is above 75 % (i.e.,Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 8 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



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

Equivocal up to the limit of concentrations tested = there are no data points at or above a
test chemical concentration of 10

− 6 

## M and either a binding curve can be fit but ≤50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be fit and the lowest average percent bind ing 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 :

B max 

## : maximum specific binding number (fmol ER/100 μg cytosol protein) measures the concentration of active receptor sites
K d 

## : dissociation constant ( nM), measures the affinity of the receptor for its natural ligand
IC 50 

## : concentration of the test substance (M) at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity ( RBA % ): ( IC 50 

## of 17 β

- estradiol ÷ IC 50 

## of test substance) × 100

II. RESULTS

A. SATURATION BINDING EXPERIMENT : Summary data pertaining to the saturation binding e xperiment were reported separately in the study profile submitted by the test order recipient. Individual data on saturation binding w ere not reported. Saturation binding

e xperiment parameters are presented in Table 5. The K d 

## for [ 3 

## H]

- 17β -estradiol was 0.1032
nM and t he B max 

## (nM) was 0.07097 for the prepared rat uterine cytosol used in these experiments. The K d 

## for the run was within the expected range of 0.03 to 1.5 nM. The B max 

## was also within the expected range of 10

- 150 fmol/100 μg protein. Non

- specific, specificEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 9 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## and total binding curves for [ 3 

## H] -estradiol to the ER are shown in Figure 1. The data produced a linear Scatchard plot (Figure 2).

TABLE 5. Saturation Binding Experiment of 17 β

- estradiol with Estrogen Receptor from Rat Uterine Cytosol

Parameter Run 1 Run 2 Run 3 Runs 1

- 3

R 2 

### (unweighted) 0.967 NR NR NR

B max 

### (nM) 0.07097 NR NR NR

B max 

### (fmol/100 μg protein) 59.28 NR NR NR

K d 

### (nM) 0.1032 NR NR NR

a Data were not included in the study report, but were reported in the study profile submitted separately.
b Only a single run of the saturation b inding e xperiment was conducted on the batch of cytosol used for this

c ompetitive b inding e xperiment.

R 2 

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



## F IGURE 1. Binding of [ 3 

## H] -17 β -Estradiol to the ER during the Saturation Binding Experiment.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 10 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## F IGURE 2. Scatchard Plot of the Binding of [ 3 

## H] -17 β -E stradiol to the ER.

B. COMPETITIVE BINDING EXPERIMENT : The results from the three c ompetitive binding e xperiments are summarized in Table 6 and presented graphically in Figures 3-5. No precipitation was observed at any concentration tested

. The mean specific binding in the presence of 2,4- D was >9 5% at concentrations ≤ 10

− 4 

## M in all three runs. The estimated

mean l og IC 50 

## and RBA was not calculated for 2,4-D as the percent binding inhibition did not reach 50% for any run.

The estimated mean l og IC 50 

## for the natural ligand, 17 β -estradiol, and the weak positive control (19-norethindrone ) was

− 9.0 and

− 5.5 M, respectively. The mean RBA was 0. 034 %
for 19-norethindrone. As the lowest average percent binding in the presence of 2,4-D was ≥ 95% at concentrations up to 10 −4 

## M, 2,4-D is classified as not interactive (0) in this assay (Table 7).Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 11 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



### TABLE 6. Competitive Binding Assay of 2,4

- D with Estrogen Receptor from Rat Uterine Cytosol a



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



### r 2 

### (unweighted), 17 β

- estradiol 0.9990 0.99 9 7 0.9995 0.9990

- 0.9997

19

- norethindrone 0.9999 0.9997 0.9962 0.9962

- 0.9999 2,4

- D 0.5875 NC 0.7841 0.5875

- 0.7841 Log IC 50 

### (M), 17 β

- estradiol

− 8.9 4 8

− 8.942

− 8.961

− 8.951 ± 0.006

19

- norethindrone

− 5.462

− 5.435

− 5.534

− 5.4 7 7 ± 0.030 2,4

- D NC NC NC NC

IC 50 

### (M), 17 β

- estradiol 1.1 3 × 10

− 9 

### 1.14 × 10

− 9 

### 1. 10 × 10

− 9 

### 1.12 × 10

− 9 

### ± 0.01 × 10

- 9



### 19

- norethindrone 3.4 5 × 10

− 6 

### 3.76 × 10

− 6 

### 2.92 × 10

− 6 

### 3.33 × 10

− 6 

### ± 0.25 × 10

- 6


### 2,4

- D NC NC NC NC Log RBA (%), 19

- norethindrone

− 3.5

− 3.5

− 3.5

− 3.5 ± 0.0 2,4

- D NC NC NC NC RBA (%), 19

- norethindrone 0.033 0.031 0.032 0.03 2 ± 0.001 2,4

- D NC NC NC NC

a Data were obtained from page 33 of the study report.
b The range is reported for r 2 

### ; the mean ± SE is reported for all other parameters.

r 2 

### Goodness of fit RBA (%) R elative binding affinity
NA Not applicable. r 2 

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

TABLE 7. Binding Classification of 2,4

- D with Estrogen Receptor a



### Run 1 2 3 Mean c 

### Binding Classification d 

### Classification category value b 

### 0 0 0 0 Not Interactive

a Data were obtained from pages 28

- 30 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 Mean of three runs expressed to the tenths place
d Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5



## FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test Compound (Run 1)

.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 12 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## FIGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test Compound (Run 2).

FIGURE 5. Percentage E2 Bound to the Estrogen Receptor in the Presence of Test Compound (Run 3).

C. PERFORMANCE CRITERIA : To ensure that the competitive binding assay function ed properly, each run was evaluated using the following criteria (Table 8) :Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 13 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



### TABLE 8. Criterion a 

### Tolerance Limit(s) Value Yes No

17 β

- estradiol fitted curve parameters Log e 

### residual SD [Log e 

### (SyX)] ≤2.35

− 0.11 to 0.54 X Top (% binding) 94 to 111 95 to 99 X Bottom (% binding)

− 4 to 1

− 0.9 to

− 0.4 X Hill Slope (log 10 

### (M)

− 1 

### )

− 1.1 to

− 0.7

− 1.08 to

− 0.98 X Weak Positive control ( 19

- norethindrone ) fitted curve parameters b


### Log e 

### residual SD <2.60

− 0.52 to 1.10 X Top (% binding) 110 to 90 89 to 100 X Bottom (% binding) 1 to

- 5

− 1.2 to 1.7 X Hill Slope (log 10 

### (M)

− 1 

### )

- 1.1 to

- 0.7

− 1.2 to

− 0.99 X

Solvent concentration

Ethanol ≤3% <3% X Negative control (octyltriethoxysilane) does not displace more
than 25% of [ 3 

### H]

- 17 β

- estradiol from the ER on average across all concentrations ≤25% ≤13.5 X

a Data were obtained from page 33 of the study report.
b The EPA Guideline does not define a set of tolerance limits for 19

- norethindrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19

- norethindrone to be an acceptable weak positive control.
NA Not applicableEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 14 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## Additionally, the curve for the reference material showed that increasing concentrations of unlabeled 17β

- estradiol displaced [ 3 

## H]

- 17β -estradiol in a manner consistent with one- site binding, as indicated by a Hill slope of approximately

− 1.0. The recommended ranges of the weak positive control 19-norethindrone were occasional outside of the ranges for the top and bottom of curves established for norethynodrel. The fitted curve parameters demonstrate that 19-norethindrone is an acceptable weak positive control in the subject assay.

The percent binding of 2,4

- D at th is top plateau 98.8 – 103.5% was within 25 percentage points of the value for the lowest concentration of the estradiol standard 93.5 – 98.1%. Examination across the runs indicated consistency of the Hill slope, placement along the X

axis, and top and bottom plateaus.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Based on the combined responses in each of three independent estrogen receptor binding assays, it was determined that 2,4-D had no appreciable effect on the binding of the reference estrogen at any concentration, up to
10 −4 

## M. The results of the in vitro estrogen receptor binding assay using rat uterine cytosol indicate that, under the conditions of this study, 2,4- D was negative ( not interactive ) for estrogen receptor at concentrations up to 10 −4 

## M.

B. AGENCY COMMENTS : The highest concentration of 2,4- D tested in this assay was 10 -4 

## M. The Test Guideline recommends testing up to 10 -3 

## M unless there is evidence of insolubility. The justification provided in the study report for lowering the top concentration was based on toxicokinetic data generated in the rat. The Test Guideline recommends testing up to 10

- 3 

## M to adequately assess the potential for the test chemical to
interact with the ER in mammalian as well as non

- mammalian taxa. The justification for lowering the top concentration is inadequate since it does not apply to non- mammalian taxa.

Summary data pertaining to the saturation binding experiment were r eported separately in the study profile submitted by the test order recipient; individual data on saturation binding were not reported. The K d 

## for [ 3 

## H]

- 17β -estradiol was 0.1032 nM and the B max 

## (nM) was 0.07097 for the prepared rat uterine cytosol used in these experiments. The K d 

## for the run was within the expected range of 0.03 to 1.5 nM, and the B max 

## was within the expected range of 10

- 150 fmol/100 μg protein. The data produced a linear Scatchard plot (Figure 2).

The mean specific binding in the presence of 2,4- D was ≥ 95% at concentrations ≤ 10

− 4 

## M in
all three runs. The estimated mean l og IC 50 

## and RBA was not calculated for 2,4- D as the percent binding inhibition did not reach 50% for any run

The estimated mean l og IC 50 

## for the natural ligand, 17 β -estradiol, and the weak positive control (19-norethindrone ) was −9.0 and

− 5.5 M, respectively. The mean RBA was 0.034%
for 19 -norethindrone. All performance criteria were met for 17 β

- estradiol and octyltriethoxysilane. Examination of the data for the reference ligand and the weak positive control across the runs indicated consistency of the Hill slope, placement along the X- axis, and top and bottom plateaus.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 15 of 15 2,4

- D/ 030001 O CSPP 89 0.1250 / OECD None



## 2,4- D was tested over a concentration range that fully defin ed the top of the curve. The percent binding at this top plateau 98.8 – 103.5% was within 25 percentage points of the
value for the lowest concentration of the estradiol standard 93.5 – 98.1%. Therefore, based on the combined responses in each of three independent estrogen receptor binding assay runs, it was determined that 2,4-D was not interactive with the estrogen receptor at concentrations up to 10 −4 

## M.

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

• The highest concentration of 2,4- D tested in this assay was 10 -4 

## M. The Test Guideline recommends testing up to 10 -3 

## M unless there is evidence of insolubility. The justification provided in the study report for lowering the top concentration was based on toxicokinetic data generated in the rat. The Test Guideline recommends testing up
to 10

- 3 

## M to adequately assess the potential for the test chemical to interact with the ER
in mammalian as well as non- mammalian taxa.

# DATA EVALUATION RECORD

2,4- DICHLOROPHENOXYACETIC ACID (2,4

- D)

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-26-2012 (MRID 48614304)

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: 2/19 /2012

Secondary Reviewer : Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 3/2 6 /2012

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

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

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

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathway s. 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).2

, 4-D/030001 Estrogen Receptor Transcriptional Activation (2012) I Page 1 of8
OCSPP 890.1300/ OECD 455



## Primary Reviewer: Ray Kent, Ph.D.


## Health Effects Division Secondary Reviewer: Minerva Mercado, Ph.D.


## Health Effects Division

## Signature:**~ ~@** 

# Date:**~** Signatur&

_. Date: 

## cP 

# - To::;s=

Template version 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line

, HeLa-9903);

OCSPP 890.1300; OECD 455.



## PC CODE: 030001

TXR#: 0052104



## TEST MATERIAL (PURITY): 2,4-D (98.5% a.i.)



## SYNONYMS: (2,4-Dichlorophenoxy) acetic acid 

## DP BARCODE: D398640



## CAS No.: 94-75-7



## CITATION: LeBaron

, M.J., Kah

, H.L. (2011). Evaluation of2,4-Dichlorophenoxy Acetic
Acid (2.4-D) in an In Vitro 

## Estrogen Receptor Transcriptional Activation Assay
in Human Cell Line HeLa-9903. Toxicology & 

## Environmental Research and Consulting

, Midland

, MI. Laboratory Report No.: 111043

, October 17, 2011.

MRID 48614304. Unpublished.



## SPONSOR: Industry Task Force 

## II on 2,4-D Research Data, c/o McKenna Long & 

## Aldridge

LLP

, 1900 K Street NW

, Washington, D.C.



## TEST ORDER#: CON-03001-01



## EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48614304), hERa-HeLa-9903 cells cultured in vitro 

## were exposed to 2,4-D (98.5% a.i., Lot #2006
2433 8006 USA) at logarithmically increasing concentrations from 10

- 10 

## to 10

- 4 

## M in DMSO (0.1 % ) for 24 hours

. A total of four separate runs were performed. Each run was performed using
96-well plates and each 2,4-D concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent 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

.

2,4-D was tested up 10

- 4 

## M based on solubility

, cytotoxicity and in vivo 

## toxicokinetic analysis.
There were deviations from expected performance criteria for all of the four reference chemicals,
but these deviations do not affect the interpretation of this study. The RPCMax was <0% for the
first run , 8.8% for the second run , 5.3% for the third run, and 7.0% for the fourth run ; the associated
PCMax was 10

- 4 

## M for runs 1-3 and 10- 5 

## M for run 4. Because the RPCMax < PC10 in all assay runs , 2,4-D was considered negative for estrogen receptor transcriptional activation in this test system.

This assay satisfies the EDSP Tier I Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890

. 1300)

.



## COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality

statements were provided.Estrogen Receptor Transcriptional Activation ( 2012 ) / Page 2 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance : 2,4

- D
Description: Technical, off

- white powder
Source (Catalog #) : Nufarm Americas ( Not Reported )
Lot/Batch #: 2006 2433 8006 USA Purity: 98.5 % Solubility : Up to 315 mg/L in water, soluble in DMS O up to 0.1M Volatility : 1.9 × 10

- 5 Pa at 25 ° C Stability: 2 year shelf life Storage conditions: Ambient Vapor pressure : 

### 1.9 x 10

- 5 

### Pa at 25°C

CAS #: 94

- 75

- 7
Structure :



## 2. Reference substances
17 β

- estradiol (strong estrogen; positive control)
Supplier : Sigma, St. Louis, MO Catalogue and Batch #: Cat# E

- 8875, Lot# 079 K 0131 Purity: 100 %
CAS # : 50

- 28

- 2



## 17α

- estradiol (weak estrogen)
Supplier: Sigma, St. Louis, MO Catalogue and Batch #: Cat # E

- 8750, Lot # 029K4116 Purity: ≥ 99.5%
CAS # : 57

- 91

- 0



## Corticosterone (negative compound)
Supplier: Sigma, St. Louis, MO Catalogue and Batch #: Cat # C

- 2505, Lot # 010M2010 Purity: 100%
CAS # : 50

- 22

- 6



## 17α

- methyltestosterone (very weak agonist)
Supplier: Sigma, St. Louis, MO Catalogue and Batch #: Cat # M

- 7252, Lot # 060M1543V Purity: 99%
CAS # : 58

- 18

- 4



## 3. Vehicle(s)

Solvent: DMS O

, Sigma

- Aldrich, Cat # 276855

Solvent control (final conc e n tration ): 0.1%Estrogen Receptor Transcriptional Activation ( 2012 ) / Page 3 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



## B. METHODS

1. C ell C ulture : Stably

- transfected hER α

- HeLa -9903 cell s were obtained from the Japanes e Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma infection by ATCC (method not reported) . Cells were maintained in Eagles Minimum Essential Medium without phenol red, supplemented with 60 mg/L kanamycin and 10%
dextran

- coated charcoal

- treated fetal b ovine serum (DCC- FBS ; Hyclone Laboratories, Inc.

