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.Apr. 2010 

The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects 

Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays 

submitted in response to the agency's Test Order for 2,4-D: Test Order# CON-030001-1. 

Page 1 of2 

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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)).

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

EPA MRID Number 48317002

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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: 02/24/2012

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'

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

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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 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.

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EXECUTIVE SUMMARY

The 21-day assay of 2,4-D (purity 98.6%) on amphibian 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 Day 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). 

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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 Effects1,2 in the Amphibian 

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

Treatment 

(mg a.i./L) 

[mean-measured]

NF Developmental 

Stage 

Hind Limb 

Length3

Asynchronous 

Development 

Thyroid Gross 

and 

Histopathology 

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

0.273 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). 

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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 

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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. 

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B. Test Organism

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: South African Clawed 

Frog 

EPA recommends African clawed frog 

(Xenopus laevis). Western [Africa] clawed 

frog Silurana (Xenopus) tropicalis may be 

used as an alternate species1; however, a 

list of all of the necessary protocol 

deviations to accommodate this species is 

recommended for inclusion in the study 

report. The guideline recommends that the 

performance criteria used to support the 

reliability of the test be identified.

Species scientific name: Xenopus laevis Species strain (if stated): 

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

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as inhouse stock? Yes Breeding pairs were ordered from Xenopus Express (Brooksville, Florida). 

Adult male and female frogs were injected with human chorionic gonadotropin at least twelve hours before the desired breeding events. 

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

No Not reported 

Acclimation period for parental frogs (if applicable): 

 days

Details on parental feeding: Not reported Details on parental health: Not reported 

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Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? No Not 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 (if applicable): 

Not reported 

Number of eggs sampled per spawn:

Not reported 

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

be initiated with larvae at Nieuwkoop - 

Faber (NF) developmental stage 51 (17 

days post-fertilization).

Age at test initiation: 18 days postfertilization (dpf) Test initiation began when tadpoles were 16 days old (discrepancy)Mean total length at test initiation (if reported): 

mm Not reported 

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

mm : mm Not reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the optional size selection method used? 

No Not reported 

Details on larval selection: Not reported Loading 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 America Iodide concentration in diet (if known):

53.7 ug/g 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

least twice per day.

Details on feeding regime: The feeding regimen 

was the same as guideline recommendations (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 

increase from 30 mg/larva/day at test 

initiation (Study Day 0-4) to 80 mg/larva/day in the last week of the test 

(Study Day 15-21).

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: flow-through EPA recommends the use of a flowthrough system.Type of flow-through dilution system (if applicable):

continuous-flow diluter system

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

32 mL/min ± 3 (complete volume turnover 

approximately every 2.7 hours for each replicate test vessel) 

Recommended flow-through rate is 25 

mL/min (complete volume replacement 

ca. every 2.7 hrs).

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

Stainless steel mixing chamber was used.

The dilution water flow and resulting test solution volume in the mixing chamber was maintained using a stainless steel float valve. The test solution was gravity fed 

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

recommended but not required; 2) 

Aeration is not recommended for mixing; 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

from the mixing chamber and split equally to replicate test vessels (contained in a temperature controlled water trough) via a Teflon manifold with Teflon delivery tubing. The diluter system was calibrated prior to test initiation, and diluter operation was monitored at least twice daily throughout the test. If there was an indication that the diluter calibration had changed (e.g., diluter malfunction, change in water quality or measured concentrations), calibration of necessary diluter components was checked and 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 static renewal (if applicable):

 NA If static renewal is used, EPA 

recommends 24-hr renewal; renewal 

period is recommended not to exceed 72 

hours.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? 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 water Water was obtained from the upper 

Saginaw Bay of Lake Huron off Whitestone Point by the City of Midland Water Treatment Plant and supplied to The Dow Chemical Company prior to municipal treatment for human consumption. Water was limed and flocculated with ferric chloride, sand-filtered, pH-adjusted with gaseous 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes Laboratory dilution water is routinely 

analyzed for pesticides, organics, metals, and other inorganics twice per year. In particular, chlorate, perchlorate, and iodide levels are also determined. 

Iodide supplementation in water?

No If reconstituted water is used or if 

background levels of iodide in natural 

water are less than 0.5 

μg/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 together 

with clear silicone adhesive

EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass 

vessels or glass vessels with stainless 

steel frames are acceptable examples).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel size: 30 x 14.5 x 20 cm deep Fill volume: 5.2 L Additional details on exposure system: 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 64 76 67 7 days 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.0 7.8 7 days 

EPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials 

should not exceed 0.5.

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Dissolved oxygen (mg/L) 5.3 7.9 7 days 

EPA recommends dissolved oxygen 

(DO) >3.5 mg/L (>40% air saturation). 

OECD recommends DO concentration 

>3.5 mg/L (>40% air saturation).

Temperature 21.9 22.5 7 days 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide <LOQ <LOQ 

EPA recommends aquatic iodide range 

0.5 - 10 

μg/L (supplemental iodide 

should not exceed 2 

μg/L).

Ammonia <0.1 0.24 4 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 quality 

Fluoride 0.1 0.1 Perchlorate <0.2 <0.2 Chlorate <10 <10 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline 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.

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 D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? No If yes, what was the method for determining the highest test concentration 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: NA Life stage: NA Test duration: NA Additional details: NA 

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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

The solubility limit and other reference studies were used to select the highest test concentration.

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

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

concentration is lowest.

Reference study citation (if applicable):

Morgan et al., 1996; Palmer and Krueger, 1997a; Palmer and Kreuger, 1997b; Alexander et al., 1985 

Separation of test concentrations:

0.1-0.4 EPA recommends that the maximum 

concentration separation be 0.1 and the 

minimum be 0.33. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of test concentrations: 4 EPA recommends a minimum of 3 

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

No "Standards were not corrected for 

purity." (Appendix B) 

Indicate the type of values presented for measured concentrations: 

Average measured 

Limit 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 reported Frequency 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

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 test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 

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

 NA EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD recommends 

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

Review. Aquatic Toxicology, 76, pp.69–92. 

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Parameter Value(s) Details or Remarks Guideline 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? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

 NA 

Photoperiod: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 12:12 

(light:dark).

Light intensity at water's surface: 

0.618-0.882 Klux EPA recommends light intensity 0.6 - 2 

Klux (at water's surface).

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: Information on test solution appearance 

did not appear to be reported in the 

report. 

Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with 2,4-D. 

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV)

≤20%.

Treatment 1 0.4 0.273 55.67 

Treatment 2 4 3.24 21.60 Treatment 3 40 38.0 16.29 Treatment 4 100 113 16.90 

Abbreviations: CV Coefficient of variation. 

LOQ = 0.120 mg a.i./L 

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 E. Observations

Biological Endpoints: Mortality (throughout study); NF stage and asynchronous development; wet weight, snout-vent length, hind limb length (Day 

7 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 (Days 7 and 

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

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

pathology observations.

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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 71 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. 

 

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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 

Stage1 # Asynchronous n 

Median 

Stage1 # 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 

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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: 

SVL1 n Mean(mm) 

±SD 

HLL: 

SVL1

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). 

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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 Weight1

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. 

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Table 13: Gross Pathology and Histopathology of the Thyroid Gland in South African Clawed Frog.

