2,4-D Human Health Risk Assessment DP No. D389455 

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: August 8, 2013 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: 2,4-D. Human Health Risk Assessment for a Proposed Use of2,4-D Choline on 

Herbicide-Tolerant Com and Soybean 

PC Code: 03000 I, 05 1505 

Decision No.: 447951 . 442244 

Petition No.: NA 

Risk Assessment Type: Single Chemical 

TXRNo.: NA 

MRIDNo.: NA 

DP Barcode: 0389455 

Registration No.: 62719-AUU, 62719-AGO 

Regulatory Action: Section 3 

CaseNo.: NA 

CAS No.: 94-75-7 

40 CFR: 40 CFR 180.142 

FROM: Alexandra LaMay, Biologist t:/1 -/ # · -

Linda 

Danette 

Taylor, 

Drew, 

Toxicologist 

Chemist ~ 

(;7~~4 r-et'-d). / L 

Risk 

Health 

Assessment 

Effects Division 

Branch 

(7509P) 

V NIT ~ 

THROUGH: ldeliz Negron Encarnacion, Chern).sJ c=. 

Michael Metzger, Branch Chief fV(t 

Risk Assessment Branch V Nil 

Health Effects Division (7509P) 

TO: Michael Walsh 

Registration Division (7505P) 

Page 1 of96 
2,4-D Human Health Risk Assessment DP No. D389455 

1.0 Executive Summary ........................................................................................................... 4 

2.0 HED Recommendations .................................................................................................... 7 

2.1 Data Deficiencies ............................................................................................................ 7 

2.2 Tolerance Considerations .............................................................................................. 7 

2.2.1 Enforcement Analytical Method ............................................................................... 7 

2.2.2 Recommended Tolerances ......................................................................................... 7 

2.2.3 Revisions to Petitioned-For Tolerances .................................................................... 8 

2.2.4 International Harmonization ..................................................................................... 8 

2.3 Label Recommendations ................................................................................................ 8 

2.3.1 Recommendations from Residue Reviews ................................................................ 8 

2.3.2 Recommendations from Occupational Assessment ................................................. 8 

2.3.3 Recommendations from Residential Assessment ..................................................... 9 

3.0 Introduction ........................................................................................................................ 9 

3.1 Chemical Identity ........................................................................................................... 9 

3.2 PhysicaVChemical Characteristics ............................................................................. 10 

3.3 Pesticide Use Pattern .................................................................................................... 10 

3.4 Anticipated Exposure Pathways ................................................................................. 11 

3.5 Consideration of Environmental Justice .................................................................... 11 

4.0 Hazard Characterization and Dose-Response Assessment .......................................... 12 

4.1 Toxicology Studies Available for Analysis ................................................................. 12 

4.2 Absorption, Distribution, Metabolism, & Elimination (ADME) ............................. 13 

4.2.1 Dermal Absorption ................................................................................................... 13 

4.3 Toxicological Effects .................................................................................................... 13 

4.4 Safety Factor for Infants and Children (FQPA Safety Factor) ............................... 14 

4.4.1 Completeness of the Toxicology Database .............................................................. 14 

4.4.2 Evidence of Neurotoxicity ........................................................................................ 15 

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal.. ...... 15 

4.4.4 Residual Uncertainty in the Exposure Database ................................................... 15 

4.5 Toxicity Endpoint and Point of Departure Selections .............................................. 15 

4.5.1 Dose-Response Assessment ...................................................................................... 16 

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment ..... 17 

4.5.3 Cancer Classification and Risk Assessment Recommendation ............................ 17 

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk 

Assessment ............................................................................................................................... 18 

5.0 Dietary Exposure and Risk Assessment ......................................................................... 20 

5.1 

5.1.1 

5.1.2 

5.1.3 

5.1.4 

5.2 

5.3 

5.4 

5.4.1 

5.4.2 

5.4.3 

Metabolite/Degradate Residue Profile ....................................................................... 20 

Summary of Plant and Animal Metabolism Studies ............................................. 20 

Summary of Environmental Degradation .............................................................. 20 