, Logan, Utah Lot# not reported), in an incubator under 5% CO 2 

## at 37o C. Upon reaching 75-
90% confluence , cells were subcultured at least twice prior to exposure to the test material.

2. Transcriptional Activation Assay s: For each test, cells were plated at a density of 1
- 1.5×10 4 

## cells /100 μL medium/well in a 96

- well plate and allowed to attach for at least 3
hours. G rowth media was replaced with media containing serial log dilutions of 2,4-D in
DMSO (0.1% total final concentration ). Cells were incubated for approximately 24 hours at approximately 37° C. Cytotoxicity was determined by a modified MTT cell viability assay

, when stock solutions were diluted with tre atment media, after addition to the cell culture plate and after the 24 -hour treatment period. Transcriptional activation of the estrogen receptor (firefly luciferase activity) was determined using a standard assay kit (Promega, Madison, WI). Chemilumine scence was measured using a Packard TopCount NXT
luminescence counter with a detection limit of ~5 cells in 100 μ L medium.

a. P reliminary T est : A preliminary test evaluating concentrations ranging from 10

− 4 

## 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 : It was stated that laboratory validation assays with 10 proficiency chemicals were performed to confirm the responsiveness of the ER transcriptional activation assay. These non- GLP unpublished results were reported to
have demonstrated lab oratory proficie ncy.

c. Reference Chemicals : To ensure th e stability of the response from the cell line, six concentrations of each of the following reference chemicals were included on each plate in the current assay, along with the test chemical :

Reference Chemical CAS No. Concentration R ange Class 17β

- estradiol 50

- 28

- 2 10

− 14 to 10

− 8 Strong estrogen

17α

- estradiol 57

- 91

- 0 10

− 12 to 10

− 6 Weak estrogen

Corticosterone 50

- 22

- 6 10

− 10 to 10

− 4 Negative compound

17α

- methyltestosterone 58

- 18

- 4 10

− 11 to 10

− 5 Very weak agonist



## 3. Data analysis: To obtain t he relative transcriptional activity to the 1 nM E2 positive control (PC) , the luminescence signals from the concurrent plate were analyzed by subtracting the
mean value of the vehicle control from each well value to norm alize the data ; each
normalized value was then divid ed by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to express relative transcriptional activity as a percentage of the PC. Graph Pad Prism v. 5.0 (GraphPad Software, Inc., La Jolla, CA) was
used to calculate the EC 50 

## , PC 10 

## , PC 50 

## , RPC Max 

## , and PC Max 

## for 2,4-D when applicable . T he
test material was defined as negative for inducing estrogen receptor transcriptional
activation if the RPC Max 

## < PC 10 

## in at least 2 of 2 (or 2 of 3) runs. Log EC 50 

## and Hill slop eEstrogen Receptor Transcriptional Activation ( 2012 ) / Page 4 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



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

w ere calculated for the luminescence data triplicates. Concentrations showing > 20% cytotoxicity or evidence of insolubility were excluded from analyses.

4. Definitions

EC 50 

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

PC 10 

## = concentration of a test chemical at which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

## = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

## = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

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



## II. RESULTS

A. PRELIMINARY TEST : 2,4- D was relatively non

- toxic and freely soluble; therefore the highest concentration was set at 10

− 4 

## M based on the in vivo 2,4-D plasma concentrations
that exceeded the threshold for linear toxicokinetics. These data were provided in an appendix to the report. No solubility or cytotox icity issues were noted at the concentrations
tested. Based on these results, logarithmically increasing concentrations from 10

− 10 

## to 10

− 4 

## M were selected for the assay.

TABLE 1

. Preliminary T est for Solubility , C ytotoxicity , and Concentration

- Selection for 2,4

- D a
Concentration (M) % Viability Comments

10

− 4 119.8 Toxicokinetic

- derived limit dose

10

− 5 123.6

10

− 6 133.1

10

− 7 132.0

10

− 8 115.1

10 −9 130.4

10 −10 117. 6

E2 at 1 nM 130.8

VC b 100.0

a Data were obtained from page 51 of the study report.

b Vehicle Control



## B. Positive and Negative Reference Chemicals

1. Proficiency Chemicals : The responsiveness of cells to the required proficiency chemicals was not reported , but it was stated that the proficiency validation assays were conducted, and proficiency was demonstrated

.Estrogen Receptor Transcriptional Activation ( 2012 ) / Page 5 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



### TABLE 2 . Proficiency Chemicals a
Compound Expected R esponse Lab R esponse Diethylstilbes t rol Positive Not reported

17 α

- Ethynyl estradiol Positive Not reported

Hexestrol Positive Not reported

Genistein Positive Not reported

Estrone Positive Not reported Butyl paraben Positive Not reported
1, 3, 5

- Tris(4

- hydroxyphenyl)benzene Positive Not reported Dibutyl phthalate Negative Not reported

Atrazine Negative Not reported

Corticosterone Negative Not reported



## 2. Reference Chemicals : Values derived from the concentration response curve ( e.g ., l og PC 50, 

## log PC 10, 

## log EC 50, 

## Hill slope) for the four concurrently run reference materials are included in
Table 3. There were deviations from expected performance criteria for all of the four
reference chemicals. The l og EC 50 

## was higher than the expected value and the Hill slope lower than the expected value for 17 β -estradiol. The l og PC 50 

## and l og PC 10 

## for 17 α

- estradiol
and 17 α

- methyltestosterone were lower than expected, as was the Hill slope for 17 α

estradiol. For 17α

- methyltestosterone, the mean RPC Max 

## was 90.8% for the first run, 152.8% for the second run, 83.3% for the third run, and 67.4% for the fourth run. For corticosterone

, the mean RPC Max 

## was 6.3% for the first run, 35.5% for the second run, 7.0% for the third run, and 5.4% for the fourth run. The deviations from the expected values do not negatively affect the interpretation of this study. Perform ance criteria values below the validated ranges usually indicate increased sensitivity of the assay compared to validation experiments.

TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical
Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 3 Run 4 Yes No 17β

- estradiol Log PC 50 −11.4 to −10.1

− 10.8

− 11.1

− 10.9

− 10.7 X Log PC 10 <−11

− 11.9

− 12.6

− 12.7

− 12.5 X Log EC 50 −11.3 to −10.1

− 9.4

− 10.4

− 10.7

− 10.6 Run 1 Hill Slope 0.7 to 1.5 0.3 0.3 0.7 0.8 X Test range 10 −14 to 10 −8 M 10 −14 to 10 −8 M X

17α

− estradiol Log PC 50 −9.6 to −8.1

− 9.3

− 10.1

− 9.0

− 9.0 X Log PC 10 −10.7 to −9.3

− 10.6

− 10.9

− 10.6

− 10.9 X Log EC 50 −9.6 to −8.4

− 8.7

− 9.6

− 8.8

− 8.6 X Hill Slope 0.9 to 2.0 0.5 1.1 0.7 0.6 X Test range 10 −12 to 10 −6 M 10 −12 to 10 −6 M X

Corticosterone Test range 10 −10 to 10 −4 M 10 −10 to 10 −4 M X

17α

− methyltestosterone Log PC 50 −6.0 to −5.1

− 7.0

− 8.8

− 7.5

− 6.5 X Log PC 10 −8.0 to −6.2

− 8.8

− 9.8

− 9.2

− 9.2 X Test range 10 −11 to 10 −5 M 10 −11 to 10 −5 M X

a Data were obtained from page 27 of the study report

.



## C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls : Data for the vehicle and positive controls are included in
Table 4. The overall mean TA value for the vehicle control was 1513-3035 arbitrary lightEstrogen Receptor Transcriptional Activation ( 2012 ) / Page 6 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



## units, and the overall mean TA value for the positive control was 12128-19639 (not reported for Run 4). The induction for the positive control ranged from 4.7- to 8.2-fold. The mean normalized value for the positive control was 10912-16604. The PC 50 

## (50% of the maximum response) for E2 in this assay is 6212.5-9819.5 and the PC 10 

## (10% of the maximum response) is 1243-1964.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a
Sample Vehicle Control Positive Control b Normalized Positive Control b

Runs Mean SD Mean SD Fold Induction c Mean SD

1 2743 248 15564 1604 5.7 12821 NR

2 1513 290 12425 1958 8.2 10912 NR

3 3035 271 19639 1116 6.5 16604 NR

4 2603 NR 12128 d 915 d 4.7 d 12158 NR

a Data were obtained from page 47, 49 and 53 of the study report

. b Positive control was 17 β

- estradiol (E2) at 1 nM.

c Fold

- induction = (mean TA of PC)/(mean TA of VC)

d Calculated by the reviewer from data on page 49 of the study report NR Not reported



## 2. Test Material : Relative (to the PC) transcriptional activation at each concentration of the test chemical during the three assay runs is presented in Table 5. The concentration

response bar graphs depicting fold induction of relative transcriptional activation are presented in Figure 1 below. The RPC M ax 

## was <0% for the first run, 8.8% fo r the second
run 5.3% for the third run and 7.0% for the fourth run ; the associated PC M ax 

## was 10

− 4 

## M for
Runs 1-3 and 10 -5 

## M for Run 4. Because the RPC Max 

## < PC 10 

## in all four runs, 2,4-D was considered negative for estrogen receptor transcriptional activation in this test system

.

TABLE 5. Relative Transcriptional Activation (RTA) of 2,4

- D a

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

Run 1 Run 2 Run 3 Run 4 Conc. (M) Mean SD Mean SD Mean SD Mean SD

10 −4 −7.2 3.8 8.8 1.0 5.3 1.1 4.4 4.8

10 −5 −4.6 4.1 3.3 4.1 1.7 1.0 7.0 3.6

10 −6 −10.1 0.9 −0.6 1.8 −6.6 0.4 2.4 0.3

10 −7 −8.9 3.4 −2.1 2.0 −6.0 3.6 −1.4 2.3

10 −8 −8.8 5.0 −2.5 1.4 −5.8 5.4 −1.1 2.7

10 −9 −6.2 4.8 −3.2 0.9 −7.5 4.7 0.1 1.9

10 −10 −1.9 3.6 −0.8 1.2 −2.5 3.8 4.3 1.1
Log EC 50 b NA NA NA NA Hill Slope b NA NA NA NA

RPC Max 0 b 8.8 5.3 7.0

PC Max NA 10 −4 10 −4 10 −5

PC 50 NA NA NA NA

PC 10 NA NA NA NA

a Data were obtained from page 54 of the study report.

b Value of RPC M ax less than or equal to 0
NA Not ApplicableEstrogen Receptor Transcriptional Activation ( 2012 ) / Page 7 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



## Figure 1. Fold Induction of Relative Transcription Activation (RTA) of 2,4-D Compared to the Positive Control.

VC= Vehicle Control PC= Positive Control (1 nM E2)



## 3. Performance C riteria : The results of the laboratory proficiency test were not reported.
There were minor deviations from expected performance criteria for all of the four reference chemicals. The Log EC 50 

## was higher than the expected value and the Hill slope lower than the expected value for 17 β -estradiol. The l og PC 50 

## and l og PC 10 

## for 17 α -estradiol and 17 α

methyltestoste rone were lower than expected, as was the Hill slope for 17 α

- estradiol. The minor deviations from the expected values do not negatively affect the interpretation of this study. M ean l uciferase activity was 4.7 to 8.2- fold . The fold-induction corresponding to the
PC 10 

## of the concurrent PC was greater than 1+2 SD s of the vehicle control on all plates. Variability was minimal , and results were reproducible among runs.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS ’ CONCLUSIONS : Based on the combined responses in each of four independent estrogen receptor transactivation assays, it was determined that 2,4- D treatment did not result in ER- mediated transcriptional activation at any concentration, including the
toxicokinetic

- derived limit, in this assay s ystem (10

− 4 

## M). The highest concentration of 2,4 D was base d on in vivo toxicokinetic analyses; concentrations higher than 10

− 4 

## M were not considered relevant for use in this assay as they are substantially above the inflection point for linear toxicokinetics

.

B. AGENCY COMMENTS : 2,4-D was tested up to the toxicokinetic- derived limit, 10

− 4 

## M. The laboratory proficiency assays were not reported . The only deviation was a weak positive response in one run in response to corticosterone. There wer e minor deviations from expected performance criteria for all of the four reference chemicals, but these minorEstrogen Receptor Transcriptional Activation ( 2012 ) / Page 8 of 8 2,4

- D/030001 O CSPP 89 0.1300 / OECD 455



## deviations do not negatively affect the interpretation of this study. The RPC Max 

## was <0% for the first run, 8.8% for the second run 5.3% for the third run and 7.0% for the fourth run;
the associated PC M ax 

## was 10 −4 

## M for Runs 1-3 and 10 -5 

## M for Run 4. Because the RPC Max 

## < PC 10 

## in all four runs, 2,4-D was considered negative for estrogen receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES : The following deficiencies were noted:

• The laboratory proficiency assays were not included in the study report, but were sent in an accompanying study profile template.