Treatment

(mg a.i./L)

[meanmeasured]

Diagnostic Observations1

Severity

Thyroid Gland 

Hypertrophy 

Thyroid Gland 

Atrophy 

Follicular Cell 

Hypertrophy 

Follicular Cell 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

0 20 20 20 20 20 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.

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Table 14: Additional Thyroid Gland Histopathology Observations in South African Clawed Frog. 

Treatment 

(mg a.i./L) 

[time-weighted, 

mean-measured] 

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence

Negative Control 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

0.273 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

3.24 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[time-weighted, 

mean-measured] 

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence

38.0 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

113 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 

Abbreviations: NA Not applicable. 

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

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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 Signs1

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. 

 

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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. 

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 Conclusions: 

The only statistically-significant effect was for Day 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. 

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Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with 2,4-D. 

Treatment 

(mg a.i./L) 

[mean-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

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

Median p Median p

% 

Diff. 

p 

% 

Diff. p

% 

Diff. 

p 

% 

Diff. p

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 NA 59 NA NA NA NA NA NA NA NA NA NA 

0.273 54 0.48 59 0.75 0 NA -3.7 0.25 0 NA 0 NA No 

3.24 54 >0.99 59 0.94 0 NA -3.7 0.25 0 NA 0 NA No 

38.0 54 >0.99 59 0.59 0 NA -3.7 0.26 0 NA 0 NA No 

113 54 >0.99 58 0.17 0 NA -14.8 0.02 0 NA 0 NA No 

Statistical Test Mann-Whitney

JonckheereTerpstra NA Jonckheere- Terpstra NA NA NA 

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Negative values indicate reductions or decreases relative 

to the negative control. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 Hind-limb length is normalized to snout-vent length (SVL). 

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Table 17: Growth Endpoints1,2 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. 

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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. 

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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. 

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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 Metamorphosis (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. 

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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 

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 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 

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 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 . . 

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

Page 45 of 221
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 

Page 46 of 221
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 . . 

Page 47 of 221
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 

Page 48 of 221
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 . . 

Page 49 of 221
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 

Page 50 of 221
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 . . . . . . . . 

Page 51 of 221
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 

Page 52 of 221
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 . . . . . . 

Page 53 of 221
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 

Page 54 of 221
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 

Page 55 of 221
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 

Page 56 of 221
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 

Page 57 of 221
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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 

Page 58 of 221
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 

Page 59 of 221
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 

Page 60 of 221
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 

Page 61 of 221
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 

Page 62 of 221
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) 

Page 63 of 221
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. 

Page 64 of 221
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 65 of 221
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 104 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 binding 

Page 66 of 221
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 15

2,4-D/ 030001 OCSPP 890.1150/ 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 competitive binding experiments, therefore, the log IC50 and 

relative binding affinity (RBA) for 2,4-D could not be calculated. The log IC50 values for R1881 alone and the positive control, 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 classified as a Non-binder in the Androgen 

Receptor Binding Assay. 

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

Assay (OCSPP 890.1150). 

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided in 

the study report. 

Page 67 of 221
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 15

2,4-D/ 030001 OCSPP 890.1150/ 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 (expiry 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 # : 50-02-2

Page 68 of 221
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 15

2,4-D/ 030001 OCSPP 890.1150/ 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 [3H]-R1881 was not 

adjusted for decay over time, and serial dilutions in TEDG buffer were prepared to achieve 

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

determine total binding. To determine non-specific binding, solutions of non-labeled R1881 

were prepared in a similar manner to achieve concentrations that were 100-fold greater than 

each respective radiolabeled concentration, resulting in final concentrations in cytosol of 25, 

50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, the radiation found in 

7.5, 15, 21, 30, or 45 μL of 10 nM [3

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

Page 69 of 221
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 15

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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 nM 

radiolabeled R1881. The optimal protein concentration was determined to be 1.97 mg 

protein/assay tube, which resulted in the binding of 8.6-9.0% of the total radioactivity 

added. As indicated in the guideline for acceptable assay performance the receptor 

concentration bound less than 25 to 35% of the radiolabeled R1881. Cytosolic protein used 

in this assay was thawed fresh for this experiment at ~4°C and maintained at ~4°C during 

the binding assay. Each run contained three concurrent replicates at each concentration, 

resulting in the 72 samples depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Run a, b

Total Binding Non-Specific Binding Radioligand alone

Tubes 1-24 c Tubes 25-48 d Tubes 49-72 e

[

3

H]-R1881

Final conc. (nM)

[

3

H]-R1881

Final conc. (nM)

R1881

Final conc. (nM)

[

3

H]-R1881

Initial conc. (nM)

[

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 triamcinolone acetonide and 7.5-45 μL [3H]-R1881. Samples were dried, 

and 300 μL of prostate cytosol were added.

d Tubes 25-48 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [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, [3H]-R1881, and/or R1881, the tubes were 

dried, dissolved in diluted prostate cytosol (300 μL), and incubated for approximately 16 

hours at 2-8°C. Samples were maintained at temperatures of ~4°C except during whole rack 

vortexing. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to 

each tube and vortexed once every 5 minutes for 20 minutes. The samples were then 

centrifuged, and the supernatant was aspirated and discarded. The samples were washed 3 

times in 50 mM TRIS buffer. Following the last wash and decanting of the Tris buffer, 

pellets were then extracted by addition of 2 ml ethanol. The samples were vortexed 3 times 

at 5 minute intervals. Samples were maintained on ice at all times between vortexing. Each 

ethanol supernatant was then decanted into a scintillation vial, and the radiation was 

quantified by liquid scintillation counting. A total of 4 runs were performed on 2 batches of 

cytosol with similar results. For the batch of cytosol used for the competitive assay, 2 runs 

were performed, 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.

Page 70 of 221
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3. Competitive Binding Experiment: A summary of the assay conditions for the competitive 

binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol

Concentration of radioligand 1 nM

Optimization of receptor concentration Sufficient to bind 4.3-5.2% 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 41 of the study report; protein concentrations (μg/tube) were not 

reported.

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

the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures 

the binding of a single concentration of [3

H]-R1881 (specific activity of 85.1 Ci/mmol) to 

the AR in the presence of increasing concentrations 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. Each assay consisted of three independent runs on three different days. 

For each run, a set of duplicate blanks and triplicate tubes with 1 μM R1881 (non-specific 

binding, NSB) were run at the beginning and end of each run. Each run also included 

triplicate samples 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). 

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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 37-42 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, with 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 [3H]-R1881. 

4. Data Analysis: The top and bottom of the curve, Hill slope, inhibition concentration (IC50), 

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. 

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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% activityb Equivocal

1

Average lowest portion of curves across 

runs is greater than 75% activityb

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 

judged to be untestable.

b. Descriptors for receptor binding

Bmax: maximal binding capacity

Kd: dissociation constant

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

by the competitor

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

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment 

parameters are presented in Table 6. The dissociation constant (Kd) for [3H]-R1881 was 

0.4641, and the estimated Bmax (nM) was 0.06392 for the batch of prostate cytosol that 

was used for this study. The Kd was below the recommended range reported in the test 

guideline (0.685-1.57 nM). Confidence in these numbers is high according to the 

goodness of fit (R2

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

TABLE 6. Saturation Binding Experiment of R-1881 with Androgen Receptor from Rat Prostate 

Cytosola

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

R2 (unweighted) 0.9937 0.9871 ND 0.9871-0.9937

Bmax (nM) 0.06194 0.06590 ND 0.06392

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

Kd (nM) 0.4359 0.4922 ND 0.4641

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

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

c The range of R2 is reported and the mean is reported for the other parameters.