Comparison of Metabolic Pathways ....................................................................... 21 

Residues of Concern Summary and Rationale ...................................................... 21 

Food Residue Profile .................................................................................................... 22 

Water Residue Profile .................................................................................................. 23 

Dietary Risk Assessment .............................................................................................. 24 

Description of Residue Data Used in Dietary Assessment .................................... 24 

Percent Crop Treated Used in Dietary Assessment .............................................. 25 

Acute Dietary Risk Assessment ............................................................................... 25 

Page 2 of 96 
2,4-D Human Health Risk Assessment DP No. D389455

Page 3 of 96

5.4.4 Chronic Dietary Risk Assessment........................................................................... 25

5.4.5 Cancer Dietary Risk Assessment............................................................................. 26

5.4.6 Summary Table......................................................................................................... 26

6.0 Residential (Non-Occupational) Exposure/Risk Characterization ............................. 26

6.1 Residential Handler Exposure .................................................................................... 27

6.2 Post-Application Exposure .......................................................................................... 28

6.4 Residential Risk Estimates for Use in Aggregate Assessment.................................. 32

6.5 Residential Bystander Post-application Inhalation Exposure (Volatilization)....... 34

6.6 Spray Drift.................................................................................................................... 35

7.0 Aggregate Exposure/Risk Characterization.................................................................. 36

7.1 Acute Aggregate Risk................................................................................................... 36

7.2 Short-Term Aggregate Risk ........................................................................................ 36

7.3 Intermediate-Term Aggregate Risk ........................................................................... 36

7.4 Chronic Aggregate Risk .............................................................................................. 37

7.5 Cancer Aggregate Risk ................................................................................................ 37

8.0 Cumulative Exposure/Risk Characterization ............................................................... 37

9.0 Occupational Exposure/Risk Characterization ............................................................ 37

9.1 Short-/Intermediate-Term Handler Risk................................................................... 37

9.2.1 Dermal Post-application Risk.................................................................................. 40

9.2.2 Inhalation Post-application Risk............................................................................. 40

10.0 References......................................................................................................................... 40

Appendix A. Toxicology Profile and Executive Summaries .................................................. 42

A.1 Toxicology Data Requirements................................................................................... 42

A.2 Toxicity Profiles............................................................................................................ 43

A.3 Hazard Identification and Endpoint Selection .......................................................... 50

A.4. HEC/HED Calculations................................................................................................. 54

A.5 Executive Summaries................................................................................................... 56

Appendix B. Metabolism Summary Table ............................................................................... 88

Appendix C. Physical/Chemical Properties............................................................................. 91

Appendix D. Review of Human Research................................................................................ 92

Appendix E. Occupational Exposure/Risk Summary Tables................................................ 93

Appendix F. Residue Summary Table ..................................................................................... 94

Appendix G. International Residue Limits .............................................................................. 96
2,4-D Human Health Risk Assessment DP No. D389455

Page 4 of 96

1.0 Executive Summary

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. It is a longstanding active ingredient (ai) registered for a variety of 

food/feed uses. Permanent tolerances for 2, 4-D are established under 40 CFR 180.142 for a 

wide variety of crops and livestock commodities.

Proposed Use: The proposed use is for 2,4-D choline use on herbicide-tolerant corn and 

soybean. The soybean contains the inserted aryloxyalkanoate dioxygenase-12 (AAD-12) gene

and is identified as containing DAS-68416-4. The corn contains the inserted aryloxyalkanoate 

dioxygenase-1 (AAD-1) gene and is identified as containing DAS-40278-9. Expression of the 

proteins encoded by the genes results in a trait that increases the herbicide tolerance of corn and 

soybean to 2,4-D via increased metabolism through a pathway involving the metabolite 2,4-

dichlorophenol (2,4-DCP). 