• 2,4- D was not tested up to the limit dose, but rationale was provided to support the
concentrations tested.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 1 of 86

Version: 22 September 2011 

### Data Requirement: EPA DP Barcode 388580

OECD Data Point

EPA MRID 48317001

EPA Guideline 890.1350

Fish Short-Term Reproduction Assay

Test Material: 2,4-Dichlorophenoxyacetic acid Purity (%): 98.6%

Common Name 2,4-D

Chemical Name IUPAC

CAS Name

CAS No. 94-75-7

Synonyms 2,4-D acid

EPA PC Code 030001

Primary Reviewer: Patience Browne Signature:

USEPA/OCSPP/OSCP Date: 06/25/2012

Additional Reviewer: Alicia Korol Signature: No longer with EPA

USEPA/OCSPP/OPP/EFED/ERB1 Date: 06/14/2011

Additional Reviewer: Amy Blankinship Signature:

USEPA/OCSPP/OPP/EFED/ERB3 Date : 12/28/2012

Final Additional Reviewer: Robin Sternberg Signature:

USEPA/OCSPP/OPP/EFED/ERB1 Date: 05/27/2015

Date Evaluation Completed: 05/27/2015 Digitally signed by ROBIN

STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126

Date: 2015.06.01 13:17:23 -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 15:14:20 -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 10:04:22 -04'00'

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 2 of 86

Version: 22 September 2011 

### CITATION: Marino, T.A., K.K. Coady, L.K. Sosinski, J. Thomas. 2010, DICHLOROPHENOXYACETIC ACID:

A FISH SHORT-TERM REPRODUCTION ASSAY USING THE FATHEAD MINNOW, Pimephales promelas

, Toxicology and Environmental Research and Consulting, The Dow Chemical Company. and Midland,

Michigan 48674, Laboratory ID: 101026, Industry Task Force II on 2,4-D Research Data c/o McKenna

Long & Aldridge LLP, Washington, D.C, Completed on 06 December 2010.

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

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

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

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

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

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

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

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

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

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

preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines,

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 3 of 86

Version: 22 September 2011 

### EXECUTIVE SUMMARY

The 21-day short-term reproduction assay of 2,4-D with fathead minnow ( Pimephales promelas ) was conducted

under flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each group; 4

groups/treatment; ca . 6 months old) were exposed to 2,4-D (98.6% purity) at nominal concentrations of 0

(negative control), 0.400, 4.00, 40.0, and 100 mg a.i./L concentrations with corresponding mean-measured

concentrations of <0.10 (<LOQ, negative control), 0.245, 3.14, 34.0, and 96.5 mg a.i./L, respectively. The

test system was maintained at 24.5 to 25.2 o 

### C and a pH of 7.02 to 7.76.

The single mortality observed during the assay occurred in a female in the high treatment group. There were

no significant differences for male or female body weight or length relative the negative control. At test

termination, observations of secondary sex characteristics were observed in the negative control and treated

groups; no treatment-related effects were reported. Clinical signs included the loss of an eye, ascites, and

scoliosis (bent tail) which were observed in single fish in the negative control or treatment groups.

Spawning occurred in the negative control at least ev ery 4 days in 3 of the 4 replicates, and mean fecundity

was 29.8 eggs/female/day/replicate; fertility in the negative control was 96.9%. Fecundity was significantly

decreased (Jonckheere-Terpstra; p<0.05) by 34% in the high treatment group with a non-significant (p>0.05)

concentration-dependent trend of decreased fecundity in the lower treatment groups compared to the negative

control. There were no significant differences for fertility between the 2,4-D treatments and the negative control.

There were no significant differences (p>0.05) bet ween the 2,4-D treatment groups and the negative control

for male or female gonado-somatic index (GSI) or plas ma vitellogenin (VTG). There was also no significant

difference (p>0.05) for male nuptial tubercle scores; no tubercles were observed for females. No apparent

treatment-related histopathological effects were observed in males and females. Although not concentrationdependent, an increase in the number of female ovaries that were observed as Stage 2 (compared to Stage

3 or 4) was reported for the 2,4-D treatments compared to the negative control. Plasma sex steroid

concentrations were not reported.

The performance and validity criteria were met in this study with the exception that the coefficient of variation

(CV) for the mean-measured concentration of the lowest treatment group was 43%, exceeding the guideline

criterion of < 20%. This deviation did not impact the interpretation of the study.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 4 of 86

Version: 22 September 2011 

### 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: 3.7 g for males, 1.9 g for females

Mean length at test initiation: Not reported

Test type: Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 5 of 86Version: 22 September 2011Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with 2,4-D.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.245NoNoNoNoNoNoNoNoNoNoNANANANA3.14NoNoNoNoNoNoNoNoNoNoNANANANA34.0NoNoNoNoNoNoNoNoNoNoNANANANA96.5YesNoNoNoNoNoNoNoNoNoNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 6 of 86

Version: 22 September 2011 

### I. MATERIALS AND METHODS

Guideline Followed: This study was conducted in accordance with the Endocrine Disruptor

Screening Program Guidelines, OCSPP (form. OPPTS) 890.1350: Fish

Short-Term Reproduction Assay, Environmental Protection Agency (EPA)

740-C-09-007, October 2009. Supporting guidance documents used

include: Organization for Economic Cooperation and Development (OECD)

Guideline for Testing of Chemicals No. 229: “Fish Short Term Reproduction

Assay”, 2009; “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, 2002; and “Guidance

Document on Aquatic Toxicity Testing of difficult Substances and Mixtures”,

OECD Series on Testing and Assessment. No. 23. 2000. Deviations from

the OCSPP 890.1350 include the following:

1. The coefficient of variation (CV) for the mean-measured concentration exceeded 20% in the 0.245

mg a.i/L treatment group.

2. Ammonia levels were detected at 0.24 mg/L, exceeding guideline recommendations of ≤ 1 μ g/L.

3. Organic carbon levels were not reported in the routine laboratory dilution water solute analysis.

4. OCSPP guidelines recommend chlorine levels at <10 μ g/L; chlorine in the dilution water was not

detectable at a level of 0.20 mg/L and it is unknown whether levels exceeded 10 μ g/L.

These deviations do not impact the interpretation of the study.

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

statements were provided. All phases of this study were conducted in

accordance with the GLP Principles of the USEPA – FIFRA GLPs Title 40

CFR, Part 160 – Federal Insecticide Fungicide Rodenticide Act (FIFRA),

Good Laboratory Practice Standards, Final Rule; OECD Series on principles

of Good Laboratory Practice and Compliance Monitoring, Number 1. OECD

Principles on Good Laboratory Practice (as revised 1997)

ENV/MC/CHEM/(98)17; and European Community (EC) – European

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 7 of 86

Version: 22 September 2011 

### Parliament and council Directive 2004/10/EC (O.J. No. L 50/44,

20/02/2004).

A. Test Material 2,4-Dichlorophenoxyacetic acid

Description: Water Solubility = 569 mg/L

log Kow = 2.81

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. : Lot No. 2006 2433 8006-USA

Purity: 98.6%

Impurities: None identified

Stability of Compound: The measured 2,4-D concentrations in the replicates across the treatments

ranged from 28.0 to 105% of nominal concentrations. Measured

concentrations were highly variable, particularly at lowest test concentrations,

and the study report attributed this to possible microbial metabolism in the

tanks even though there was regular cleaning of the tanks. The CVs for the

replicates in the lowest treatment group ranged from 38-49%. The mean

measured concentrations were 0.245, 3.14, 34.0, and 96.5 mg a.i./L 2,4-

D which was 61.3, 78.5, 85.0, and 96.5% of nominal concentrations,

respectively.

Storage Conditions of

Test Chemicals: Not reportedData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 8 of 86Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):not reportedWere fish obtained from a singlelaboratory stock?not reportedTest fish were obtained from the commercialsupplier, New England Bioassay,Manchester, CT. EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameasdefinitive test?Yeswater quality, temperature, and lighting werethe same as test conditions EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:30 days EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 9 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:For 2 weeks out of the 30 days period,mortality was reported at a rate of 1.8% 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 reco mmends 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:thawed, frozen brineshrimp 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, UTFrequency of feeding:at least 2 times/day1.25-2.0 ml shrimp/replicate vesselData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 10 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding:Feeding start date not reported, but fishwere fed during the holding/acclimation,pre-exposure, and exposure periods. Foodwas withheld 12 hours prior to testtermination.Fish food tested free of relevantcontaminants.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:5.5 months6 months at beginning of exposure periodEPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at test initiation (if determined):3.7 gMean calculated from all males selected forthe test EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex.Range of individual weights(males) at testinitiation (if determined):individual data not providedrange of individual weights were kept within±20% of the mean It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Mean weight of females at test initiation (if determined):1.9 gMean calculated from all females selectedfor the testData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 11 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsRange of individual weights (females) at test initiation (if determined):Individual data notprovidedrange of individual weights were kept within±20% of the meanMean length of males at test initiation (if determined):Not reportedMean length of females at test initiation (if determined):Not reportedDuration of pre-exposure phase:14 days EPA recommends a minimum of 14 days.Were pre-exposure conditions identical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:32The 32 test vessels included 12 test vesselsmore than the 20 that would be needed forthe definitive test. EPA recommends that additional tanks set up at the beginning of pre - exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 12 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsPre-exposure fecundity:>15 eggs/female/reproductive day/replicatemean = 28.8 eggs/female/day; range from5.6 to 53.2 eggs/female/day across allreplicates 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 pre-exposure:>2 times in 7 daysThe top 20 performing spawning groups(>15 eggs/female/reproductive day) wereused in the exposure period and were assigned to concentration levels via completerandomized block design. EPA recommends that spawning occur at least

twice in the 7 days prior to the definitive test.Details on pre-exposure:Spawning substrate were inspected and thenumber of eggs laid and eggs found to beinfertile were recorded dailyData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 13 of 86Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Fflow-through EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilutionsystem:Continuous flow diluterA continuous-flow diluter (syringe pumpdelivery) system delivered stock solution to amixing chamber where the solution was mixedwith dilution water. The test solution was splitequally to replicate test vessels. Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:45 ± 4.5 mL/min Recommended flow-through rate is 45

mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Correctionsand Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 14 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:Diluter system was calibrated prior to exposureperiod. For concentrations measured in testvessels, mixing cells, and stock solutions, thepercent of target concentration ranged from108% to 121%. Within that range, the flowsplitting accuracy of the concentrations for eachstock solution-test vessel pairs were within 10%of each other.Homogeneity of test concentration was analyzedin onevessel at the 0.40 and 100 mg a.i./Ltreatment levels. Of the 4 samples taken ateach treatment level, the %CV for the 0.40 and100 mg a.i./L levels was 0.646% and 0.348%,respectively.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamb er is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within

10%.Aeration?not reported EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 15 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Natural waterDilution water originated from Lake Huron whichwas supplied to the Dow Chemical Companyvia the City of Midland Water Treatment Plant.The water from the lake was limed andflocculated with ferric chloride and then pumpedto the laboratory. Before use in the lab, thewater was sand-filtered, pH-adjusted withgaseous CO2, carbon-filtered, and UVirradiated. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival

at least as good as indicated in the test

guideline.Was dilution water analyzed forpesticides, heavy metals, andother contaminants?YesDilution water is bi-annually analyzed forpesticides, organics, metals and otherinorganics.Test vessel type/materials:Glass sealed together withclear silicone adhesiveEPA 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:29 cm x 20 cm x 25 cm EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:10 Lapproximate fill volume; water depth 13 cm EPA recommends 10 L solution per tank.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 16 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSpawning substrate material:PVC pipe, cut lengthwise 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:9 cm9 cm long, 10 cm diameterAdditional details on exposuresystem:Temperature was measured in one test vessel through the exposure period. Temperature, DO, and pH were measured in each test vessel at Day 0 andweekly thereafter during the assay (Table 5). Hardness, alkalinity, and conductivity were measured from a sample in the controland highest exposurelevel solutions at Day 0 and weekly thereafter (Table 5).Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.5 25.2 25.01Weekly EPA recommends temperature 25±1 oC; interreplicate and inter -treatment differentials should not exceed 1 o C.pH7.027.767.381Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)6.20 8.29 7.391Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 17 of 86Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTotal alkalinity (mg/L as CaCO3)30 46 36.51Weekly EPA recommends total alkalinity >20 mg/L

as CaCO 3.Hardness [mg/L as CaCO3]626863.51WeeklyTotal organic carbon (mg/L)<2mg/LOnce EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (g/L)<0.10 mg/L(as N) EPA recommends that unionized ammonia in the dilution water be <1 g/L.Residual chlorine (g/L) <200 Biannually EPA recommends that residual chlorine in dilution water be <10 g/L.Other: Ammonia(mg/L)240biannually 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.Other: Conductivity (μmhos/cm)177207188.51weekly1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levelsData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 18 of 86Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?No EPA recommends conducting a range-finder if

96 -hour LC

50 data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the rangefinder?NA EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is appropriate.Species:NALife stage:NA EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the

test.Test duration:NA EPA recommends a 96-hour exposure.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 19 of 86Version: 22 September 2011Additional details:NA EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a

concentrati on-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the

derived 96 -hr LC

50, may be selected as the highest exposure concentration in the 21-

day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 20 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:Reference studies withfathead minnowThe nominal test concentrations were basedon two available 2,4-D studies with thefathead minnow with reported LC50values of320 mg a.i./L (Alexander et al.,1985) and133 mg a.i./L (Mayer and Ellersieck, 1986).From these two lethal endpoints, theexperimental highconcentrations could beestimated at 100 and 40 mg a.i./L,respectively, as specified in the 890.1350guideline. Because of the uncertaintybetween the two concentrations, both wereselected as highconcentrations and 4concentrations were used for theexperimental test. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Alexander et al.,1985;Mayer and Ellersieck,1986Separation of test concentrations:0, 0.4, 4.0, 40, 100mg/L EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 21 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of test concentrations:4 EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?Not reportedIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ):0.1 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):not reportedFrequency of measurement:0, 7, 14, and 21 days It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 22 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre -exposure. Tanks are then ranked according to pre -exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoWas a positive control used?NoData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 23 of 86Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsPhotoperiod:16 hrs light :8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:591 - 770 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Information regarding test solutionappearance during the study did not appearto be reported.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 24 of 86Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with 2,4-D.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0.00<0.10NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV)≤ 20%.Treatment 10.4000.24543.3161.3% nominalTreatment 24.003.1413.378.5% nominalTreatment 340.034.07.085.0% nominalTreatment410096.54.596.5% nominalAbbreviations:CVCoefficient of variation.1Study authors reported a dramatic decrease in measured concentrations between Days 7 and 14 due to a suspected increase in microbial populations capable ofmetabolizing 2,4-D. Though this occurred in all concentrations, the effect was proportionately greater on the lowest nominal concentration. The %CVs were >20% foreach time point in this treatment group, though authors report that the variation in concentration was consistent among the replicates and thus the overall exposurewas consistent among replicates.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 25 of 86

Version: 22 September 2011 

### E. Observations

Biological Endpoints: Mortality, external abnormalities, abnormal behavior relative to controls,

fecundity, fertilization, secondary sex characteristics, full body weight and

length, gonadal status (GSI and histology), nuptial tubercle scoring, and

vitellogenin concentrations. Observations of appearance were made at test

termination, than fish were euthanized, weighed and measured. Blood was

collected from the caudal peduncle using heparinized capillary tubes. Fish

viscera were removed and fixed in Davidson’s solution then preserved in

formalin. Gonads were removed, weighed, placed into a plastic tissue

cassette and then Davidson’s fixative solution. Vitellogenin blood plasma

levels were measured with the fathead minnow VTG ELISA kit obtained from

Biosense Laboratories, Bergen, Norway. In addition to test samples, each

ELISA plate contained 6 calibration standards and 2 non-specific binding

assay blanks. Plasma steroid concentrations were not measured.