R2 Goodness of fit for curve calculated for specific binding, 

ND Not determined

Figure 1 illustrates the non-specific, specific, and total binding curves for [3H]-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 values indicated acceptable performance of 

Page 73 of 221
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 15

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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.

FIGURE 1. Binding of [3H]-R1881 to the Androgen Receptor during the Saturation 

Binding Experiment.

FIGURE 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/100

μg)

Specific binding Run 2

(fM/100μM)

Total Run 2

Nonspecific Run 2

Specific Binding Run 1

(fM/100μg)

Total Run 1

Nonspecific Run 1

Scatchard

0 1 2 3 4

0

2

4

6

8

run 2

run 1

Specific Bound (fM/100μg)

Specific Bound/Free

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B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive 

binding experiments are summarized in Tables 7 and 8 and shown graphically in Figures 3 

and 4. The estimated mean log IC50 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 IC50s 

for R1881 and the weak positive control (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. 

R2 Goodness of fit 

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

RBA (%) Relative 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 97 103 101 82 to 114

Log IC50 (M) -8.993 -8.986 -8.956 -8.978 ---

IC50 (M) 1.017e-009 1.032e-009 1.106e-009 1.052e-009

Hill slope -1.0 -0.9 -1.0 -1.0 -1.2 to -0.8

R2 0.9998 0.9999 1.000 1.000 ---

RBA (%) 100 100 100 100 100

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 99.0 102.1 99.7 ±1.3 ---

10-7 96.5 98.0 104.7 99.7±2.5 ---

10-6 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 27.6 ±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 IC50 (M) -4.379 -4.440 -4.416 -4.412 ---

IC50 (M) 4.177e-005 3.630e-005 3.840e-005 3.871e-005

Hill slope -1.0 -1.1 -1.0 -1.0 -1.4 to -0.6

R2 0.9998 0.9999 0.9995 1.000 ---

RBA (%) 0.0024 0.0028 0.0029 0.0027 ---

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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. 

R2 Goodness of fit 

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

RBA (%) Relative binding affinity

Collective Responses of the Independent AR Binding Assays 

The collective 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 dexamethasone 

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

10-11 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 99.22 104.25 102.2 ± 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

10-5 102.33 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

(97.9 to 101.5)

n/a

Top (%)

(95% CI)

~-146.6

(very wide)

n/a 105.1

(103.8 to 106.4)

n/a

Log IC50 (M)

(95% CI)

n/a n/a n/a n/a

IC50 (M)

(95% CI)

n/a n/a n/a n/a

Log EC50 (M)

(95% CI)

~-13.15

(very wide)

n/a -8.363

(-9.431 to -7.294)

n/a

EC50 (M)

(95% CI)

~7.139e-014

(very wide)

n/a 4.339e-009 

(3.707e-010 to 

5.078e-008)

n/a

Hill slope

(95% CI)

~-0.8113

(very wide)

n/a 1.939

(-3.018 to 6.895)

n/a

R2 0.6318 n/a 0.9371 n/a

Data Interpretation negative negative negative n/a

RBA (%) n/a n/a n/a n/a

Page 76 of 221
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 15

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variability between assays, but the overall robustness of the responses for R1881 (strong positive 

control/standard curve) and dexamethasone (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 valid.

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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 #3 

Page 78 of 221
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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 competitive binding assay 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 Hill 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 Kd (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 30, 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 reported

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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 concentrations up to 10−4 M.

B. AGENCY COMMENTS: 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 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. Specific 

binding was >75% at all concentrations tested (10−11 to 10−4 M). An IC50 and RBA could 

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

The estimated average log IC50s for R1881 and the weak positive control (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, 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. 

Page 80 of 221
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: 3/17/2012

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: 3/26/2012

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. 

Page 81 of 221
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 82 of 221
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 

@/;911< 

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 aromatase activity. 2,4-D was 

tested at logarithmic concentrations from 10- 10 M to 10-4 M in three independent runs. 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. The sponsor considered 

concentrations higher than 10-

4 M to be not relevant for testing in this assay as they are 

substantially above the inflection point for linear toxicokinetics (See Appendix B of the AR 

binding assay study report, MRID 48614301). 

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

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

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- 10 to 

10- 5 M) using a known inhibitor ( 4-hydroxyandrostenedione; 4-0H ASDN), and the test 

chemical series (10- 10 to 10-4 M) with 3 repetitions per concentration. 

Page 83 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

Aromatase activity in the full activity controls ranged from 0.131 to 0.244 nmol∙mgprotein−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 2nd and 3rd 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 IC50, 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 the 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 IC50 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 (R2

) 

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 

activity 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 range).

Based on the data from the average response curve, 2,4-D is classified as a Non-inhibitor of 

aromatase activity in this assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Aromatase assay (OCSPP 

890.1200). 

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

Assurance statements were provided. 

Page 84 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: 2,4-D

Description: Off-white powder 

Source: Nufarm Americas, Inc. (Burr Ridge, IL)

Lot/Batch #: 2006 2433 8006-USA

Purity: 98.5% a.i.

Volatility: Not reported

Storage conditions: Ambient

Stability: Not reported

Solvent: Ethanol

Solubility (in test solvent): Soluble up to 0.01 M

Highest Concentration Tested: 10−4 M in all runs

Stock Solution Preparation

Methodology: Dissolved the test material in ethanol.

Molecular weight: 221.0

CAS #: 94-75-7

Structure:

2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS # : 63-05-8

Source: Steraloids, Inc. (Cat. # A6030100)

Lot/Batch #: L1627 

Purity: 98.4%

3. Radiolabeled Substrate: 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: 26.3 Ci/mmol

Radiochemical Purity (In-lab 

determination): Not reported

4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN) 

CAS # : 566-48-3

Source: Sigma-Aldrich (Cat. # F2552)

Lot/Batch #: 081K2133 

Purity: 99.6%

5. Solvent (Vehicle Control): Ethanol

Source:

Lot/Batch #:

Sigma Aldrich (Cat. # E7023)

72596PMV

Justification for choice of solvent: Not provided. Ethanol is a Guideline preferred solvent.

Concentration 

(% of total volume in assays): 1% v/v

Page 85 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

6. Test Microsomes: 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 Components 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 inhibition 

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 recombinant 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. 

TABLE 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

[3H]ASDN 2 μM

Propylene Glycol 100 μL 

Temperature 37°C

Incubation Time 15 min

2. Suitability Assessments: 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. Aromatase 

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 Assay: 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 the 

Page 86 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 12

2,4-D / 030001 OCSPP 890.1200 / 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 3H2O 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 Proficiency: 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 Proficiency: Raw data were not provided, however, 

summary data of proficiency exercised were equivocal. Relative aromatase activity in the 

presence of prochloraz, a known aromatase inhibitor indicated dose-dependent inhibition. 

Aromatase activity appeared to be unaffected 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 of Proficiency of New Technician for Conducting Assay (when 

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

reported. The positive control data for slope, top and bottom percent, and log IC50 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 Limita Actual Upper Limita

Slope −1.2 −0.8 –1.1 –0.78

Top (%) 90 110 91 100

Bottom (%) −5 +6 –8.2 –2.6

Log IC50 (M) −7.3 −7.0 –7.3 –7.2

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

b. Proficiency Chemicals: 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). 