For corn, the proposed use is for pre-plant, pre-emergence, or post-emergence (when corn is no 

later than the V8 growth stage or 48 inches tall) application to herbicide-tolerant field corn at a 

maximum single application rate of 1.0 lb acid equivalent (ae)/acre (A). For soybean, the 

proposed use is for pre-plant, pre-emergence, or post-emergence (when soybean is no later than 

the R2, or full flowering, growth stage) application to soybean at a maximum single application 

rate of 1.0 lb ae/A. For post-emergence applications for both crops, a maximum of two 

applications are permitted with a minimum of 12 days between applications. The maximum 

seasonal application rate is 3 lbs ae/A/season for both crops. The directions indicate no more 

than one pre-emergence application and no more than two post-emergence applications per use 

season. Applications are to be made using groundboom equipment. Aerial application and 

chemigation are prohibited. The proposed preharvest interval (PHI) for corn and soybeans is 30 

days. 

Hazard Characterization: The toxicology database is adequate to assess this proposed use. The 

toxicity profile of 2,4-D shows that the principal toxic effects are changes in the kidney, thyroid, 

liver, adrenal, eye, and ovaries/testes in the rat following exposure to 2,4-D via the oral route at 

dose levels above the threshold of saturation of renal clearance. No systemic toxicity was 

observed in rabbits following repeated exposure via the dermal route at dose levels up to the 

limit dose. Neurotoxicity was observed in the acute neurotoxicity study in rats at the high dose. 

In an extended 1-generation reproductive toxicity study in rats, reproductive toxicity, 

developmental neurotoxicity, and immunotoxicity were not observed, and the thyroid effects 

observed at dose levels up to/approaching renal saturation were considered treatment-related, 

although not adverse. Maternal and developmental toxicity were observed at high dose levels 

exceeding the threshold of saturation of renal clearance. There are no residual uncertainties for 

pre- and/or postnatal toxicity. HED recommends that the 10X FQPA Safety Factor (for the 

protection of infants and children) be reduced to 1X. 2,4-D is not acutely (lethal) toxic via the 

oral, dermal, and inhalation routes, is not a dermal irritant or a dermal sensitizer, but it is a severe 

eye irritant. 2,4-D has been classified as a Category D chemical, i.e., not classifiable as to 

human carcinogenicity.

Dietary Exposure and Risk Assessment: Acute and chronic aggregate (food + dietary drinking
2,4-D Human Health Risk Assessment DP No. D389455

Page 5 of 96

water) exposure and risk assessments were conducted using the Dietary Exposure Evaluation 

Model software with the Food Commodity Intake Database (DEEM-FCID) Version 3.16. This 

software uses 2003-2008 food consumption data from the U.S. Department of Agriculture’s 

(USDA’s) National Health and Nutrition Examination Survey, What We Eat in America, 

(NHANES/WWEIA). 

The estimated drinking water concentrations (EDWCs) were estimated using the Pesticide Root 

Zone Model/Exposure Analysis Modeling System (PRZM/EXAMS) models for surface water 

and Screening Concentration In GROund Water model (SCI-GROW) for ground water. For 

ground water, the maximum concentration detected from monitoring data was 15 μg/L. For 

surface water, the 1-in-10 year acute (peak) and annual average concentrations are 136 and 23

μg/L, respectively. EDWCs of 136 ppb and 23 were used in the acute and chronic analyses, 

respectively. 

The acute dietary exposure assessment assumed tolerance-level residues except soybeans, 100% 

crop treated (CT) for all commodities, empirical and default processing factors as appropriate, 

and incorporated the drinking water estimates described above. The resulting acute food plus 

drinking water risk estimates are not of concern to HED (≤100% of the acute population adjusted 

dose, aPAD) at the 95th percentile of the exposure distribution for the general population and all 

population subgroups. The resulting acute risk estimate for children 1 to 2 years old (the 

subgroup with the greatest exposure) was 14% of the aPAD at the 95th percentile of the exposure. 

The chronic dietary exposure assessment assumed tolerance-level exposure values for all 

commodities except soybean, 100 %CT information for all commodities, empirical and default 

processing factors as appropriate, and incorporated drinking water estimates as described above. 

The resulting chronic risk estimates are not of concern to HED for the general population and all 

population subgroups. The most highly exposed population was children 1 to 2 years old, 

utilizing 15% of the chronic PAD (cPAD). 