Were raw (individual) data provided? Yes, with the exception of the re-analysis of female VTG

levels and the coloration/appearance of females. Data were also provided

in spreadsheet form via email.

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 17 β -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.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 26 of 86

Version: 22 September 2011 

### II. RESULTS AND DISCUSSION

A. Results

Only one fish from the 96.5 mg a.i./L treatment group died during the test. No mortalities occurred

in the control fish.

Table 9: Adult Fish Survival in Fathead Minnow.

Treatment

(mg a.i./L)

[mean- measured] Males Females

n # Surviving % Survival n # Surviving % Survival

Negative Control

(<LOQ) 8 8 100 16 16 100

0.245 8 8 100 16 16 100

3.14 8 8 100 16 16 100

34.0 8 8 100 16 16 100

96.5 8 8 100 16 15 93.8

n = number of individuals per treatment at test initiation.

LOQ=0.10 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 27 of 86Version: 22 September 2011Mean male body weight ranged from 3.57 g (negative control) to 3.93 g (34 mg a.i./L), and mean female body weight ranged from 1.59 g (negative control) to 1.62 g (3.14,34, and 96.5 mg a.i./L). Mean male length ranged from 51.5 mm (negative control) to 54.5 mm(34 mg a.i./L), and mean female length ranged from 42.1 mm (3.14 mga.i./L) to 42.5 mm (96.5 mg a.i./L).Table 10: Size at Test Termination in Fathead Minnow.Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDNegative Control(<LOQ)43.570.1414 1.590.169451.51.54442.41.370.24543.720.30841.640.074453.01.37442.70.563.1443.690.14441.620.173452.20.89442.10.9234.043.930.48841.620.173454.52.20442.41.4796.543.730.40841.620.082453.82.45442.50.78SDStandard deviation.n = number of independent replicates per treatment.LOQ=0.10 mg a.i./L

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 28 of 86

Version: 22 September 2011 

### Fecundity ranged from 12.5 to 38.6 eggs/female/reproductive data across all treatment groups, and

fertilization success as a percentage of embryos to unfertilized eggs ranged from 91.7 to 99.4%.

Table 11: Fecundity and Fertilization Success in Fathead Minnow.

Treatment

(mg a.i./L)

[mean-measured] Fecundity 1 Fertilization Success (%) 2

Mean ± SD Mean ± SD

Negative Control (<LOQ)) 29.8 6.4 96.9 0.50

0.245 29.0 6.47 96.3 3.08

3.14 25.9 5.27 96.1 1.54

34.0 22.9 8.72 97.7 0.78

96.5 19.7 5.97 95.8 2.68

LOQ=0.10 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.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 29 of 86

Version: 22 September 2011 

### Male media tubercle scores ranged from 31 (negative control and 0.245 mg a.i./L) to 35 (3.14 mg a.i./L).

No tubercles were noted in females.

Table 12: Nuptial Tubercle Score in Fathead Minnow.

Treatment

(mg a.i./L)

[mean-measured] Males Females 1

n Median Tubercle

Score 2 n Median Tubercle

Score

Negative Control (<LOQ) 4 31 4 0

0.245 4 31 4 0

3.14 4 35 4 0

34.0 4 33 4 0

96.5 4 34 4 0

n = number of independent replicates per treatment.

LOQ=0.10 mg a.i./L

1 The study authors reported that no tubercles were observed on female fish.

2 Mean tubercle scores: 32, 31, 34, 33, and 35 for the negative control and mean-measured 0.245, 3.14, 34.0, and

96.5 mg a.i./L treatment levels, respectively.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 30 of 86

Version: 22 September 2011 

### Mean male GSI ranged from 1.2 to 1.3%, and mean female GSI ranged from 11.2% (3.15 mg a.i./L) to 13.3%

(96.5 mg a.i./L).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Mean GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Negative Control (<LOQ) 4 1.2 0.1 4 11.9 1.7

0.245 4 1.2 0.3 4 12.9 2.3

3.14 4 1.2 0.1 4 11.2 2. 6

34.0 4 1.2 0.2 4 11.8 3.4

96.5 4 1.3 0.1 4 13.3 2.0

n = number of independent replicates per treatment.

LOQ=0.10 mg a.i./L

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.



### The study author reports that there were no effects of 2,4-D on the histology or germ cell distribution

(staging) of either the testes or ovaries of the test fish. All histopathological findings were considered basal

variation unassociated with 2,4-D exposure due to a lack of a concentration-response relationship. Although

not concentration-dependent, an increase in the number of female ovaries that were observed as Stage 2

(compared to Stage 3 or 4) was noted by the reviewer for the 2,4-D treatments compared to the negative

control. A summary of the reported stage in the ovaries is shown in Table 14b.

The study report did not include observations of decreased proportion of spermatagonia, increased vascular

or interstitial proteinaceous fluid, asynchronous gland development, altered proportions of spermatocytes or

spermatids, or granulomatous inflammation. There was a single observation of mineralization of the

seminiferous tubule/duct (severity grade 1) in one control male. Macrophages (histiocytes; severity grade

1) were observed in female fish in the controls and at all treatment levels.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 31 of 86

Version: 22 September 2011 

### Table 14a: Gonadal Staging in Fathead Minnow.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Median Stage 1 n Median Stage 2

Negative Control (<LOQ) 8 3 4 4

0.245 8 4 4 4

3.14 8 3 4 3

34.0 4 3 4 3

96.5 4 3 4 4

n = total number of animals per treatment upon which observations were made at test termination.

LOQ=0.10 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.



### Table 14b. Ovary staging (from study report)

Ovary Control 0.245 mg a.i./L 3.14 mg a.i./L 34 mg a.i./L 96.5 mg a.i./L

Examined 16 16 16 16 15

Stage 2 0 3 4 6 3

Stage 3 6 2 6 3 4

Stage 4 10 11 6 7 8

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 32 of 86

Version: 22 September 2011 Table 15: Gonadal Histopathology in Male Fathead Minnow.

Treatment

(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of

Testis-Ova Mineralization of

seminiferous

tubule Increased

Testicular

Degeneration Interstitial Cell

Hypertrophy/

Hyperplasia

n Incidence n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 0 8 8 8 8 8 7 8 8 8 8

1 8 0 8 0 8 1 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

0.245 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

3.14 0 8 7 8 8 8 8 8 8 8 8

1 8 1 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

34.0 0 8 7 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 1 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0 8 0

96.5 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

LOQ=0.10 mg a.i./L

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 33 of 86

Version: 22 September 2011 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 34 of 86Version: 22 September 2011Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow.Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations2SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationn1Incidencen1Incidencen1Incidencen1Incidencen1IncidenceNegative Control(<LOQ)0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA0.2450NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 35 of 86Version: 22 September 2011Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations2SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationn1Incidencen1Incidencen1Incidencen1Incidencen1Incidence3.140NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA34.00NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 36 of 86Version: 22 September 2011Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations2SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationn1Incidencen1Incidencen1Incidencen1Incidencen1Incidence96.50NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAAbbreviations:NANot applicable.LOQ=0.10 mg a.i./L1Number of individual fish observed2Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. SeeAppendix E of the test guideline for reference.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 37 of 86

Version: 22 September 2011 

### Table 17: Gonadal Histopathology in Female Fathead Minnow.

Treatment

(mg a.i./L)

[mean-measured] Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation

n Incidence n Incidence n Incidence

Negative Control

(<LOQ) 0 16 9 16 16 16 16

1 16 7 16 0 16 0

2 16 0 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

0.245 0 16 8 16 16 16 16

1 16 5 16 0 16 0

2 16 3 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

3.14 0 16 10 16. 16 16 16

1 16 3 16 0 16 0

2 16 2 16 0 16 0

3 16 1 16 0 16 0

4 16 0 16 0 16 0

34.0 0 16 7 16 16 16 16

1 16 5 16 0 16 0

2 16 3 16 0 16 0

3 16 1 16 0 16 0

4 16 0 16 0 16 0

96.5 0 15 11 15 15 15 15

1 15 3 15 0 15 0

2 15 1 15 0 15 0

3 15 0 15 0 15 0

4 15 0 15 0 15 0

LOQ=0.10 mg a.i./L

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 38 of 86

Version: 22 September 2011 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.



### Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow.

Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 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

0.245 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

3.14 0 16 16 16 16 16 15 16 16

1 16 0 16 0 16 1 16 0

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

34.0 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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 39 of 86

Version: 22 September 2011 Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

96.5 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

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

LOQ=0.10 mg a.i./L

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 40 of 86

Version: 22 September 2011 

### Mean male plasma vitellogenin ranged from 0.909 ng/mL (34 mg a.i./L) to 1.77 ng/mL (0.245 mg a.i./L).

Mean female plasma vitellogenin ranged from 19.8×10 6 

### ng/mL (3.14 mg a.i./L) to 55.1×10 6 

### ng/mL (0.245

mg a.i./L).

Table 19: Plasma Vitellogenin in Fathead Minnow.

Treatment

(mg a.i./L)

[mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean

(ng/mL plasma) ±SD n Mean

(ng/mL plasma) ±SD

Negative Control (<LOQ) 4 1.34 1.73 4 27.6×10 6 21.5×10 6

0.245 4 1.77 1.25 4 55.1×10 6 47.5×10 6

3.14 4 0.930 0.777 4 19.8×10 6 7.69×10 6

34.0 4 0.909 0.591 4 21.0×10 6 14.4×10 6

96.5 4 1.29 0.983 4 31.6×10 6 12.7×10 6

SD Standard deviation.

n = number of independent replicates per treatment.

LOQ=0.10 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 41 of 86Version: 22 September 2011Plasma testosterone and plasma 17β-estradiol in male and females were not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17β-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control(<LOQ)NAND NDNAND NDNANDNDNANDND0.245NANDNDNANDNDNANDNDNANDND3.14NANDNDNANDNDNANDNDNANDND34.0NANDNDNANDNDNANDNDNANDND96.5NANDNDNANDNDNANDNDNANDNDAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.10 mg a.i./LNo abnormal behavior was observed among control or treatment fish, such as hyperventilation, loss of equilibrium, uncoordinatedswimming, atypicalquiescence, and feeding abstinence. It was reported that one female fish in the 0.245 mg a.i./L treatment group lost an eye during the exposureData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 42 of 86Version: 22 September 2011period which could have been from breeding activity and/or colliding with breeding substrate. Another female fish in the 3.14 mg a.i./L treatmentgroup exhibited ascites ( i.e., accumulation of fluid in the coelomic cavity). Scoliosis, or bent tail, was displayed by one fish in the 96.5 mg a.i./Ltreatment group and one fish in the control group. Vertical banding was observed in both control and treatment group female test fish, but notreatment-related effects were observed. The raw data for this banding was not reported.Table 21: Secondary Sex Characteristics and ClinicalSigns in Fathead Minnow at Test Termination.Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceNegative Control (<LOQ)NoneNANANoneNANA0.245NoneNANANoneNANA3.14NoneNANANoneNANA34.0NoneNANANoneNANA96.5NoneNANANoneNANAAbbreviations:NANot applicable.LOQ=0.10 mg a.i./L

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 43 of 86

Version: 22 September 2011 

### B. Study Author’s Analysis and Conclusions

The study author reported the following statistical methodology:

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, 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

concentration-response. If the endpoint response was not consistent with a monotone concentrationresponse, 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. Where non-normality or variance heterogeneity was observed normalizing and/or

variance stabilizing transformations was applied. If the data were 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-Whitney-Wilcoxon 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. A treatment effect for tubercle score was determined using the multiquantal

Jonckheere-Terpstra test.

Female plasma VTG was reanalyzed by the study authors in an effort to demonstrate proficiency in the

method as outlined in the 890.1350 guideline. Results of the reanalysis are presented in Appendix II to

this DER.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 44 of 86

Version: 22 September 2011 

### The enzyme-linked immunosorbent assay (ELISA) test was used to analyze the vitellogenin (VTG) levels

in male and female test fish blood plasma. An ELISA reanalysis of VTG concentrations in female fish

demonstrated the ability of using the ELISA in accordance with the methods recommended by the 890.1350

guideline; insufficient male plasma was available for reanalysis. For the primary test, the correlation

coefficient (R 2 

### ) for the calibration curves were all >0.99 (meeting 890.1350 standards); the concentrations

for each standard fell between 82.1 and 115% of nominal concentrations (meeting 890.1350 standards);

and the average absorbance measured for the non-specific binding assay blanks was less than 0.06

absorbance units (highest recommended level by 890.1350 guidelines) although one plate produced an

average absorbance unit value of 0.067.

For the reanalysis of female VTG samples, the R 2 

### were all >0.99 and the concentrations for each standard

fell between 70-120% of nominal (850.1350 recommended range) with the exception of one standard with

a recovery of 144%. Two aliquots of each sample dilution were analyzed on the same ELISA plate (well

duplicates) to verify the co-efficient of variation. It was reported that 74 of the 79 samples did not differ by

more than 20%, and all were less than 31%, and thus these results indicated acceptable method precision.

The actual response in the well-duplicate samples was greater than the primary concentration analysis. The

study author indicated that it has been shown that concentrations of VTG may increase when re-analyzed

following a freeze-thaw cycle (Brodeur, et al ., 2006).

C. Reviewer’s Analysis and Conclusions

Statistical Methods: Statistical analyses were recalculated by the reviewer following decision logic provided

in test guidelines. Continuous data were tested for normality using Shapiro-Wilks test and for homogeneity

of variance using Levene’s test. Data that demonstrated a monotonic trend were analyzed using JonckheereTerpstra according the 890.1350 guideline flowchart and using Williams test according to EFED policy.

Data that met the assumptions of normality and homogeneity of variance were then analyzed using Dunnett’s

test if the data did not demonstrate a monotonic concentration response. Data that failed the assumption of

normality or homogeneity of variance were analyzed using the Mann-Whitney-Wilcoxon test. Mortality was

assessed using the Fisher’s exact test with a Bonferroni-Holm adjustment.