TABLE 3. Proficiency Chemicals

Compound CAS# Class Concentrationsa

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. 

Page 87 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

5. Determination of Aromatase Activity with Test Chemical(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 responses (87 and 114%) 

from the beginning and end, respectively, of Run 1 that were outside of the guideline 

recommended range of 90 to 110%. 

 

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions 

(Tubes) Description Reference or Chemical (M)

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

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

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

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

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

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

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

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

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

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

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, [3H]ASDN, and NADPH. 

C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatase activity (nmol/mg protein/min) and 

percent control for the positive control and test chemical. The following raw data and 

calculated endpoints for each run were included in the report (Table 5). 

TABLE 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 minute

Page 88 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

2. Statistical Methods: 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 IC50 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 regression 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% activityb

Equivocal

Average lowest portion of curves across runs is greater 

than 75% activityb

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 110% of the average full activity, with the exception of 

the 2nd and 3rd 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), 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 

(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 overall 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 IC50, and coefficient of variation for replicates of each concentration, with the following 

Page 89 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 12

2,4-D / 030001 OCSPP 890.1200 / 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). 

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 Variance

Page 90 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

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

C. TEST SUBSTANCE: For 2,4-D, aromatase activity averaged 0.195±0.045 nmol∙mgprotein−1

∙min−1 at the lowest tested concentration (10−10 M) and 0.168±0.025 nmol∙mgprotein−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%. 

Page 91 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12

2,4-D / 030001 OCSPP 890.1200 / OECD None

FIGURE 2. Inhibition Response Curves for 2,4-D From Each Test Run. 

FIGURE 3. Mean Inhibition Response Curve for 2,4-D. 

The data for 2,4-D were modeled for Run 1, and the goodness of fit (R2

) value was 0.83; 

however, data from Runs 2 and 3 were not amenable to modeling. The average dosePage 92 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 11 of 12

2,4-D / 030001 OCSPP 890.1200 / 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 IC50 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, the estimated log IC50 averaged −7.23 M and the slope was −0.92. 

Confidence in the mean log IC50 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 Percent of Control) From Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean SD %CV

Log IC50 (M)

2,4-D NA NA NA NA NA NA

4-OH ASDN −7.2 −7.3 −7.2 −7.23 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 32 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. 

Therefore, 2,4-D is classified 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 2nd and 3rd 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 (10−5 M). These results were 

within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, 

and coefficient of variation for replicates of each concentration, with the following 

Page 93 of 221
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12

2,4-D / 030001 OCSPP 890.1200 / 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 modeled for Run 1, and the goodness 

of fit (R2) 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 IC50 averaged −7.23 M and the slope was −0.92. 

Confidence in the mean log IC50 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 

Page 94 of 221
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. 

Page 95 of 221
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 96 of 221
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: ~ 

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Signature: .. ~ A} 

Date: G ftqfta= 

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. 

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In the competitive binding experiment, no 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 log IC50 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 IC50 for the natural ligand, 17β-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 17β-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. Based on the results from the three runs, 2,4-D is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

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

assay (OCSPP 890.1250).

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

statements were provided. 

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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 27, 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: 17β-estradiol

Supplier: Sigma, St. Louis MO

Catalog #: E8875

Batch #: 098K1372

Purity: 100%

CAS #: 50-28-2

4. Radioactive ligand: [

3

H]-17β-estradiol

Supplier: Perkin-Elmer, Boston MA

Catalog #: NET517001MC

Batch#: 639068

Radiochemical purity: >97%

Specific activity: 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 #: 68-22-4

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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 or 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) Binding Experiment: A saturation binding experiment 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. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat uterine cytosol

Concentration of radioligand (as serial dilutions) 0.03-3.0 nM

Concentration of non-labeled ligand (100X [radioligand]) 3.0-300 nM

Concentration of receptor Sufficient to bind 40.77% of radioligand 

at 0.03 nMb 

Temperature ~2-8 oC

Incubation time ~16 hours

Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM

Glycerol 10%

Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM

a Data were not included in the study report, but were reported in the study profile submitted separately.

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

including minimal ligand depletion, indicated acceptable performance in the assay.

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The specific activity of the stock [3H]-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 

concentrations 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 the 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

350 μL 300 μL --- TEDG + PMSF buffer

50 μL 50 μL 50 μL [

3

H]-17β-estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17β-estradiol (8 serial dilutions,

100x each respective labeled concentration) f

100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.)

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

a Data were not included in the study report, but 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 300 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 times as follows: 

TEDG +PMSF buffer was added, vortexed, centrifuged for 10 min at 1000 x g, and the 

supernatant decanted and discarded. After washing, ethanol 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 saturation 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 Binding Experiment: A summary of the experimental conditions for the 

competitive binding experiment is presented in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind 6.75-7.15% of 

radioligand

Concentration of test substance (as serial dilutions) 10-11 to 10-4 mM

Temperature 4-8 °C 

Incubation time 16-20 hours 

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

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM 

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

The solubility of 2,4-D in ethanol was evaluated visually. On the day of the assay, the 

specific activity of the stock solution [3

H]-17β-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 achieve the concentrations 

shown in Table 4. Each assay consisted of three runs performed on separate days. Each run 

included three replicates of each test substance at each concentration, plus four blank and six 

samples of the master mix to determine full radioactivity, resulting in a total of 112 samples.

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TABLE 4. Molar (M) concentrations in Competitive 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 pages 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+PMSF 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]-17β-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 radioassayed by 

scintillation counting. The temperature was maintained at 4±2 °C throughout the assay prior 

to extraction with ethanol.

C. DATA ANALYSIS: For the competitive binding experiment, 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 log IC50 as a parameter to be estimated. Estimates of model parameters 

[e.g., log IC50, IC50, Hillslope, R2

, 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 was first individually classified as follows:

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

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

Not interactive = there are usable data points at or above 10−6 M and either the lowest point 

on the fitted response curve within the range of the data is above 75% (i.e., 

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<25% of the radiolabeled estradiol has been displaced from the ER) or a 

binding curve cannot be fitted and the lowest average percent binding among 

concentration groups in the data is above 75%. 

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

test chemical concentration of 10−6 M and either a binding curve can be fit but 

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

binding curve cannot be fit and the lowest average percent binding among 

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value

Interactive 2

Equivocal 1

Not interactive 0

Equivocal up to the limit of concentrations tested “missing”

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

following ranges: 

Test Material Classification Numerical Range

Interactive average ≥1.5

Equivocal 0.5≥ average <1.5

Not interactive average <0.5

Equivocal up to the limit of concentrations tested “missing”

b. Descriptors for receptor binding: 

Bmax: maximum specific binding number (fmol ER/100 μg cytosol protein) measures the 

concentration of active receptor sites

Kd: dissociation constant (nM), measures the affinity of the receptor for its natural ligand

IC50: concentration of the test substance (M) at which 50% of the radioligand is displaced 

from the receptor

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

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Summary data pertaining to the saturation 

binding experiment were reported separately in the study profile submitted by the test order 

recipient. Individual data on saturation binding were not reported. Saturation binding 

experiment parameters are presented in Table 5. The Kd for [3H]-17β-estradiol was 0.1032 

nM and the Bmax (nM) was 0.07097 for the prepared rat uterine cytosol used in these 

experiments. The Kd for the run was within the expected range of 0.03 to 1.5 nM. The Bmax

was also within the expected range of 10-150 fmol/100 μg protein. Non-specific, specific 

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and total binding curves for [3H]-estradiol to the ER are shown in Figure 1. The data 

produced a linear Scatchard plot (Figure 2). 