Residential (Non-Occupational) Exposure and Risk Assessment: There are registered uses of 

2,4-D on ornamental turf including golf courses and parks as well as aquatic uses; therefore, 

residential handler exposure and post-application exposure to treated turf and aquatic sites is 

possible. As the Residential Standard Operating Procedures (SOPs) were updated in 2012, these 

scenarios have been re-assessed. There is no potential hazard via the dermal route for 2,4-D, 

therefore the handler assessment included only the inhalation route of exposure and the postapplication assessment included only the inhalation and incidental oral route of exposure. The 

residential handler and post-application risk estimates are not of concern for 2,4-D for all 

scenarios and all routes of exposure. 

An increase in use of 2,4-D is anticipated in the near future due to several factors (e.g., resistance 

traits in certain crops and weed resistance management). Exposure to drift and volatilization, 

and the appropriate available data, were considered in this assessment due to the anticipated 

market expansion. Concerning spray drift, the residential postapplication exposure assessment 

for registered use as direct application to turf is protective of potential deposition on turf from 

spray drift for the proposed use of 2,4-D choline on herbicide-tolerant corn and soybean. The 

potential exposure to vapor phase 2,4-D residues emitted from treated fields for the proposed 
2,4-D Human Health Risk Assessment DP No. D389455

Page 6 of 96

uses of 2,4-D choline has been evaluated in this assessment. Volatilization modeling was 

completed using Probabilistic Exposure and Risk model for FUMigants (PERFUM) as well as 

chemical- and formulation-specific flux data. The results indicate that volatilization of 2,4-D

from treated crops does occur and could result in bystander exposure to vapor phase 2,4-D. 

Results of PERFUM modeling, however, indicate that airborne concentrations, even at the edge 

of the treated fields, are not of concern. 

Aggregate Risk Estimates: The acute aggregate risk assessment includes only food and water 

exposure. The resulting acute food plus drinking water risk estimates are not of concern to HED 

(≤100% aPAD) at the 95th percentile of the exposure distribution for the general population and

all population subgroups.

The short-term aggregate risk assessment includes food, water, and residential exposure. The 

resulting short-term aggregate risks are not of concern to HED (MOEs > LOC of 100) for adults 

and children.

There are no intermediate-term residential exposure to 2,4-D; therefore the intermediate-term 

aggregate risk assessment includes only food and drinking water exposure. Furthermore, the 

chronic aggregate risk assessment includes only food and water exposure. The chronic food plus

drinking water risk estimates are not of concern to HED for the general population and all 

populations subgroups.

Occupational Exposure and Risk Assessment: Occupational handlers may apply 2,4-D choline 

with groundboom equipment. There is no potential hazard via the dermal route for 2,4-D, 

therefore the occupational handler assessment included only the inhalation route of exposure. 

Occupational handler inhalation risk estimates are not of concern (i.e., MOEs > LOC of 300) for 

all scenarios for use of 2,4-D choline on herbicide-tolerant corn and soybean. At baseline 

personal protective equipment (PPE) (i.e., no respirator), the occupational handler inhalation 

MOE is 4,900 for mixer/loaders and 3,200 for applicators using groundboom equipment. 

There is no potential hazard via the dermal route for 2,4-D; therefore, a quantitative occupational 

post-application dermal risk assessment was not completed. Furthermore, a quantitative postapplication inhalation risk assessment was not performed for workers at this time; although the 

assessment was not performed, other exposure scenarios are expected to be protective of 

potential worker post-application inhalation exposure. The minimum Worker Protection 

Standard (WPS) restricted entry interval (REI) of 48 hours is adequate to protect agricultural 

workers from post-application exposures to 2,4-D. 

Environmental Justice Considerations: Potential areas of environmental justice concerns, to 

the extent possible, were considered in this human-health risk assessment, in accordance with 

U.S. Executive Order 12898, “Federal Actions to Address Environmental Justice in Minority 

Populations and Low-Income Populations.