Conclusions: Fecundity was significantly decreased in the highest concentration tested, 96.5 mg a.i./L

compared to the control. There was no significant difference for fertilization success (# embryos/# eggs

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 45 of 86

Version: 22 September 2011 

### x 100), male tubercle score, male and female condition index (whole body length/weight), male and female

GSI, or male and female VTG levels. No signs of abnormal behavior, appearance, or secondary sex

characteristics in the treatment groups relative to the control. Additionally, the study author reports that there

were no effects of 2,4-D on the histology or germ cell distribution (staging) of either the testes or ovaries

of the test fish. While not concentration-dependent, an increase in the number of female ovaries that were

observed as Stage 2 (compared to Stage 3 or 4) was reported for the 2,4-D treatments compared to the

control; the number of Stage 2 ovaries was 0, 3, 4, 6, and 3 for the control, 0.245, 3.14, 34.0 and 96.5

mg a.i./L treatment group, respectively.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 46 of 86Version: 22 September 2011Table 22: Reproductive and HPG Endpoints1,2,3for Male Fathead Minnow in the FSTRA with 2,4-D.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p%Diff.pNegative Control(<LOQ)31 NA NA NANA NANA NA NA NANA0.245310.9952.290.998No32.10.954NANANANA3.14350.925-1.251.000No-30.30.962NANANANA34.0330.978-1.251.000No-32.10.954NANANANA96.5340.7488.320.817No-3.61.000NANANANAStatistical TestDunnett’s4Dunnett’sNADunnett’sNANANANot applicableLOQ=0.10 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3For percent (%) difference, positive values indicate an increase relative to the negative control, and negative values indicate a decrease relative to the negative control.4Mean tubercle scores: 32, 31, 34, 33, and 35 for the negativecontrol and mean-measured 0.245, 3.14, 34.0, and 96.5 mg a.i./Ltreatment levels, respectively.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 47 of 86Version: 22 September 2011Table 23: Reproductive and HPG Endpoints1,2,3for Female Fathead Minnow in the FSTRA with 2,4-D.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2%Diff.p%Diff.pMedianp%Diff.pEffect?(Yes/No)%Diff.p%Diff.p% Diff.pNegative Control(<LOQ)NANANANA0NANANANANANANANANANA0.245-2.60.332-0.640.9750NA8.210.945No99.60.494NANANANA3.14-130.210-0.830.9430NA-6.110.980No-28.40.889NANANANA34.0-230.0880.770.9540NA-1.131.00No-23.50.678NANANANA96.5-340.031-1.160.8390NA11.50.847No14.60.494NANANANAStatistical TestJonckheereTerpstraDunnett’s NA Dunnett’s NAMann-WhitneyNA NAAbbreviations:Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.10 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3For percent (%) difference, positive values indicate an increase relative to the negative control, and negative values indicate a decrease relative to the negative control.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 48 of 86

Version: 22 September 2011 

### Table 24: Growth Endpoints 1,2,3 

### in the Fish Short-Term Reproduction Assay (FSTRA) with 2,4-D.

Treatment

(mg a.i./L)

[mean-measured] Body Weight Length

Males Females Males Females

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

Negative Control (<LOQ) NA NA NA NA NA NA NA NA

0.245 4.20 0.914 2.67 0.979 2.96 0.579 0.59 0.992

3.14 3.36 0.958 2.04 0.992 1.26 0.959 -0.71 0.984

34.0 10.01 0.380 2.04 0.992 5.82 0.097 -0.12 1.000

96.5 4.48 0.895 1.57 0.997 4.32 0.273 0.18 1.000

Statistical Test Dunnett’s Dunnett’s Dunnett’s Dunnett’s

LOQ=0.10 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 For percent (%) difference, positive values indicate an increase relative to the negative control, and negative values

indicate a decrease relative to the negative control.



### E. Study Deficiencies

Several deviations are listed in Section I. Materials and Methods of this DER. One test performance

criterion was not met; the CV for the mean-measured concentration of the lowest treatment group was 43%

which is greater than the guideline criterion of < 20%. These deviations did not impact the interpretation of

this study.

F. Reviewer’s Comments

Two replicate spawning groups with performance ranking within the top 20 breeding groups were

inadvertently excluded from assignment to the exposure period test vessels. The study author reported that

this deviation did not impact the experimental test because the 20 spawning groups used for the exposure

period met the minimum criteria as outlined in the 890.1350 guideline.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 49 of 86

Version: 22 September 2011 

### Ammonia and chlorine levels were measured in dilution water biannually. Ammonia levels were detected at

0.24 mg a.i./L, exceeding guideline recommendations of ≤ 1 μ g/L. Additionally, OCSPP guidelines

recommend chlorine levels <10 μ g/L but chlorine in the dilution water was only quantified at a level <200

μ g/L (chlorine was not detectable at 200 μ g/L). Organic carbon levels in dilution water were not measured.

The study author’s rationale for the decrease in test concentrations in the test vessels but not the stock

solutions was the marked increase in growth and waste production from Day 7 to Day 14 which increased

microbial populations. Test material microbial biodegradation had a greater percentage effect in the two

lower treatment groups than the two highest treatment groups. The study author reported that biodegradation

is known to occur under the conditions in the current study (Sinton et al., 1986). It was noted that the

variability in 2,4-D concentrations in the stock solutions might have occurred because the large stock

volumes were not being continuously mixed during their use.

The reviewer reports the gonad histopathological results as they were reported by the study author (Table

6). The stage and severity grading of the gonads was conducted in accordance with the procedures

described in the 890.1350 guidelines. Gonad sections were examined under a light microscope by an

American College of Veterinary Pathologists board-certified veterinary pathologist.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 50 of 86

Version: 22 September 2011 

### III. REFERENCES

Alexander, H.C., Gersich, F.M., and Mayes, M.A. (1985). Acute Toxicity of Four Phenoxy Herbicides to Aquatic

Organisms. Bull. Env. Contam. Toxicol. 35

, 314-321.

Brodeur, J.C., Woodburn K.B., Zhang F., Bartels M.J., and Klecka G.M. (2006). Plasma sampling and freezing

procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow

* Pimephales promelas ). Env. Toxicol. Chem. 25 , 337-348.

Hansch, C., Leo, A., and Hoekman, D. (1995). Exploring QSAR Hydrophobic, Electronic and Steric Constants,

ACS, Washington, D.C.

Hopkins, D.L. (1987). 2,4-Dichlorophenoxyacetic Acid: Determination of the water solubility. Report of

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland Michigan.

Lezotte F., Van Hoven R., and Nixon, W. (2002). Determination of Water Solubility of 2,4-D Acid by the

Shake Flask Method: Lab Project Number: 467C-103: 467/121801/7840/SUB467. Unpublished study

prepared by Wildlife International, Ltd. MRID No. 45692501.

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.

Mayes, M. A., Gorzinski, S. J., Potter, R. B., and Richardson, C. H. (1990). 2,4-Dichlorophenoxyacetic acid:

Evaluation of the Toxicity to Early Life Stages of the Fathead Minnow, Pimephales promelas. Report of

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Sinton, G. L., Fan, L. T., Erickson, L. E., and Lee, S. M. (1986). Biodegradation of 2,4-D and related

xenobiotic compounds. Enzyme Microb. Technol. 8, 395-403.

Wo, C. (2008). Certificate of Analysis for 2,4-D Acid, Lot# 2006 2433 8006-USA. Study number 26625,

PSL Reference No. 081210-2H. Eurofms|Product Safety Laboratories, Dayton, New Jersey.Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 51 of 86Version: 22 September 2011APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:EndpointMonotonic?Parametric?890.1350EFEDCommentsSurvivalOverall:NoNAFisher’s Exact Testn.s p>0.05Fisher’s Exact Testn.s p>0.05No significant effect on overall survivalFemale body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectFemale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectFemale VTGYesYesMann Whitney;n.s. p>0.05Mann Whitney;n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale VTGYesNoDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectFemale GSIYesYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale GSINoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectFemale tuberclescoreNANANANANo scoreData Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-DEPA MRID Number 48317001Page 52 of 86Version: 22 September 2011EndpointMonotonic?Parametric?890.1350EFEDCommentsMale tubercle scoreYesYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectFecundityNoYesJonckheere-Terpstra:dose 3 p=0.011Williams;dose 3 p=0.031890.1350 and EFED same conclusions, effectat dose 3FertilityYesNoDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectF testosteroneNANANANANAM testosteroneNANANANANAF estradiolNANANANANAM estradiolNANANANANA

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 53 of 86

Version: 22 September 2011 

### Appendix II: Reanalysis of Female Plasma VTG.

Table: Plasma Vitellogenin in Fathead Minnow (Results of Reanalysis of Female Samples).

Treatment

(mg a.i./L)

[measured] Plasma Vitellogenin (VTG)

Females

n Mean

(ng/mL plasma) ±SD

Negative Control 4 59.8x10 6 26.6x10 6

0.245 4 74.8x10 6 31.0x10 6

3.14 4 55.7x10 6 18.8x10 6

34.0 4 58.7x10 6 33.7x10 6

96.5 4 92.5x10 6 27.6x10 6

SD Standard deviation.

n = number of independent replicates per treatment

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 54 of 86

Version: 22 September 2011 

### Appendix III: Reviewer’s Statistical Output.

Fisher's Exact Test

============================================================================
NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 16 0 16

0.245 16 0 16

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

TOTAL 32 0 32

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

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 16. Since b is greater than 11.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================
NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 16 0 16

3.14 16 0 16

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

TOTAL 32 0 32

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

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 16. Since b is greater than 11.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 55 of 86

Version: 22 September 2011 

### NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 16 0 16

34.0 16 0 16

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

TOTAL 32 0 32

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

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 16. Since b is greater than 11.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================
NUMBER OF

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

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

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

CONTROL 16 0 16

96.5 15 1 16

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

TOTAL 31 1 32

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

Critical Fisher's value (16,16,16) (alpha=0.05) is 11.0. b value is 15. Since b is greater than 11.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 56 of 86

Version: 22 September 2011 

### Summary of Fisher's Exact Tests

---------------------------------------------------------------------------- NUMBER NUMBER SIG
GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 16 0

1 0.245 16 0

2 3.14 16 0

3 34.0 16 0

4 96.5 16 1

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

test for fish screen study - TEST DATA 2 4 D 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

0.952 0.390 2.884 0.059 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.59 0.17 0.08 10.51 1.33, 1.86 Dose1 4 1.64 0.07 0.04 4.48 1.52, 1.75 Dose2 4 1.63 0.17 0.09 10.65 1.35, 1.90 Dose3 4 1.63 0.17 0.09 10.52 1.35, 1.90 Dose4 4 1.62 0.08 0.04 5.11 1.49, 1.75

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

Ctrl 1.53 1.47 1.84 . .

Dose1 1.61 1.58 1.74 102.67 -2.67

Dose2 1.64 1.43 1.79 102.04 -2.04

Dose3 1.63 1.45 1.80 102.04 -2.04

Dose4 1.62 1.52 1.72 101.57 -1.57

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

4 15 0.05 0.994

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

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 57 of 86

Version: 22 September 2011 

### Ctrl 1.59 . 1.62 . . . . . .

Dose1 1.64 0.979 1.62 0.692 . . . . .

Dose2 1.63 0.992 1.62 0.727 1.000 . . . .

Dose3 1.63 0.992 1.62 0.746 1.000 1.000 . . .

Dose4 1.62 0.997 1.62 0.750 1.000 1.000 1.000 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 0.38 0.984

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 1.53 . .

Dose1 1.61 0.343 0.877

Dose2 1.64 1.000 0.722

Dose3 1.63 0.889 0.593

Dose4 1.62 0.676 0.579

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

4 15 0.05 0.994

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 -1.59 . -1.59 . . . . . .

Dose1 -1.64 0.979 -1.63 0.441 . . . . .

Dose2 -1.63 0.992 -1.63 0.470 1.000 . . . .

Dose3 -1.63 0.992 -1.63 0.487 1.000 1.000 . . .

Dose4 -1.62 0.997 -1.63 0.497 1.000 1.000 1.000 . .

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

4 0.38 0.984

MannWhit - testing each trt median signif. different from control

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 58 of 86

Version: 22 September 2011 

### Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -1.53 . .

Dose1 -1.61 0.343 0.123

Dose2 -1.64 1.000 0.278

Dose3 -1.63 0.889 0.407

Dose4 -1.62 0.676 0.421

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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
0.970 0.762 2.364 0.100 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.57 0.14 0.07 3.99 3.34, 3.80 Dose1 4 3.72 0.31 0.15 8.32 3.23, 4.21 Dose2 4 3.69 0.14 0.07 3.91 3.46, 3.92 Dose3 4 3.93 0.49 0.24 12.44 3.15, 4.70 Dose4 4 3.73 0.41 0.20 10.91 3.08, 4.38

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

Ctrl 3.53 3.45 3.77 . .

Dose1 3.69 3.38 4.12 104.20 -4.20

Dose2 3.65 3.57 3.90 103.36 -3.36

Dose3 3.85 3.45 4.57 110.01 -10.01

Dose4 3.75 3.25 4.17 104.48 -4.48

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

4 15 0.61 0.660

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 59 of 86

Version: 22 September 2011 

### p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 3.57 . 3.73 . . . . . .

Dose1 3.72 0.914 3.73 0.826 . . . . .

Dose2 3.69 0.958 3.73 0.855 1.000 . . . .

Dose3 3.93 0.380 3.73 0.870 0.895 0.842 . . .

Dose4 3.73 0.895 3.73 0.879 1.000 1.000 0.911 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 1.83 0.766

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 3.53 . .

Dose1 3.69 0.678 0.718

Dose2 3.65 0.283 0.830

Dose3 3.85 0.413 0.920

Dose4 3.75 0.678 0.841

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

4 15 0.61 0.660

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

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

Ctrl -3.57 . -3.57 . . . . . .

Dose1 -3.72 0.914 -3.71 0.340 . . . . .

Dose2 -3.69 0.958 -3.71 0.364 1.000 . . . .

Dose3 -3.93 0.380 -3.83 0.186 0.895 0.842 . . .

Dose4 -3.73 0.895 -3.83 0.190 1.000 1.000 0.911 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 1.83 0.766

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 60 of 86

Version: 22 September 2011 

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -3.53 . .

Dose1 -3.69 0.678 0.282

Dose2 -3.65 0.283 0.170

Dose3 -3.85 0.413 0.080

Dose4 -3.75 0.678 0.159

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

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

Test Stat P-value Test Stat P-value

0.939 0.225 1.586 0.229 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 42.43 1.33 0.67 3.14 40.30, 44.55 Dose1 4 42.68 0.57 0.28 1.33 41.77, 43.58 Dose2 4 42.13 0.92 0.46 2.19 40.66, 43.59 Dose3 4 42.38 1.49 0.74 3.51 40.01, 44.74 Dose4 4 42.50 0.81 0.41 1.91 41.21, 43.79

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

Ctrl 42.20 41.10 44.20 . .

Dose1 42.65 42.10 43.30 100.59 -0.59

Dose2 42.45 40.80 42.80 99.29 0.71

Dose3 42.25 40.90 44.10 99.88 0.12

Dose4 42.85 41.30 43.00 100.18 -0.18

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

4 15 0.14 0.966

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 61 of 86

Version: 22 September 2011 

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

Ctrl 42.43 . 42.55 . . . . . .

Dose1 42.68 0.992 42.55 0.651 . . . . .

Dose2 42.13 0.984 42.33 0.565 0.948 . . . .

Dose3 42.38 1.000 42.33 0.583 0.994 0.997 . . .