TABLE 5. Saturation Binding Experiment of 17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosol

Parameter Run 1 Run 2 Run 3 Runs 1-3

R2 (unweighted) 0.967 NR NR NR

Bmax (nM) 0.07097 NR NR NR

Bmax (fmol/100 μg protein) 59.28 NR NR NR

Kd (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 binding experiment was conducted on the batch of cytosol used for this

competitive binding experiment.

R2 Goodness of fit for curve calculated for specific binding

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

Experiment.

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FIGURE 2. Scatchard Plot of the Binding of [3H]-17β-Estradiol to the ER.

B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive 

binding experiments 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 >95% at concentrations ≤10−4 M in all three runs. The estimated 

mean log IC50 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 IC50 for the natural ligand, 17β-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).

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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 ± SEb

r

2 (unweighted), 17β-estradiol 0.9990 0.9997 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 IC50 (M), 17β-estradiol −8.948 −8.942 −8.961 −8.951 ± 0.006

19-norethindrone −5.462 −5.435 −5.534 −5.477 ± 0.030

2,4-D NC NC NC NC

IC50 (M), 17β-estradiol 1.13 × 10−9 1.14 × 10−9 1.10 × 10−9 1.12×10−9 ± 0.01×10-9

19-norethindrone 3.45 × 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 0.032 ± 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 r2

; the mean ± SE is reported for all other parameters.

r

2 Goodness of fit

RBA (%) Relative binding affinity

NA Not applicable. r2 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).

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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 functioned

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

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TABLE 8. Criterion a Tolerance 

Limit(s) Value Yes No

17β-estradiol fitted curve parameters

Loge residual SD [Loge (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 (log10(M)−1) −1.1 to −0.7 −1.08 to −0.98 X

Weak Positive control (19-norethindrone) fitted curve parametersb

Loge 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 (log10(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]-17β-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 applicable

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Additionally, the curve for the reference material showed that increasing concentrations of 

unlabeled 17β-estradiol displaced [3H]-17β-estradiol in a manner consistent with one-site 

binding, as indicated by a 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 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%. 

Examination across the runs indicated consistency of the Hill slope, placement along the Xaxis, 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 

10−4M. 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 10−4M. 

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 reported separately in 

the study profile submitted by the test order recipient; individual data on saturation binding 

were not reported. The Kd for [3

H]-17β-estradiol was 0.1032 nM and the Bmax (nM) was 

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

was within the expected range of 0.03 to 1.5 nM, and the Bmax 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 log IC50 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 IC50 for the natural ligand, 17β-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 17β-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. 

Page 110 of 221
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 15

2,4-D/ 030001 OCSPP 890.1250/ OECD None

2,4-D was tested over a concentration range that fully defined 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. 

Page 111 of 221
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: 3/26/2012

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. 

Page 112 of 221
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 113 of 221
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: ~ ~@ 

Signatur& Date: ~ _. 

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. 

Page 114 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 2 of 8 

2,4-D/030001 OCSPP 890.1300/ 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 DMSO 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

17β-estradiol (strong estrogen; positive control)

Supplier: Sigma, St. Louis, MO

Catalogue and Batch #: Cat# E-8875, Lot# 079K0131

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: DMSO, Sigma-Aldrich, Cat # 276855

Solvent control 

(final concentration):

0.1%

Page 115 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 3 of 8 

2,4-D/030001 OCSPP 890.1300/ OECD 455

B. METHODS

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

Collection of Research Bioresources 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 bovine serum (DCC-FBS; Hyclone Laboratories, Inc., 

Logan, Utah Lot# not reported), in an incubator under 5% CO2 at 37o C. Upon reaching 75-

90% confluence, cells were subcultured at least twice prior to exposure to the test material. 

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1 -

1.5×104 cells/100 μL medium/well in a 96-well plate and allowed to attach for at least 3

hours. Growth 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 treatment 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). Chemiluminescence was measured using a Packard TopCount NXT 

luminescence counter with a detection limit of ~5 cells in 100μL medium.

a. Preliminary Test: 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 laboratory proficiency. 

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

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

in the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class

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

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

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

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

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

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

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

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

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

percentage of the PC. Graph Pad Prism v. 5.0 (GraphPad Software, Inc., La Jolla, CA) was 

used to calculate the EC50, PC10, PC50, RPCMax, and PCMax for 2,4-D when applicable. The 

test material was defined as negative for inducing estrogen receptor transcriptional 

activation if the RPCMax < PC10 in at least 2 of 2 (or 2 of 3) runs. Log EC50 and Hill slope 

Page 116 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 4 of 8 

2,4-D/030001 OCSPP 890.1300/ OECD 455

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

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

cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions

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

(bottom) and maximum (top) response 

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

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

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

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

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

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

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS

A. PRELIMINARY TEST: 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 cytotoxicity 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 Test for Solubility, Cytotoxicity, and Concentration-Selection for 2,4-Da

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

VCb 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.

Page 117 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 5 of 8 

2,4-D/030001 OCSPP 890.1300/ OECD 455

TABLE 2. Proficiency Chemicals a

Compound Expected Response Lab Response

Diethylstilbestrol Positive Not reported

17α-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., log PC50, 

log PC10, log EC50, 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 log EC50 was higher than the expected value and the Hill slope 

lower than the expected value for 17β-estradiol. The log PC50 and log PC10 for 17α-estradiol 

and 17α-methyltestosterone were lower than expected, as was the Hill slope for 17αestradiol. For 17α-methyltestosterone, the mean RPCMax 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 RPCMax 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. Performance criteria values below the validated ranges 

usually indicate increased sensitivity of the assay compared to validation experiments.

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 3 Run 4 Yes No

17β-estradiol

Log PC50 −11.4 to −10.1 −10.8 −11.1 −10.9 −10.7 X

Log PC10 <−11 −11.9 −12.6 −12.7 −12.5 X

Log EC50 −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 PC50 −9.6 to −8.1 −9.3 −10.1 −9.0 −9.0 X

Log PC10 −10.7 to −9.3 −10.6 −10.9 −10.6 −10.9 X

Log EC50 −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 PC50 −6.0 to −5.1 −7.0 −8.8 −7.5 −6.5 X

Log PC10 −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 light 

Page 118 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 6 of 8 

2,4-D/030001 OCSPP 890.1300/ 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 PC50 (50% of the 

maximum response) for E2 in this assay is 6212.5-9819.5 and the PC10 (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 4.7d 12158 NR a Data were obtained from page 47, 49 and 53 of the study report. b Positive control was 17β-estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC) d 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 concentrationresponse bar graphs depicting fold induction of relative transcriptional activation are

presented in Figure 1 below. 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 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-Da

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 EC50b NA NA NA NA

Hill Slopeb NA NA NA NA

RPCMax 0b 8.8 5.3 7.0

PCMax NA 10−4 10−4 10−5

PC50 NA NA NA NA

PC10 NA NA NA NA

a Data were obtained from page 54 of the study report. b Value of RPCMax less than or equal to 0 

NA Not Applicable

Page 119 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 7 of 8 

2,4-D/030001 OCSPP 890.1300/ 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 Criteria: 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 EC50 was higher than the expected value and the Hill slope lower than 

the expected value for 17β-estradiol. The log PC50 and log PC10 for 17α-estradiol and 17αmethyltestosterone were lower than expected, as was the Hill slope for 17α-estradiol. The 

minor deviations from the expected values do not negatively affect the interpretation of this 

study. Mean luciferase activity was 4.7 to 8.2-fold. The fold-induction corresponding to the 

PC10 of the concurrent PC was greater than 1+2 SDs 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 system (10−4 M). The highest concentration of 

2,4 D was based 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, 10−4M. 