1

” 

Review of Human Research: This risk assessment relies in part on data from studies in which 

adult human subjects were intentionally exposed to a pesticide to determine their inhalation 

 

1

http://www.hss.energy.gov/nuclearsafety/env/guidance/justice/eo12898.pdf.
2,4-D Human Health Risk Assessment DP No. D389455

Page 7 of 96

exposure. Appendix D provides additional information on the review of human research used to 

complete the 2,4-D risk assessment. There is no regulatory barrier to continued reliance on these 

studies, and all applicable requirements of EPA’s Rule for the Protection of Human Subjects of 

Research (40 CFR Part 26) have been satisfied.

2.0 HED Recommendations

HED recommends for a registration for the use of 2,4-D choline on herbicide-tolerant corn and 

soybean. Additional data are needed as outlined in section 2.1 below. The specific tolerance 

recommendations are discussed in 2.2, and label modifications are discussed in section 2.3.

2.1 Data Deficiencies

None.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

Adequate analytical methods are available for data collection and the enforcement of plant 

commodity tolerances. Task Force II submitted an adequate GC/ECD enforcement method for 

plants (designated as EN-CAS Method No. ENC-2/93) which has been independently validated 

and radiovalidated. An enforcement method was submitted for determination of 2,4-D in 

livestock commodities, which has been adequately radiovalidated. The Agency concluded that 

the methods are adequate; all data requirements have been satisfied. The 10/97 edition of FDA 

PAM Volume I, Appendix I indicates that 2,4-D is partially recovered (50-80%) using 

Multiresidue Methods Section 402 E1 and 402 E2.

2.2.2 Recommended Tolerances

Tolerances for 2,4-D residues are listed in 40 CFR §180.142 with the following definition:

“Tolerances are established for residues of the herbicide, plant regulator, and fungicide 2,4-D, 

including its metabolites and degradates, in or on the commodities in the table below. 

Compliance with the tolerance levels is to be determined by measuring residues of 2,4-D (2,4-

dichlorophenoxyacetic acid), both free and conjugated, determined as the acid”.

No new tolerances are required to support the requested use of 2,4-D choline on the herbicide 

resistant AAD-1 corn and AAD-12 soybean. However, revisions to current tolerances are 

required for international harmonization (see discussion below). The existing livestock 

commodity tolerances are adequate to cover the use on feedstuffs of AAD-1 field corn. A 

livestock feeding/grazing restriction for the soybean use is proposed; therefore, the current 

tolerances for residues of 2,4-D on soybean feedstuffs are not being reevaluated at this time.

The currently established tolerances for residues of 2,4-D on corn and soybean commodities are 

listed in the table below. 

Table 2.2.3. Tolerance Summary for 2,4-D
2,4-D Human Health Risk Assessment DP No. D389455

Page 8 of 96

Commodity Established/Proposed 

Tolerance (ppm)

Recommended

Tolerance (ppm)

Comments

Correct Commodity Definition

Corn, field, forage 6.0 10

Corn, field, grain 0.05 0.05

Corn, field, stover 50 50

Soybean, seed 0.02 0.01

2.2.3 Revisions to Petitioned-For Tolerances

The petitioner proposed that existing tolerances on corn and soybean commodities are adequate

to cover the use of 2,4-D choline on herbicide-tolerant corn and soybean. HED has determined 

that the existing tolerances for residues of 2,4-D on corn and soybean commodities are adequate 

to cover the proposed use of 2,4-D choline on AAD-1 corn and AAD-12 soybean. However, 

revisions to current tolerances are required for international harmonization. The proposed 

tolerances are different from those recommended by HED for corn forage and soybean seed. In 

order to harmonize with the Codex MRLs for corn forage and soybean seed (see discussion 

below), the tolerance for corn forage (6.0 ppm) will be increased to 10 ppm and the tolerance for 

soybean seed (0.02 ppm) will be decreased to 0.01 ppm. 