Dose4 42.50 1.000 42.33 0.595 0.999 0.987 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

4 1.03 0.905

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 42.20 . .

Dose1 42.65 0.678 0.718

Dose2 42.45 1.000 0.442

Dose3 42.25 0.889 0.463

Dose4 42.85 0.676 0.566

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

4 15 0.14 0.966

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 -42.43 . -42.40 . . . . . .

Dose1 -42.68 0.992 -42.40 0.597 . . . . .

Dose2 -42.13 0.984 -42.40 0.632 0.948 . . . .

Dose3 -42.38 1.000 -42.40 0.651 0.994 0.997 . . .

Dose4 -42.50 1.000 -42.50 0.605 0.999 0.987 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
4 1.03 0.905

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 62 of 86

Version: 22 September 2011 

### 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 -42.20 . .

Dose1 -42.65 0.678 0.282

Dose2 -42.45 1.000 0.558

Dose3 -42.25 0.889 0.537

Dose4 -42.85 0.676 0.434

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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
0.938 0.222 1.295 0.316 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 51.55 1.54 0.77 2.98 49.10, 54.00 Dose1 4 53.08 1.35 0.67 2.53 50.93, 55.22 Dose2 4 52.20 0.91 0.45 1.74 50.75, 53.65 Dose3 4 54.55 2.21 1.11 4.06 51.03, 58.07 Dose4 4 53.78 2.46 1.23 4.58 49.85, 57.70

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

Ctrl 51.55 49.70 53.40 . .

Dose1 52.60 52.10 55.00 102.96 -2.96

Dose2 52.00 51.40 53.40 101.26 -1.26

Dose3 54.85 52.00 56.50 105.82 -5.82

Dose4 54.50 50.30 55.80 104.32 -4.32

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

4 15 1.80 0.181

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 63 of 86

Version: 22 September 2011 

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

Ctrl 51.55 . 53.03 . . . . . .

Dose1 53.08 0.579 53.03 0.927 . . . . .

Dose2 52.20 0.959 53.03 0.944 0.955 . . . .

Dose3 54.55 0.097 53.03 0.953 0.769 0.378 . . .

Dose4 53.78 0.273 53.03 0.958 0.980 0.726 0.971 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 6.12 0.190

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 51.55 . .

Dose1 52.60 0.235 0.926

Dose2 52.00 0.580 0.745

Dose3 54.85 0.125 0.966

Dose4 54.50 0.235 0.975

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

4 15 1.80 0.181

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 -51.55 . -51.55 . . . . . .

Dose1 -53.08 0.579 -52.64 0.241 . . . . .

Dose2 -52.20 0.959 -52.64 0.257 0.955 . . . .

Dose3 -54.55 0.097 -54.16 0.035 0.769 0.378 . . .

Dose4 -53.78 0.273 -54.16 0.036 0.980 0.726 0.971 . .

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 64 of 86

Version: 22 September 2011 

### 4 6.12 0.190

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 -51.55 . .

Dose1 -52.60 0.235 0.074

Dose2 -52.00 0.580 0.255

Dose3 -54.85 0.125 0.034

Dose4 -54.50 0.235 0.025

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL
Williams Dose3

Jonckheere Dose3

test for fish screen study - TEST DATA 2 4 D ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

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

Test Stat P-value Test Stat P-value
0.932 0.172 4.120 0.019 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 27595000 21470025 10735012 77.80 -6568601,61758601 Dose1 4 55085000 47464483 23732241 86.17 -2.044E7,1.3061E8 Dose2 4 19765000 7687585 3843793 38.89 7532336,31997664 Dose3 4 21102500 14406782 7203391 68.27 -1821905,44026905 Dose4 4 31632500 12736128 6368064 40.26 11366478,51898522

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

Ctrl 20850000 10090000 58590000 . .

Dose1 47025000 9630000 1.1666E8 199.62 -99.62

Dose2 20845000 9690000 27680000 71.63 28.37

Dose3 14660000 12450000 42640000 76.47 23.53

Dose4 29195000 18870000 49270000 114.63 -14.63

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

Numerator df Denominator df F-stat P-value

4 15 1.30 0.315

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 65 of 86

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 27595000 . 41340000 . . . . . .

Dose155085000 0.373 41340000 0.852 . . . . .

Dose219765000 0.976 24166667 0.532 0.316 . . . .

Dose321102500 0.988 24166667 0.550 0.350 1.000 . . .

Dose431632500 0.998 24166667 0.561 0.682 0.960 0.974 . .

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

4 3.43 0.489

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 20850000 . .

Dose1 47025000 0.494 0.807

Dose2 20845000 0.889 0.442

Dose3 14660000 0.678 0.257

Dose4 29195000 0.494 0.655

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

4 15 1.30 0.315

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 -2.76E7 . -2.76E7 . . . . . .

Dose1-5.509E7 0.373 -3.19E7 0.479 . . . . .

Dose2-1.977E7 0.976 -3.19E7 0.510 0.316 . . . .

Dose3 -2.11E7 0.988 -3.19E7 0.527 0.350 1.000 . . .

Dose4-3.163E7 0.998 -3.19E7 0.538 0.682 0.960 0.974 . .

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 66 of 86

Version: 22 September 2011 

### Degrees of Freedom TestStat P-value

4 3.43 0.489

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -2.085E7 . .

Dose1 -4.703E7 0.494 0.193

Dose2 -2.085E7 0.889 0.558

Dose3 -1.466E7 0.678 0.743

Dose4 -2.92E7 0.494 0.345

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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

0.904 0.048 2.073 0.135 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1337.00 1735.75 867.87 129.82 -1424.96, 4098.96 Dose1 4 1766.25 1249.84 624.92 70.76 -222.52, 3755.02 Dose2 4 932.00 779.35 389.67 83.62 -308.11, 2172.11 Dose3 4 907.75 589.11 294.55 64.90 -29.65, 1845.15 Dose4 4 1288.50 981.96 490.98 76.21 -274.01, 2851.01

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

Ctrl 492.00 424.00 3940.00 . .

Dose1 1769.00 447.00 3080.00 132.11 -32.11

Dose2 807.50 153.00 1960.00 69.71 30.29

Dose3 750.50 380.00 1750.00 67.89 32.11

Dose4 1177.00 360.00 2440.00 96.37 3.63

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

4 15 0.38 0.819

Dunnett - testing each trt mean signif. different than control

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 67 of 86

Version: 22 September 2011 

### 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 1337.00 . 1551.63 . . . . . .

Dose1 1766.25 0.954 1551.63 0.691 . . . . .

Dose2 932.00 0.962 1042.75 0.456 0.835 . . . .

Dose3 907.75 0.954 1042.75 0.472 0.821 1.000 . . .

Dose4 1288.50 1.000 1042.75 0.482 0.974 0.991 0.989 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 1.44 0.837

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 492.00 . .

Dose1 1769.00 0.678 0.718

Dose2 807.50 0.889 0.500

Dose3 750.50 0.889 0.390

Dose4 1177.00 0.889 0.447

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

4 15 0.38 0.819

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 -1337.00 . -1235.75 . . . . . .

Dose1-1766.25 0.954 -1235.75 0.636 . . . . .

Dose2 -932.00 0.962 -1235.75 0.671 0.835 . . . .

Dose3 -907.75 0.954 -1235.75 0.690 0.821 1.000 . . .

Dose4-1288.50 1.000 -1288.50 0.674 0.974 0.991 0.989 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 68 of 86

Version: 22 September 2011 

### Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 1.44 0.837

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 -492.00 . .

Dose1 -1769.00 0.678 0.282

Dose2 -807.50 0.889 0.500

Dose3 -750.50 0.889 0.610

Dose4 -1177.00 0.889 0.553

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

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

Test Stat P-value Test Stat P-value
0.928 0.143 0.624 0.652 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 11.94 1.65 0.83 13.83 9.31, 14.57 Dose1 4 12.92 2.29 1.15 17.74 9.27, 16.57 Dose2 4 11.21 2.58 1.29 22.98 7.11, 15.31 Dose3 4 11.81 3.44 1.72 29.16 6.33, 17.29 Dose4 4 13.32 2.02 1.01 15.16 10.10, 16.53

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

Ctrl 11.59 10.48 14.12 . .

Dose1 13.27 10.12 15.04 108.21 -8.21

Dose2 11.81 7.89 13.34 93.89 6.11

Dose3 12.77 6.88 14.81 98.87 1.13

Dose4 13.71 10.52 15.33 111.49 -11.49

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

4 15 0.48 0.748

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 69 of 86

Version: 22 September 2011 

### 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 11.94 . 12.43 . . . . . .

Dose1 12.92 0.945 12.43 0.696 . . . . .

Dose2 11.21 0.980 12.11 0.659 0.861 . . . .

Dose3 11.81 1.000 12.11 0.678 0.966 0.997 . . .

Dose4 13.32 0.847 12.11 0.690 0.999 0.750 0.906 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 2.60 0.627

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 11.59 . .

Dose1 13.27 0.678 0.718

Dose2 11.81 0.678 0.330

Dose3 12.77 0.678 0.463

Dose4 13.71 0.494 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

4 15 0.48 0.748

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 -11.94 . -11.94 . . . . . .

Dose1 -12.92 0.945 -11.98 0.574 . . . . .

Dose2 -11.21 0.980 -11.98 0.608 0.861 . . . .

Dose3 -11.81 1.000 -11.98 0.627 0.966 0.997 . . .

Dose4 -13.32 0.847 -13.32 0.301 0.999 0.750 0.906 . .

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 70 of 86

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

4 2.60 0.627

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -11.59 . .

Dose1 -13.27 0.678 0.282

Dose2 -11.81 0.678 0.670

Dose3 -12.77 0.678 0.537

Dose4 -13.71 0.494 0.213

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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

0.944 0.289 2.115 0.129 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.20 0.14 0.07 11.96 0.97, 1.43 Dose1 4 1.23 0.22 0.11 18.26 0.87, 1.59 Dose2 4 1.19 0.10 0.05 8.43 1.03, 1.35 Dose3 4 1.19 0.23 0.11 19.11 0.83, 1.55 Dose4 4 1.30 0.10 0.05 7.61 1.14, 1.46

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

Ctrl 1.25 1.00 1.31 . .

Dose1 1.30 0.92 1.41 102.29 -2.29

Dose2 1.21 1.05 1.29 98.75 1.25

Dose3 1.21 0.94 1.39 98.75 1.25

Dose4 1.31 1.19 1.40 108.32 -8.32

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

4 15 0.33 0.856

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 71 of 86

Version: 22 September 2011 

### 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 1.20 . 1.22 . . . . . .

Dose1 1.23 0.998 1.22 0.651 . . . . .

Dose2 1.19 1.000 1.22 0.686 0.996 . . . .

Dose3 1.19 1.000 1.22 0.705 0.996 1.000 . . .

Dose4 1.30 0.817 1.22 0.717 0.972 0.867 0.867 . .

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

4 1.55 0.817

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 1.25 . .

Dose1 1.30 0.678 0.718

Dose2 1.21 0.780 0.384

Dose3 1.21 0.889 0.444

Dose4 1.31 0.494 0.703

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

4 15 0.33 0.856

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 -1.20 . -1.20 . . . . . .

Dose1 -1.23 0.998 -1.20 0.586 . . . . .

Dose2 -1.19 1.000 -1.20 0.620 0.996 . . . .

Dose3 -1.19 1.000 -1.20 0.639 0.996 1.000 . . .

Dose4 -1.30 0.817 -1.30 0.281 0.972 0.867 0.867 . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 72 of 86

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
4 1.55 0.817

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 -1.25 . .

Dose1 -1.30 0.678 0.282

Dose2 -1.21 0.780 0.616

Dose3 -1.21 0.889 0.556

Dose4 -1.31 0.494 0.297

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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

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

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 . . , . Dose3 4 0.00 0.00 0.00 . . , . Dose4 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 . .

Dose3 0.00 0.00 0.00 . .

Dose4 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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 73 of 86

Version: 22 September 2011 

### . . . 1 . . . . . . .

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 . . . . . . . .

Dose3 0.00 . . . . . . . .

Dose4 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

4 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 .

Dose3 0.00 1.000 .

Dose4 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

. . . 1 . . . . . . .

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

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

Ctrl 0.00 . . . . . . . .

Dose1 0.00 . . . . . . . .

Dose2 0.00 . . . . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 74 of 86

Version: 22 September 2011 

### Dose3 0.00 . . . . . . . .

Dose4 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
4 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 .

Dose3 0.00 1.000 .

Dose4 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL
Williams Dose1

Jonckheere Dose1

test for fish screen study - TEST DATA 2 4 D 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
0.963 0.608 0.683 0.615 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 32.00 4.02 2.01 12.56 25.60, 38.40 Dose1 4 31.25 3.88 1.94 12.43 25.07, 37.43 Dose2 4 33.63 4.96 2.48 14.74 25.74, 41.51 Dose3 4 33.13 1.97 0.99 5.96 29.98, 36.27 Dose4 4 34.50 3.08 1.54 8.93 29.60, 39.40

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

Ctrl 31.25 28.00 37.50 . .

Dose1 31.00 27.00 36.00 97.66 2.34

Dose2 34.50 27.00 38.50 105.08 -5.08

Dose3 32.50 31.50 36.00 103.52 -3.52

Dose4 34.25 31.00 38.50 107.81 -7.81

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 75 of 86

Version: 22 September 2011 

### 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

4 15 0.48 0.749

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

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

Ctrl 32.00 . 32.90 . . . . . .

Dose1 31.25 0.995 32.90 0.719 . . . . .

Dose2 33.63 0.925 32.90 0.754 0.891 . . . .

Dose3 33.13 0.978 32.90 0.772 0.950 1.000 . . .

Dose4 34.50 0.748 32.90 0.783 0.732 0.997 0.984 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 2.41 0.661

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 31.25 . .

Dose1 31.00 0.889 0.386

Dose2 34.50 0.678 0.722

Dose3 32.50 0.491 0.745

Dose4 34.25 0.346 0.879

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

4 15 0.48 0.749

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

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

Ctrl -32.00 . -31.63 . . . . . .

Dose1 -31.25 0.995 -31.63 0.642 . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 76 of 86

Version: 22 September 2011 

### Dose2 -33.63 0.925 -33.38 0.388 0.891 . . . .

Dose3 -33.13 0.978 -33.38 0.401 0.950 1.000 . . .

Dose4 -34.50 0.748 -34.50 0.241 0.732 0.997 0.984 . .

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

4 2.41 0.661

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 -31.25 . .