The laboratory proficiency assays were not reported. The only deviation was a weak 

positive response in one run in response to corticosterone. There were minor deviations 

from expected performance criteria for all of the four reference chemicals, but these minor 

Page 120 of 221
Estrogen Receptor Transcriptional Activation (2012) / Page 8 of 8 

2,4-D/030001 OCSPP 890.1300/ OECD 455

deviations do not negatively 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 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.

Page 121 of 221
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'

Page 122 of 221
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.

Page 123 of 221
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 25.2o

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 every 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) between the 2,4-D treatment groups and the negative control 

for male or female gonado-somatic index (GSI) or plasma 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. 

Page 124 of 221
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-through 

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Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with 2,4-D.

Treatment 

(mg a.i./L)

[meanmeasured]

Fecundity 

Fert. 

Success 

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

M F M F 

M F M F M F M F

0.245 No No No No No No No No No No NA NA NA NA 

3.14 No No No No No No No No No No NA NA NA NA 

34.0 No No No No No No No No No No NA NA NA NA 

96.5 Yes No No No No No No No No No NA NA NA NA 

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

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

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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 μg/L. 

3. Organic carbon levels were not reported in the routine laboratory dilution water solute analysis. 

4. OCSPP guidelines recommend chlorine levels at <10 μg/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 μg/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 

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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 reported

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

promelas).

Species scientific name: Pimephales promelasSpecies strain (if stated): not reportedWere fish obtained from a single laboratory stock?

not reported Test fish were obtained from the commercial 

supplier, New England Bioassay, Manchester, CT.

EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test?

Yes water quality, temperature, and lighting were 

the 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details 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 recommends 

that fish be held another 7 days. If mortalities 

greater than 5% occur during this extended 

acclimation period, EPA recommends that the 

fish not be used.

Type of food: thawed, frozen brine 

shrimp

EPA recommends that fish be fed frozen brine 

shrimp twice per day to promote active 

reproduction and maintain body condition.

Source of food: Brine Shrimp Direct, 

Ogden, UT 

Frequency of feeding: at least 2 times/day 1.25-2.0 ml shrimp/replicate vessel

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding: Feeding start date not reported, but fish 

were fed during the holding/acclimation, pre-exposure, and exposure periods. Food 

was withheld 12 hours prior to test termination. Fish food tested free of relevant 

contaminants.

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 5.5 months 6 months at beginning of exposure period EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at test initiation (if determined): 3.7 g Mean calculated from all males selected for the 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 test initiation (if determined): 

individual data not provided range 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 g Mean calculated from all females selected for the test 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Range of individual weights (females) at test initiation (if determined): Individual data not provided range of individual weights were kept within ±20% of the meanMean length of males at test initiation (if determined): 

Not reported 

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

Not reported 

Duration of pre-exposure phase: 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: 32 The 32 test vessels included 12 test vessels 

more than the 20 that would be needed for the 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: 2 Number of females per tank: 4 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Pre-exposure fecundity: >15 eggs/female/ 

reproductive day/replicate

mean = 28.8 eggs/female/day; range from 5.6 to 53.2 eggs/female/day across all replicates

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 days The top 20 performing spawning groups 

(>15 eggs/female/reproductive day) were used in the exposure period and were assigned to concentration levels via complete randomized 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 the 

number of eggs laid and eggs found to be infertile were recorded daily 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

system. As noted in the Corrections and 

Clarifications document1 , the use of a static 

renewal system is not recommended for this 

assay.

Type of flow-through dilution system:

Continuous flow diluter A continuous-flow diluter (syringe pump 

delivery) system delivered stock solution to a mixing chamber where the solution was mixed with dilution water. The test solution was split equally to replicate test vessels. 

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-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.

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

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

(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

Diluter system was calibrated prior to exposure period. For concentrations measured in test vessels, mixing cells, and stock solutions, the percent of target concentration ranged from 108% to 121%. Within that range, the flow splitting accuracy of the concentrations for each stock solution-test vessel pairs were within 10% 

of each other. 

Homogeneity of test concentration was analyzed in one vessel at the 0.40 and 100 mg a.i./L treatment levels. Of the 4 samples taken at each treatment level, the %CV for the 0.40 and 100 mg a.i./L levels was 0.646% and 0.348%, respectively. 

Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is 

not recommended for mixing; 3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is within 

10%.

Aeration? not reported EPA recommends aeration if dissolved oxygen 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Natural water Dilution water originated from Lake Huron which 

was supplied to the Dow Chemical Company via the City of Midland Water Treatment Plant. The water from the lake was limed and flocculated with ferric chloride and then pumped to the laboratory. Before use in the lab, the water was sand-filtered, pH-adjusted with gaseous 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 for pesticides, heavy metals, and other contaminants? 

Yes Dilution water is bi-annually analyzed for 

pesticides, organics, metals and other inorganics. 

Test vessel type/materials: Glass sealed together with 

clear silicone adhesive

EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass vessels 

or glass vessels with stainless steel frames 

are acceptable examples).

Test vessel size: 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 L approximate fill volume; water depth 13 cm EPA recommends 10 L solution per tank.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Spawning 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 cm 9 cm long, 10 cm diameter Additional details on exposure system: 

Temperature was measured in one test vessel through the exposure period. Temperature, DO, and pH were measured in each test vessel at Day 0 and weekly thereafter during the assay (Table 5). Hardness, alkalinity, and conductivity were measured from a sample in the control and highest exposure level solutions at Day 0 and weekly thereafter (Table 5).

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations

Temperature (°C) 24.5 25.2 25.01 Weekly 

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

pH 7.02 7.76 7.381 Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L) 6.20 8.29 7.391 Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations

Total alkalinity (mg/L as CaCO3) 30 46 36.51 Weekly EPA recommends total alkalinity >20 mg/L 

as CaCO3.

Hardness [mg/L as CaCO3] 62 68 63.51 Weekly Total organic carbon (mg/L) <2 mg/L Once 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) 240 biannually 

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) 177 207 188.51 weekly 

1 Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels 

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D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

96-hour LC50 data for the fathead minnow are 

unavailable.

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

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Additional details: NA EPA recommends conducting a range-finder 

with five test concentrations plus a control 

(six total treatment levels), with four 

females and two males per exposure tank 

(36 fish total). The number of mortalities 

that occur may be used to develop a 

concentration-response curve. 