2.2.4 International Harmonization

A maximum residue limits (MRL) has been established by Canada for field corn grain at 0.05 

ppm. Codex has established MRLs for maize forage (10 ppm), maize (0.05 ppm), maize fodder 

(40 ppm), and soybean seed (0.01 ppm). The U.S. tolerances for field corn grain are harmonized 

with the corresponding MRLs. The Codex MRL for field corn forage is higher than the U.S. 

tolerance; HED recommends for the tolerance for field corn forage to be increased to 10 ppm to 

allow harmonization with Codex. The U.S. tolerance for field corn stover and soybean seed are 

higher than the Codex MRLs for the corresponding commodities. As the maximum 2,4-D 

residue value was <0.01 ppm in/on all soybean seed samples in the field trials, HED 

recommends for the tolerance for soybean seed to be decreased to 0.01 ppm to allow 

harmonization with Codex.

2.3 Label Recommendations

2.3.1 Recommendations from Residue Reviews

860.1200 Directions for Use

A revised label should be submitted which specifies a minimum preharvest interval of 50 

days for soybean. The proposed label currently specifies a 30 day PHI which does not 

reflect the minimum PHI (51 days) used for soybean seed in the crop field trials.

2.3.2 Recommendations from Occupational Assessment

None.
2,4-D Human Health Risk Assessment DP No. D389455

Page 9 of 96

2.3.3 Recommendations from Residential Assessment

None.

3.0 Introduction

3.1 Chemical Identity

Table 3.1 Test Compound Nomenclature.

2,4-Dichlorophenoxyacetic acid

(2,4-D)

Common name 2,4-D acid

IUPAC name (2,4-dichlorophenoxy)acetic acid

CAS name 2-(2,4-dichlorophenoxy)acetic acid

CAS # 94-75-7

PC Code 030001

End-use product/(EP) Not applicable

2,4-D dimethylamine salt (DMA)

IUPAC name (2,4-dichlorophenoxy)acetic acid - dimethylamine (1:1)

CAS name 2-(2,4-dichlorophenoxy)acetic acid compound with N-methylethanamine (1:1)

CAS # 2008-39-1

PC Code 030019

End-use product/(EP) 3.79 lb ae/gal SL/L formulation (Weedar®64 Herbicide)

2,4-D choline

IUPAC name 2-hydroxy-N,N,N-trimethylethanaminium (2,4-dichlorophenoxy)acetate

CAS name Ethanaminium, 2-hydroxy-N,N,N-trimethyl-, 2-(2,4-dichlorophenoxy)acetic acid 

hydroxide (1:1:1)

CAS # 1048373-72-3

PC Code 051505
2,4-D Human Health Risk Assessment DP No. D389455

Page 10 of 96

Table 3.1 Test Compound Nomenclature.

2,4-dichlorophenol (2,4-DCP)

2,4-dichlorophenol

3.2 Physical/Chemical Characteristics

The vapor pressure of 2,4-D is 1.4 x 10-7 mm Hg at 25 °C. The pKa of 2,4-D is 2.73; this 

indicates that 2,4-D will exist primarily in anion form in the environment; therefore, significant 

volatilization from moist soil and water surfaces is not anticipated because anions are not 

expected to volatilize2.

2,4-D is non-persistent (t1/2=6.2 days) in terrestrial (aerobic) environments, moderately persistent 

(t1/2=45 days) in aerobic aquatic environments, and highly persistent (t1/2= 231 days) in anaerobic 

terrestrial and aquatic environments. Because 2,4-D will be anionic under most environmental 

conditions, it is expected to be mobile (Koc=61.7) in soil and aquatic environments. 

Toxicokinetic studies conducted in pregnant rats show that 2,4-D is transferred through maternal 

milk to the pups.

3.3 Pesticide Use Pattern

The end-use product relevant for this petition request for use on corn and soybean is GF-2654 

TC, an end use product containing 56.3% 2,4-D choline salt (38.4% of 2,4-D, or 3.8 lb ae/gal of 

2,4-D). 