Dose1 -31.00 0.889 0.614

Dose2 -34.50 0.678 0.278

Dose3 -32.50 0.491 0.255

Dose4 -34.25 0.346 0.121

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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

0.964 0.633 0.391 0.812 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 29.80 6.40 3.20 21.47 19.62, 39.98 Dose1 4 29.03 6.47 3.23 22.28 18.74, 39.31 Dose2 4 25.93 5.27 2.64 20.34 17.53, 34.32 Dose3 4 22.88 8.72 4.36 38.14 8.99, 36.76 Dose4 4 19.70 5.97 2.98 30.29 10.20, 29.20

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

Ctrl 28.30 24.00 38.60 . .

Dose1 29.05 22.90 35.10 97.40 2.60

Dose2 27.15 18.90 30.50 87.00 13.00

Dose3 22.85 12.50 33.30 76.76 23.24

Dose4 18.35 14.00 28.10 66.11 33.89

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 77 of 86

Version: 22 September 2011 

### ******************************************************************************** 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

4 15 1.61 0.223

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 29.80 . 29.80 . . . . . .

Dose1 29.03 0.999 29.03 0.513 . . . . .

Dose2 25.93 0.826 25.93 0.271 0.962 . . . .

Dose3 22.88 0.418 22.88 0.105 0.693 0.965 . . .

Dose4 19.70 0.146 19.70 0.031 0.322 0.683 0.959 . .

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

4 5.36 0.252

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 28.30 . .

Dose1 29.05 0.780 0.332

Dose2 27.15 0.678 0.210

Dose3 22.85 0.346 0.088

Dose4 18.35 0.156 0.011

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL
Williams Dose4

Jonckheere Dose4

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

4 15 1.61 0.223

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 -29.80 . -25.47 . . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 78 of 86

Version: 22 September 2011 

### Dose1 -29.03 0.999 -25.47 0.884 . . . . .

Dose2 -25.93 0.826 -25.47 0.907 0.962 . . . .

Dose3 -22.88 0.418 -25.47 0.919 0.693 0.965 . . .

Dose4 -19.70 0.146 -25.47 0.926 0.322 0.683 0.959 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 5.36 0.252

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 -28.30 . .

Dose1 -29.05 0.780 0.668

Dose2 -27.15 0.678 0.790

Dose3 -22.85 0.346 0.912

Dose4 -18.35 0.156 0.989

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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
0.944 0.281 2.641 0.075 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 96.93 0.50 0.25 0.52 96.13, 97.72 Dose1 4 96.30 3.08 1.54 3.19 91.41, 101.19 Dose2 4 96.13 1.54 0.77 1.60 93.68, 98.57 Dose3 4 97.68 0.78 0.39 0.80 96.43, 98.92 Dose4 4 95.80 2.68 1.34 2.79 91.54, 100.06

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

Ctrl 97.10 96.20 97.30 . .

Dose1 97.75 91.70 98.00 99.36 0.64

Dose2 96.60 93.90 97.40 99.17 0.83

Dose3 97.70 96.70 98.60 100.77 -0.77

Dose4 95.30 93.20 99.40 98.84 1.16

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 79 of 86

Version: 22 September 2011 

### ******************************************************************************** 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

4 15 0.56 0.697

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

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

Ctrl 96.93 . 96.93 . . . . . .

Dose1 96.30 0.975 96.70 0.515 . . . . .

Dose2 96.13 0.943 96.70 0.547 1.000 . . . .

Dose3 97.68 0.954 96.70 0.565 0.862 0.804 . . .

Dose4 95.80 0.839 95.80 0.296 0.996 0.999 0.677 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 3.93 0.415

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 97.10 . .

Dose1 97.75 0.343 0.877

Dose2 96.60 0.678 0.385

Dose3 97.70 0.235 0.825

Dose4 95.30 0.346 0.474

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

4 15 0.56 0.697

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

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 80 of 86

Version: 22 September 2011 

### Ctrl -96.93 . -96.45 . . . . . .

Dose1 -96.30 0.975 -96.45 0.717 . . . . .

Dose2 -96.13 0.943 -96.45 0.752 1.000 . . . .

Dose3 -97.68 0.954 -96.74 0.693 0.862 0.804 . . .

Dose4 -95.80 0.839 -96.74 0.704 0.996 0.999 0.677 . .

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

4 3.93 0.415

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 -97.10 . .

Dose1 -97.75 0.343 0.123

Dose2 -96.60 0.678 0.615

Dose3 -97.70 0.235 0.175

Dose4 -95.30 0.346 0.526

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D ANALYSIS RESULTS FOR VARIABLE VAR13 ( F VTG re-analysis (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
0.958 0.511 0.324 0.858 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 59.80 26.56 13.28 44.42 17.53, 102.06 Dose1 4 74.85 30.98 15.49 41.40 25.54, 124.15 Dose2 4 55.67 18.79 9.40 33.76 25.77, 85.57 Dose3 4 58.70 33.69 16.84 57.39 5.09, 112.31 Dose4 4 92.48 27.63 13.82 29.88 48.51, 136.45

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

Ctrl 60.45 27.03 91.27 . .

Dose1 67.38 46.50 118.13 125.16 -25.16

Dose2 59.01 32.96 71.71 93.10 6.90

Dose3 60.78 18.20 95.05 98.16 1.84

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 81 of 86

Version: 22 September 2011 

### Dose4 83.75 70.47 131.96 154.66 -54.66

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

4 15 1.21 0.346

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 59.80 . 68.30 . . . . . .

Dose1 74.85 0.860 68.30 0.750 . . . . .

Dose2 55.67 0.999 68.30 0.784 0.865 . . . .

Dose3 58.70 1.000 68.30 0.801 0.922 1.000 . . .

Dose4 92.48 0.322 68.30 0.812 0.896 0.378 0.459 . .

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

4 3.89 0.422

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 60.45 . .

Dose1 67.38 0.678 0.718

Dose2 59.01 1.000 0.442

Dose3 60.78 1.000 0.426

Dose4 83.75 0.235 0.871

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

4 15 1.21 0.346

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

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 82 of 86

Version: 22 September 2011 

### Ctrl -59.80 . -59.80 . . . . . .

Dose1 -74.85 0.860 -63.07 0.513 . . . . .

Dose2 -55.67 0.999 -63.07 0.545 0.865 . . . .

Dose3 -58.70 1.000 -63.07 0.563 0.922 1.000 . . .

Dose4 -92.48 0.322 -92.48 0.078 0.896 0.378 0.459 . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
4 3.89 0.422

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl -60.45 . .

Dose1 -67.38 0.678 0.282

Dose2 -59.01 1.000 0.558

Dose3 -60.78 1.000 0.574

Dose4 -83.75 0.235 0.129

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

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

test for fish screen study - TEST DATA 2 4 D 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

0.958 0.511 0.324 0.858 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , . Dose4 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 83 of 86

Version: 22 September 2011 

### Dose3 . . . . .

Dose4 . . . . .

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

4 15 1.21 0.346

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 . . .

Dose3 . . .

Dose4 . . .

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

4 15 1.21 0.346

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 . . .

Dose3 . . .

Dose4 . . .

Jonckheere Dose1

test for fish screen study - TEST DATA 2 4 D 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
0.958 0.511 0.324 0.858 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 84 of 86

Version: 22 September 2011 

### Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , . Dose4 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

Dose3 . . . . .

Dose4 . . . . .

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

4 15 1.21 0.346

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 . . .

Dose3 . . .

Dose4 . . .

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

4 15 1.21 0.346

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 . . .

Dose3 . . .

Dose4 . . .

Jonckheere Dose1

test for fish screen study - TEST DATA 2 4 D 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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 85 of 86

Version: 22 September 2011 

### 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 0.511 0.324 0.858 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , . Dose4 0 . . . . . , .

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

Ctrl . . . . .

Dose1 . . . . .

Dose2 . . . . .

Dose3 . . . . .

Dose4 . . . . .

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

4 15 1.21 0.346

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 . . .

Dose3 . . .

Dose4 . . .

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

4 15 1.21 0.346

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 . . .

Dose3 . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with 2,4-D

EPA MRID Number 48317001

Page 86 of 86

Version: 22 September 2011 

### Dose4 . .

. Jonckheere Dose1

# DATA EVALUATION RECORD

2,4- DICHLOROPHENOXY ACETIC ACID ( 2,4-D)

Study Type: O CSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-26-2012 (MRID 48614305)

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

Arlington, VA 22202

Prepared by
CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/14 /201 2

Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 3/22 /201 2

Program Manager: Signature: Jack D. Early, M.S. Date: 3 / 27 /201 2

Quality Assurance: Signature: Jack D. Early, M.S. Date: 3 / 27 /201 2

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

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The rob ustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each
indiv idual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).2.4-D I 030001



## Primary Reviewer: -'G=r=-=eCQg=or"-'y'-'A'-=k=erm=a=n=-------- 

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


## Health Effects Division Steroidogenesis Assay (2011) I Page I of 12
OCSPP 890

. 1550/ OECD None



## DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890

. 1550



## PC CODE: 030001 

## DP BARCODE: D398637



## TXR#: 0052104 

## CAS No.: 94- 75- 7



## TEST MATERIAL (PURITY): 2

, 4-D (98

. 5% a.i.)



## SYNONYMS: 2

, 4-dichlorophenoxyacetic acid ; 2

, 4-D acid, 2-(2

, 4-dichlorophenoxy)acetic

acid



## CITATION: LeBaron, M

. J. ; Kan, H.L; Perala, A.W. (2011) Evaluation of2

, 4- dichlorophenoxyacetic acid (2

, 4-D) in the 

## in vitro steroidogenesis assay. Toxicology & 

### Environmental Research and Consulting, The Dow Chemical Company, Midland

, MI. Laboratory Study No.: 111038, October 20

, 2011.

MRID 48614305. Unpublished

.



## SPONSOR: Industry Task Force 

## II on 2,4-D Research Data

, 1900 K Street NW, Washington

, DC



## TEST ORDER#: CON-030001-1



## EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48614305), H295R cells
cultured 

## in vitro in 24-well plates were incubated with 2,4-D (98

. 5% purity

, Lot# 2006 2433 8006-USA) at log concentrations of 10

- 10 

### to 10

- 4 

### M for 48 hours in triplicate in three independent experiments

. Dimethyl sulfoxide (DMSO) was used as the vehicle

, at a final

concentration in the assay of 0.1 %.

Testosterone and estradiol levels were measured using LC-APPi-MS/MS. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to
demonstrate that the assay responded properly to positive control agents at two concentration
levels

. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack

o f cytotoxicity

, adequate production of testosterone and estradiol, acceptable reproducibility (low

% CV)

, and appropriate induction and inhibition with positive controls

, with two exceptions. The required concentration for estradiol production (40 pg/mL) was not met in any run for blank or solvent control

, with values ranging from 26

. 7 to 38

. 3 pg/mL

, but the basal concentrations were greater than 2.5-fold of the minimum detection level (10 pg/mL) as required

. Secondly

, all threeSter oidogenesis Assay ( 2011 ) / Page 2 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## runs of 1 μM prochlora z only reduced estradiol to 0.6-fold that of the solvent control (instead of
0.5- fold).

2,4-D had no effect on testosterone production at concentrations up to 10 −4 

## M, and no effect on estradiol production at concentrations up to 10 −5 

## M. A t the highest co ncentration tested (10 −4 

## M), 2,4- D i ncrease d (p≤0.05) estradiol levels by 20% (1.2- fold) in all three runs relative to the
DMSO

- treated cells.

Based on hormone responses in each of the independent runs, 2,4- D treatment resulted in statistically significant and reproducible increases in estradiol production. 2,4- D treatment did not result in statistically significant and reproducible alterations in testosterone production.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Steroidogenesis Assay (OCSPP 890.1550).

COMPLIANCE : Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.Ster oidogenesis Assay ( 2011 ) / Page 3 of 12 2,4 -D / 030001 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 Study Director: H. L. Kan
Other Personnel: M. J. LeBaron ( lead scientist ), A. W. Perala ( analytical chemist ), B. B

. Gollapudi, ( technical reviewer ) Study Period: March 14, 2011

– October 20, 2011



## 2

. Test S ubstance : 2,4

- D
Description: O ff

- white powder
Lot # (expiration date): 2006 2433 8006

- USA (not provided) Purity: 9 8.5 % Solubility: Soluble in DMSO up to 0.1 M; up to 315 mg/L in water Vapor pressure : 1.9 × 10

- 5 Pa at 25°C Stability: 2 year shelf life ; stability under test conditions not provided Storage conditions: Ambient
CAS #: 94

- 75

- 7 Molecular weight: 221.0
Structure:



## 3

. Positive C ontrol (Inducer) : Forskolin
Description (molecular weight) : White powder (410.50)
Source: Sigma

- Aldrich (St. Louis, MO)
Lot # ( expiration d ate) : 097K50653 ( not provided ) Purity: 99 % Solubility (in s olvent): Soluble in DMSO up to 0.01 M Storage conditions: Ambient
CAS #: 66575

- 29

- 9



## 4. Positive C ontrol (Inhibitor) : Prochloraz
Description (molecular weight): Off

- w hite powder (376.67)
Source: Sigma

- Aldrich (St. Louis, MO)
Lot # (expiration date): SZE 6220X ( not provided ) Purity: 99.1 % Solubility (in solvent): Soluble in DMSO up to 0.01 M Storage conditions: Ambient
CAS #: 6 7747

- 09

- 5



## 5

. Solvent/ V ehicle C ontrol : Dimethyl Sulfoxide ( DMSO )
Description (molecular weight) : Clear liquid (78.13)
Source: Sigma

- Aldrich (St. Louis, MO)
Lot # (expiration date): 16496CPV and 68996LMV ( n ot provided ) Purity: ≥ 99 .9 % Storage conditions: Room temperature
CAS #: 67

- 68

- 5
Justification for choice of solvent: Not provided. DMSO is a Guideline recommended solvent in which 2,4

- D is reasonably soluble

. Final concentration in assay : 0.1 % v/vSter oidogenesis Assay ( 2011 ) / Page 4 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## 6

. Stock Medium : Dulbecco’s modified Eagle’s medium/ Ham’s F12
nutrient mixture

Source: Sigma

- Aldrich ( St. Louis, MO )
Lot # (expiration date): RNBB 6106 ( not provided )

Sodium bicarbonate : C omponent of stock medium
Nu

- Serum : 2.5%, BD Biosciences ; L ot # 81515 ; t ested for background hormone concentrations by performing laboratory
ITS+ premix: BD Biosciences; Lot # 88964 & 05245 Other components: 2.5 mM L

- glutamine (Life Technologies, GIBCO, Grand Island, NY ), and penicillin/streptomycin (25 IU/ mL /25 μ g/ mL ) (GIBCO).