Based upon the results, the highest 

concentration that does not result in 

increased mortality or signs of overt 

morbidity compared to controls, or 1/3 the 

derived 96-hr LC50, may be selected as the 

highest exposure concentration in the 21-

day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Reference studies with fathead minnow

The nominal test concentrations were based on two available 2,4-D studies with the fathead minnow with reported LC50 values of 

320 mg a.i./L (Alexander et al., 1985) and 

133 mg a.i./L (Mayer and Ellersieck, 1986). From these two lethal endpoints, the experimental high concentrations could be 

estimated at 100 and 40 mg a.i./L, respectively, as specified in the 890.1350 guideline. Because of the uncertainty between the two concentrations, both were selected as high concentrations and 4 concentrations were used for the experimental 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 LC50), whichever concentration is lowest.

Reference study citation (if applicable):

Alexander et al., 1985; 

Mayer and Ellersieck, 1986 

Separation of test concentrations: 0, 0.4, 4.0, 40, 100 

mg/L 

EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable1

.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of test concentrations: 4 EPA recommends a minimum of 3 

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

Not reported 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit 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 reported Frequency 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the randomized complete block design used?

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure 

fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive 

test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls.

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (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 item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate.

Was a solvent used? No Was a positive control used? No 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Photoperiod: 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 solution 

appearance during the study did not appear to be reported. 

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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with 2,4-D.

Treatment ID

Nominal 

Concentration

(mg a.i./L) 

Mean Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only)

0.00 <0.10 NA

EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

≤20%.

Treatment 1 0.400 0.245 43.31 61.3% nominal Treatment 2 4.00 3.14 13.3 78.5% nominal Treatment 3 40.0 34.0 7.0 85.0% nominal Treatment 4 100 96.5 4.5 96.5% nominal 

Abbreviations: CV Coefficient of variation. 1 Study authors reported a dramatic decrease in measured concentrations between Days 7 and 14 due to a suspected increase in microbial populations capable of 

metabolizing 2,4-D. Though this occurred in all concentrations, the effect was proportionately greater on the lowest nominal concentration. The %CVs were >20% for each time point in this treatment group, though authors report that the variation in concentration was consistent among the replicates and thus the overall exposure was consistent among replicates. 

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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 17β-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. 

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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./L

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Mean 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 mg a.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 Weight Length 

Males Females Males Females 

n

Mean

(g) 

±SD n 

Mean

(g) 

±SD n

Mean

(mm) 

±SD n

Mean

(mm) 

±SD

Negative Control 

(<LOQ) 4 3.57 0.141 4 1.59 0.169 4 51.5 1.54 4 42.4 1.37

0.245 4 3.72 0.308 4 1.64 0.074 4 53.0 1.37 4 42.7 0.56 

3.14 4 3.69 0.144 4 1.62 0.173 4 52.2 0.89 4 42.1 0.92 

34.0 4 3.93 0.488 4 1.62 0.173 4 54.5 2.20 4 42.4 1.47 

96.5 4 3.73 0.408 4 1.62 0.082 4 53.8 2.45 4 42.5 0.78 

SD Standard deviation. 

n = number of independent replicates per treatment. LOQ=0.10 mg a.i./L 

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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]

Fecundity1 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.

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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 Females1

n Median Tubercle 

Score2 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. 

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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 GSI1

(%)

±SD n Mean GSI1

(%)

±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. 

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Table 14a: Gonadal Staging in Fathead Minnow.

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

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 

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Table 15: Gonadal Histopathology in Male Fathead Minnow.

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

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 

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1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow. 

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations2

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n1 Incidence n1 Incidence n1 Incidence n1 Incidence n1 Incidence 

Negative Control 

(<LOQ) 

0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

0.245 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations2

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n1 Incidence n1 Incidence n1 Incidence n1 Incidence n1 Incidence 

3.14 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

34.0 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations2

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n1 Incidence n1 Incidence n1 Incidence n1 Incidence n1 Incidence 

96.5 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

Abbreviations: NA Not applicable.

LOQ=0.10 mg a.i./L 

1 Number of individual fish observed 2 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.

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Table 17: Gonadal Histopathology in Female Fathead Minnow. 

Treatment 

(mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

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 

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1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow.

Treatment 

(mg a.i./L)

[meanmeasured]

Additional Diagnostic Observations1

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 

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Treatment 

(mg a.i./L)

[meanmeasured]

Additional Diagnostic Observations1

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 

 

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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 19.8×106

 ng/mL (3.14 mg a.i./L) to 55.1×106 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 27.6×106 21.5×106

0.245 4 1.77 1.25 4 55.1×106 47.5×106

3.14 4 0.930 0.777 4 19.8×106 7.69×106

34.0 4 0.909 0.591 4 21.0×106 14.4×106

96.5 4 1.29 0.983 4 31.6×106 12.7×106

SD Standard deviation. 

n = number of independent replicates per treatment. 

LOQ=0.10 mg a.i./L 

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Plasma testosterone and plasma 17

β-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 17

β-estradiol (E2) 

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD 

Negative Control 

(<LOQ) 

NA ND ND NA ND ND NA ND ND NA ND ND

0.245 NA ND ND NA ND ND NA ND ND NA ND ND 

3.14 NA ND ND NA ND ND NA ND ND NA ND ND 

34.0 NA ND ND NA ND ND NA ND ND NA ND ND 

96.5 NA ND ND NA ND ND NA ND ND NA ND ND 

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

LOQ=0.10 mg a.i./L 

No abnormal behavior was observed among control or treatment fish, such as hyperventilation, loss of equilibrium, uncoordinated swimming, atypical quiescence, 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 exposure 

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period which could have been from breeding activity and/or colliding with breeding substrate. Another female fish in the 3.14 mg a.i./L treatment group 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./L 

treatment group and one fish in the control group. Vertical banding was observed in both control and treatment group female test fish, but no treatment-related effects were observed. The raw data for this banding was not reported. 

Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow at Test Termination. 

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Negative Control (<LOQ) None NA NA None NA NA 

0.245 None NA NA None NA NA 

3.14 None NA NA None NA NA 

34.0 None NA NA None NA NA 

96.5 None NA NA None NA NA

Abbreviations: NA Not applicable.

LOQ=0.10 mg a.i./L

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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. 

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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 (R2

) 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 R2

 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 

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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. 

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Table 22: Reproductive and HPG Endpoints1,2,3 for Male Fathead Minnow in the FSTRA with 2,4-D.

Treatment 

(mg a.i./L) 

[mean-measured] 

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative Control 

(<LOQ) 

31 NA NA NA NA NA NA NA NA NA NA

0.245 31 0.995 2.29 0.998 No 32.1 0.954 NA NA NA NA 

3.14 35 0.925 -1.25 1.000 No -30.3 0.962 NA NA NA NA 

34.0 33 0.978 -1.25 1.000 No -32.1 0.954 NA NA NA NA 

96.5 34 0.748 8.32 0.817 No -3.6 1.000 NA NA NA NA 

Statistical Test Dunnett’s4 Dunnett’s NA Dunnett’s NA NA 

NA Not applicable 

LOQ=0.10 mg a.i./L 

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 For percent (%) difference, positive values indicate an increase relative to the negative control, and negative values indicate a decrease relative to the negative control. 4 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. 

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Table 23: Reproductive and HPG Endpoints1,2,3 for Female Fathead Minnow in the FSTRA with 2,4-D.

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2

% 

Diff. p

% 

Diff. p Median p

% 

Diff. 

p 

Effect? 

(Yes/No) 

% 

Diff. p

% 

Diff. p % Diff. p

Negative Control 

(<LOQ) 

NA NA NA NA 0 NA NA NA NA NA NA NA NA NA NA

0.245 -2.6 0.332 -0.64 0.975 0 NA 8.21 0.945 No 99.6 0.494 NA NA NA NA 

3.14 -13 0.210 -0.83 0.943 0 NA -6.11 0.980 No -28.4 0.889 NA NA NA NA 

34.0 -23 0.088 0.77 0.954 0 NA -1.13 1.00 No -23.5 0.678 NA NA NA NA 

96.5 -34 0.031 -1.16 0.839 0 NA 11.5 0.847 No 14.6 0.494 NA NA NA NA 

Statistical Test 

JonckheereTerpstra Dunnett’s NA Dunnett’s NA Mann-Whitney NA NA 

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

LOQ=0.10 mg a.i./L

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 For percent (%) difference, positive values indicate an increase relative to the negative control, and negative values indicate a decrease relative to the negative control. 

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Table 24: Growth Endpoints1,2,3 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. 

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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 μg/L. Additionally, OCSPP guidelines 

recommend chlorine levels <10 μg/L but chlorine in the dilution water was only quantified at a level <200 

μg/L (chlorine was not detectable at 200 μg/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. 

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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. 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION: 

Endpoint Monotonic? Parametric? 890.1350 EFED Comments Survival Overall: No NA

Fisher’s Exact Test n.s p>0.05

Fisher’s Exact Test n.s p>0.05 No significant effect on overall survival

Female body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female VTG Yes Yes Mann Whitney; 

n.s. p>0.05 

Mann Whitney; n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male VTG Yes No Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female GSI Yes Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male GSI No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female tubercle score 

NA NA NA NA No score 

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Endpoint Monotonic? Parametric? 890.1350 EFED Comments Male tubercle score Yes Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Fecundity No Yes Jonckheere-Terpstra: 

dose 3 p=0.011 

Williams; dose 3 p=0.031 

890.1350 and EFED same conclusions, effect at dose 3 

Fertility Yes No Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

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

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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 59.8x106 26.6x106

0.245 4 74.8x106 31.0x106

3.14 4 55.7x106 18.8x106

34.0 4 58.7x106 33.7x106

96.5 4 92.5x106 27.6x106

SD Standard deviation. 

n = number of independent replicates per treatment 

 

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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 

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

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 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. 

 

 

 

 

 

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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 

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 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 

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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 

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 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 

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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 

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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 

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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 

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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 

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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 

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EPA MRID Number 48317001 

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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 

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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 

Page 187 of 221
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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 

Page 188 of 221
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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 

Page 189 of 221
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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 . . 

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

Page 190 of 221
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 

Page 191 of 221
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 . . 

Page 192 of 221
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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 

Page 193 of 221
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 . . . . . . . . 

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 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 

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

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PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 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 . . . . . 

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 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 

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EPA MRID Number 48317001 

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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 . . . . . . 

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 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 

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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 

Page 200 of 221
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EPA MRID Number 48317001 

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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 

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EPA MRID Number 48317001 

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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 

Page 202 of 221
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EPA MRID Number 48317001 

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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 . . . . . 

Page 203 of 221
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EPA MRID Number 48317001 

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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 . . . . . , . 

Page 204 of 221
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EPA MRID Number 48317001 

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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 

Page 205 of 221
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 . . . 

Page 206 of 221
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 

Page 207 of 221
DATA EVALUATION RECORD

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

Study Type: OCSPP 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: 3/14/2012

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. 

Page 208 of 221
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 209 of 221
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 

of 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 three 

Page 210 of 221
Steroidogenesis Assay (2011) / Page 2 of 12

2,4-D / 030001 OCSPP 890.1550/ OECD None

runs of 1 μM prochloraz 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. At the highest concentration tested (10−4

M), 2,4-D increased (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.

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis Assay 

(OCSPP 890.1550). 

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

Confidentiality statements were provided. 

Page 211 of 221
Steroidogenesis 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 Substance: 2,4-D

Description: Off-white powder

Lot # (expiration date): 2006 2433 8006-USA (not provided)

Purity: 98.5%

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 Control (Inducer): Forskolin

Description (molecular weight): White powder (410.50)

Source: Sigma-Aldrich (St. Louis, MO)

Lot # (expiration date): 097K50653 (not provided)

Purity: 99%

Solubility (in solvent): Soluble in DMSO up to 0.01 M

Storage conditions: Ambient

CAS #: 66575-29-9

4. Positive Control (Inhibitor): Prochloraz

Description (molecular weight): Off-white powder (376.67)

Source: Sigma-Aldrich (St. Louis, MO)

Lot # (expiration date): SZE6220X (not provided)

Purity: 99.1%

Solubility (in solvent): Soluble in DMSO up to 0.01 M

Storage conditions: Ambient

CAS #: 67747-09-5

5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO)

Description (molecular weight): Clear liquid (78.13)

Source: Sigma-Aldrich (St. Louis, MO)

Lot # (expiration date): 16496CPV and 68996LMV (not 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/v

Page 212 of 221
Steroidogenesis 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: Component of stock medium

Nu-Serum: 2.5%, BD Biosciences; Lot # 81515; tested 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 μg/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% CO2 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-Test 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, standards 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 derivatized 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 were dissolved in DMSO and diluted 

1:1000 in the final treatment medium. When added to the cell culture plates, these dilutions 

yielded final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μM 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 final 

concentration of DMSO in the medium was held constant at 0.1% (v/v). 

Page 213 of 221
Steroidogenesis Assay (2011) / Page 5 of 12

2,4-D / 030001 OCSPP 890.1550/ OECD None

3. Cell Plating and Preincubation: Cells were maintained in the stock medium described 

above. The hormone concentrations in undiluted Nu-Serum were 974 pg/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% CO2) 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 Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

1 2 3 4 5 6

A Blank b Blank Blank Blank + methanolc 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, two aliquots of medium were 

collected and frozen at −80°C until further processing. 

5. Cell Viability/Cytotoxicity Assay: 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 confluence, homogeneity from well-to-well, and any signs of cytotoxicity or 

Page 214 of 221
Steroidogenesis 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 Aqueous 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 Aqueous One Solution contains a tetrazolium compound [3-(4,5-

dimethylthiazol-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 proportional 

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 Measurement System: 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, derivatized with dansyl chloride, and analyzed by 

LC/APPI-MS/MS. The lower limit of quantification (LLQ) for this measurement system 

was 25 pg/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. 

Page 215 of 221
Steroidogenesis 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. Cytotoxicity was not observed at any tested 2,4-D concentration. The 

variability (CV) between 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 effect 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 μM, 2,4-D increased (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 for 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 pg/mL for 

testosterone and 10 pg/mL for estradiol.

Page 216 of 221
Steroidogenesis Assay (2011) / Page 8 of 12

2,4-D / 030001 OCSPP 890.1550/ OECD None

FIGURE 1. Change in Testosterone Production Relative to 2,4-D Concentration.

Page 217 of 221
Steroidogenesis 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. 

Page 218 of 221
Steroidogenesis 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). Viability 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 Concentration

(μM)

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) based 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%). 

Page 219 of 221
Steroidogenesis 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 pg/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-fold 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 increased (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 alteration in estradiol production. 

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Steroidogenesis 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 Guideline, 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. 

Page 221 of 221