## 7. Test Cells : H295R human adrenocortical carcinoma cells (ATCC CLR -2128; Batch # not provided) at passage 7.5 – 8.5 were incubated in the stock medium. Incubation conditions were at approximately 37° C with 5% CO 2 

## for approximately 24 hours prior to exposure.

The following performance criteria were met (indicated by an “x”):

X Cell passage identifier . Cell Passage # : 7 .5

– 8 .5

X Cells frozen down at passage 5

X Frozen cells cultured for 4 additional passages

X Total number of passages does not exceed 10



## B. METHODS

1. Pre -T est Information

a. Hormone Assay Interference Test : A separate chemical interference assay was not performed, as LC/APPI -MS/MS was used along with internal spike-in controls to measure extraction and quantitation efficiency.

b. Hormone Extraction : Briefly, s tandards and samples were vortexed with methylene chloride containing both testosterone and estradiol internal standards, and the organic phase were transferred to clean vials. The aliquots were evaporated to dryness, and the final
residue in each vial was deriv a tized with dansyl chloride in sodium bicarbonate buffer (100mM, pH 10.5). Samples were then subjected to LC/APPI

- MS/MS analysis.

c. Laboratory Proficiency Test : The Sponsor stated that Laboratory proficiency assays were performed to optimize and validate the H295R steroidogenesis assay. These non- GLP, unpublished experimental results with positive control chemicals demonstrated laboratory proficiency and validation and are reported separately (data not presented and report not cited).

2. Test Solutions : 2,4-D, forskolin, and prochloraz w ere dissolved in DMSO and diluted
1:1000 in the final treatment medium. When added to the cell culture plates, these dilutions yie lded final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μ M for prochloraz, and 10

− 4 

## , 10

− 5 

## , 10

− 6 

## , 10

− 7 

## , 10

− 8 

## , 10

− 9 

## , and 10

− 10 

## M for 2,4-D. The selected concentrations of the test material in the dosing solutions used for the treatment in a definitive assay (generally the first assay) were verified analytically. The f inal
concentration of DMSO in the medium was held constant at 0.1 % (v/v).Ster oidogenesis Assay ( 2011 ) / Page 5 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## 3. Cell Plating and P reincubation : Cells were maintained in the stock medium described
above. The hormone concentrations in undiluted Nu- Serum were 974 p g/mL for testosterone and 681 pg/mL for estradiol. H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for seven to eight additional passages prior to use in the assay. The cells were plated into wells of a 24

- well cell culture plate at a density of approximately 200,000 – 300,000 cells in 1 mL (50 – 60% confluency). The cells were then placed into an incubator (approximately 37° C /5% CO 2 

## ) for approximately 24 hours prior to chemical exposure. After a 24 -hour pre- incubation, the cells were checked microscopically for good attachment and proper morphology.

4. Exposure: The medium was removed from the cells and replaced with 1 mL of medium containing concentrations of 2,4-D (or solvent) in triplicate, according to the schematic presented in Table 1.

TABLE 1

. Dosing Schematic for the Exposure of H295R Cells to 2,4

- D (Final Concentrations in M). a

1 2 3 4 5 6

A DMSO DMSO DMSO 10

− 7 10

− 7 10

− 7

B 10

− 4 10

− 4 10

− 4 10

− 8 10

− 8 10

− 8

C 10

− 5 10

− 5 10

− 5 10

− 9 10

− 9 10

− 9

D 10

− 6 10

− 6 10

− 6 10

− 10 10

− 10 10

− 10

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



## A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s response to forskolin (an inducer 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 S chematic for the QC P late for P ositive C ontrols ( F inal C oncentrations in μM). a

1 2 3 4 5 6

A Blank b Blank Blank Blank + methanol c Blank + methanol Blank + methanol

B DMSO DMSO DMSO DMSO + methanol DMSO + methanol DMSO + methanol
C Forskolin 1 μM Forskolin 1 μM Forskolin 1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM

D Forskolin 10 μM Forskolin 10 μM Forskolin 10 μM Prochloraz 1 μM Prochloraz 1 μM Prochloraz 1 μM

a Data were obtained from page 15 of the study report.
b Blank wells received medium only.

c Methanol (MeOH) was added after the exposure was terminated and the medium was removed.



## Following dosing, the plates were incubated for approximately 48 hours under the conditions previously described. After the incubation period, t wo aliquots of medium were
collected and frozen at

− 80°C until further processing.

5. Cell V iability/ Cytotoxicity A ssay : The cell viability/cytotoxicity testing was conducted in the QC plate and in the test chemical exposure plate immediately after termination of the exposure experiments. In addition to viability/cytotoxicity testing, cells were checked for the degree of co nfluence, homogeneity from well- to -well, and any signs of cytotoxicity orSter oidogenesis Assay ( 2011 ) / Page 6 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## altered morphology. A subjective parallel visual assessment of each well was conducted.
The CellTiter 96 A queous One Solution Cell Proliferation Assay kit (Madison Wisconsin,
Cat# G3580) was used for assessment of cell viability testing, as per manufacturer’s instructions. This cell proliferation assay kit is a colorimetric modified MTT cell viability

assay. The A queous One Solution contains a tetrazolium compound [3-(4,5- dimethyl thiazol-2- yl)

- 5(3 -carboxymethoxyphenyl)-2- (4 -sulfophenyl)-2H- tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportio nal to the number of viable cells in culture. The minimum cell viability required per well was ≥80%; wells with lower viability were excluded in the final data analysis.

6. Hormone M easurement S ystem : The concentration of each hormone (testosterone and estradiol) was evaluated in triplicate in the culture medium . Hormones were extracted from the samples with methylene chloride, deriv a tized with dansyl chloride , and analyzed by
LC / APPI

- MS/MS. The lower limit of quantification (LLQ) for this measurement system was 25 p g/ mL for testosterone and 10 pg/ mL for estradiol.

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 within 30% of nominal concentration)

NA Hormone cross

- reactivity (antibody

- based assays only; ≤30% of basal production of the respective hormone)

X Solvent control within 75% range below maximum response on standard curve

NA Test compound tested for interference with measurement system



## C. DATA ANALYSIS : To evaluate the relative increase or decrease of hormone production after test chemical exposure, the results were normalized to the mean solvent control (SC) value for each assay and results were expressed as fold

- change relative to the SC in each exposure plate. All data were expressed as mean ± Standard Deviation (SD)

. Relative changes were calculated as follows:

Relative Change = Hormone concentration in each well ÷ Hormone concentration of mean solvent (vehicle) control

Prior to conducting statistical analyses, the assumptions of normality and variance homogeneity were evaluated. Homogeneity of variance were 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, then the data were transformed to approximate homogeneity or a normal distribution. If the data were homogeneous and approximately normally distributed, differences between chemical treatments and SC were analyzed 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) and if significant, was followed by the Wilcoxon rank sum test with a Bonferroni- Holm correction. Differences were considered significant at p ≤ 0.05. These statistical analyses were considered appropriate.Ster oidogenesis Assay ( 2011 ) / Page 7 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## II. RESULTS

A. TEST COMPOUND : Precipitation of the test compound was not reported at any
concentration. C ytotoxicity was not observed at any tested 2,4- D concentration

. The variability (CV) be tween the runs for the solvent controls was 4.6% (testosterone) and 14.6% (estradiol), both within the guideline recommendation of 30%. The variability (CV) within each run for the solvent controls in the testosterone and estradiol assays was 13.6 % or less, which was within the guideline recommendation of 30%.

2,4-D had no e ffect on testosterone levels at any concentration tested (Table 3, Figure 1),
and a slight effect on estradiol levels at 2,4-D concentrations of 10 μM or less. However, at
a concentration of 100 μ M, 2,4- D i n crease d (p≤0.05) estradiol levels by 20% relative to
DMSO

- treated cells in all three runs (Figure 2 ).

TABLE 3

. Mean (±SD) Hormone Concentrations Following Treatment with 2,4 -D f or 48 Hours. a

Nominal
Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD Statistical Significance b

Testosterone (pg/mL) Fold Difference

DMSO 842.0 735.0 778.0

— — — — — — 0.0001 776.0 691.7 746.3 0.9 0.9 1.0 0.9 0.0

— 0.001 805.7 696.0 752.0 1.0 0.9 1.0 1.0 0.0

— 0.01 737.7 696.3 726.7 0.9 0.9 0.9 0.9 0.0

— 0.1 752.0 647.0 745.3 0.9 0.9 1.0 0.9 0.0

— 1 796.7 665.0 734.0 0.9 0.9 0.9 0.9 0.0

— 10 787.7 684.3 747.0 0.9 0.9 1.0 0.9 0.0

— 100 714.3 610.3 728.0 0.8 0.8 0.9 0.9 0.1

— Estradiol (pg/mL) Fold Difference

DMSO 29.8 35.7 37.0

— — — — — — 0.0001 24.7 37.2 36.0 0.8 1.0 1.0 0.9 0.1

— 0.001 24.7 35.7 34.2 0.8 1.0 0.9 0.9 0.1

— 0.01 25.6 34.8 37.8 0.9 1.0 1.0 1.0 0.1

— 0.1 26.5 36.4 34.9 0.9 1.0 0.9 1.0 0.1

— 1 25.3 33.7 35.1 0.8 0.9 0.9 0.9 0.1

— 10 27.0 33.8 37.9 0.9 0.9 1.0 1.0 0.1

— 100 34.6 44.3 45.1 1.2 1.2 1.2 1.2 0.0 Runs 1, 2, 3

a Data were obtained from page 28 of the study report. The lower limit of quantification (LLQ) was 25 p g/mL for
testosterone and 10 pg/mL for estradiol.Ster oidogenesis Assay ( 2011 ) / Page 8 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## FIGURE 1. Change in Testosterone P roduction Relative to 2,4-D C oncentration.Ster oidogenesis Assay ( 2011 ) / Page 9 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## FIGURE 2. Change in Estradiol Production Relative to 2,4-D Concentration.Ster oidogenesis Assay ( 2011 ) / Page 10 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



## B. CYTOTOXICITY : 2,4-D was not cytotoxic to the H295R cells (Table 4 ). Via bility in each run ranged from 92.5–133% relative to solvent controls.

TABLE 4 . Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, or 2,4

- D for 48 Hours. a



### Compound Concen tration ( μ M) Cell Viability

Mean SD

Media NA 96.6 1.59
Media + Methanol NA 29.4 2.35
DMSO + Methanol NA 30.9 3.18
Forskolin 1 121.7 9.91
Forskolin 10 114.1 5.25
Prochloraz 0.1 110.1 9.50
Prochloraz 1 99.9 7.40
2,4

- D 0.0001 116.6 8.05 2,4

- D 0.001 110.9 5.37 2,4

- D 0.01 111.7 8.35 2,4

- D 0.1 104.9 4.92 2,4

- D 1 106.4 5.89 2,4

- D 10 110.7 5.16 2,4

- D 100 106.9 5.12

a Values were calculated by the reviewers from data obtained from page 41 of the study report.



## 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 5. The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both
blank and SC wells for testosterone (Table 6) . The required concentration for estradiol was

not met in any run for blank or SC, with values ranging from 26.7–38.3 pg/mL. Forskolin at 10 μM induced testosterone an average of 2.4-fold and estradiol 20-fold over the SC. Prochloraz at 1 μM inhibited concentrations of testosterone by 0.3-fold and estradiol by 0.6- fold compared to SC. Guideline requirements for basal hormone induction and inhibition were met for testosterone and estradiol, except that all three runs of 1 μM prochloraz only
reduced estradiol to 0.6-fold that of the SC rather than 0.5-fold.

The variability (%CV) between the runs (calculated by the reviewer) ba sed on the absolute
hormone concentrations in the SC were 4.6% for testosterone and 14.6 % for estradiol , and
within the guideline criteria of ≤30% for the assays. The %CVs within each run for the QC plates were 2.5-6.6% for testosterone and 3.8-10.1 % for estradiol, which were within the guideline criteria (≤30%).Ster oidogenesis Assay ( 2011 ) / Page 11 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



### TABLE 5 . Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for
48 Hours. a

Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD

Testosterone (pg/mL) Fold Difference (Relative to DMSO)

Blank 1041 936 1040

— — — — — DMSO 778 719 781

— — — — — 1 μM Forskolin 1377 1217 1310 1.8 1..7 1.7 1.7 0.0
10 μM Forskolin 2007 1757 1733 2.6 2.4 2.2 2.4 0.2
0.1 μM Prochloraz 515 514 534 0.7 0.7 0.7 0.7 0.0 1 μM Prochloraz 250 233 218 0.3 0.3 0.3 0.3 0.0

Estradiol (pg/mL) Fold Difference (Relative to DMSO)

Blank 26.3 34.8 37.2

— — — — — DMSO 28.5 34.2 38.3

— — — — — 1 μM Forskolin 348 366 492 12.2 10.7 12.8 11.9 1.1 10 μM Forskolin 610 626 780 21.4 18.3 20.4 20.0 1.6 0.1 μM Prochloraz 26.4 29.7 28.6 0.9 0.9 0.5 0.8 0.3
1 μM Prochloraz 17.6 19.5 21.9 0.6 0.6 0.6 0.6 0.0

a Data were obtained from page 29 of the study report. The lower limit of quantification (LLQ) was 25 p g/mL for
testosterone and 10 pg/mL for estradiol.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Based on the combined hormone responses in each of three independent H295R steroidogenesis assays, it was determined that 2,4-D treatment resulted in a statistically significant increase in estradiol production at the assay
limit

- concentration of 100 μM (10

− 4 

## M), while no effects on estradiol production were seen
at lower concentrations. There were no effects on testosterone production at any concentration of 2,4- D. The slight (1.2-fold or less) increase in estradiol production did not
meet the 1.5

- fol d cut-off criteria established in the validation program for this assay, and hence was not interpreted to be biologically relevant.

B. AGENCY COMMENTS : Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. The exceptions included: the required concentration for estradiol production (40 pg/mL) was not met in any run for blank or solvent control, with values ranging from 26.7–38.3 pg/mL; and all three runs of 1 μM prochloraz only reduced estradiol
to 0.6-fold that of the solvent control rather than 0.5-fold.

2,4-D had no effect on testosterone production at concentrations up to 10 −4 

## M, and no effect on estradiol production at concentration up to 10 −5 

## M. However, at the highest
concentration tested (10 −4 

## M), 2,4- D i ncrease d (p≤0.05) estradiol levels by 20% in all three

runs relative to the DMSO

- treated cells.

Based on hormone responses in each of the independent runs, 2,4- D treatment resulted in a statistically significant and reproducible alterati on in estradiol production.Ster oidogenesis Assay ( 2011 ) / Page 12 of 12 2,4 -D / 030001 OCSPP 890.1550/ OECD None



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

• Stability of 2,4-D in the test system was not reported.

• The report erroneously specified an LLQ of 25 mg/mL. The assay must be able to quantify 500 pg/mL according to the G uideline, and this assay was able to do so.

• There were two minor departures from the Guideline: (i) the required concentration for estradiol was not met in any run for blank or SC, ranging from 26.7 to 38.3 pg/mL rather than 40 pg/mL; and (ii) all three runs of 1 μM prochloraz only reduced estradiol
to 0.6-fold that of the SC rather than 0.5-fold.