For corn, the proposed use is for pre-plant, pre-emergence, or post-emergence (when corn is no 

later than the V8 growth stage or 48 inches tall) application to herbicide-tolerant field corn at a 

maximum single application rate of 1.0 lb acid equivalent (ae)/acre (A). For soybean, the 

proposed use is for pre-plant, pre-emergence, or post-emergence (when soybean is no later than 

the R2, or full flowering, growth stage) application to soybean at a maximum single application 

rate of 1.0 lb ae/A. For post-emergence applications for both crops, a maximum of two 

applications are permitted with a minimum of 12 days between applications. The maximum 

seasonal application rate is 3 lbs ae/A/season for both crops. The directions indicate no more 

than one pre-emergence application and no more than two post-emergence applications per use 

season. Applications are to be made using groundboom equipment. Aerial application and 

chemigation are prohibited. The proposed preharvest interval (PHI) for corn and soybeans is 30 

days. The 2,4-D product labels direct mixers, loaders, applicators and other handlers to wear 

long-sleeved shirt and long pants, shoes plus socks, chemical-resistant gloves, and protective 

 

2

http://webwiser.nlm.nih.gov/getSubstanceData.do;jsessionid=0F808AD57ADA839F338C6442E7801A53?substanc

eID=108&displaySubstanceName=Acetic%20Acid%2024-Dichlorophenoxy-

&UNNAID=&STCCID=&selectedDataMenuItemID=81
2,4-D Human Health Risk Assessment DP No. D389455

Page 11 of 96

eyewear; a chemical-resistant apron is required when mixing or loading, cleaning up spills or 

equipment, or otherwise exposed to the concentrate.

Refer to section 2.3 for recommended modifications to the proposed label.

Table 3.3. Summary of Directions for Use of 2,4-D

Applic. Timing, 

Type, and 

Equip.

Formulation 

[EPA Reg. 

No.]

Applic. 

Rate (lb 

ae/A)

Max. No. 

Applic. per 

Season

Max. 

Seasonal 

Applic. Rate 

(lb ai/A)

PHI 

(days) Use Directions and Limitations

Herbicide-Tolerant Soybeans

Groundboom 

equipment only

Preplant, 

Preemergence, 

and 

Postemergence 

Application

3.8 lb 2,4-

D/gal

 (GF-2654 

TC)

1 lb 

ae/acre 3

3 lb ae/acre 

per use 

season

30

Do not apply this product through 

any type of irrigation system. Do 

not aerially apply this product. Do 

not apply more than one 

preemergence application and no 

more than two postemergence 

applications per use season. Do not 

apply to soybeans later than the R2 

stage. 

Herbicide-Tolerant Corn

Groundboom 

equipment only

Preplant, 

Preemergence, 

and 

Postemergence 

Application

3.8 lb 2,4-

D/gal

 (GF-2654 

TC)

1 lb 

ae/acre 3

3 lb ae/acre 

per use 

season

Forage: 

30

Do not apply this product through 

any type of irrigation system. Do 

not aerially apply this product. Do 

not apply more than one 

preemergence application and no 

more that two postemergence 

applications per use season. Do not 

apply to corn later than the V8 stage 

or corn more than 48 inches tall.

3.4 Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the 

proposed new use of 2,4-D on herbicide-tolerant corn and soybean. Humans may be exposed to 

2,4-D in food and drinking water, since 2,4-D may be applied directly to growing crops and 

application may result in 2,4-D reaching surface and ground water sources of drinking water. 

There are residential uses of 2,4-D, so humans may be exposed to 2,4-D in residential or nonoccupational settings, including during pesticide application as well as potential for postapplication exposure. In an occupational setting, applicators may be exposed while handling the 

pesticide prior to application, as well as during application. There is a potential for postapplication exposure for workers re-entering treated fields. 

This risk assessment considers all of the aforementioned exposure pathways based on the 

proposed new uses of 2,4-D, but also considers the existing uses as well, particularly for the 

dietary and residential exposure assessments. 

3.5 Consideration of Environmental Justice
2,4-D Human Health Risk Assessment DP No. D389455

Page 12 of 96

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions 

to Address Environmental Justice in Minority Populations and Low-Income Populations," 

(http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf. As a part of every pesticide risk 

assessment, OPP considers a large variety of consumer subgroups according to well-established 

procedures. In line with OPP policy, HED estimates risks to population subgroups from 

pesticide exposures that are based on patterns of that subgroup’s food and water consumption, 

and activities in and around the home that involve pesticide use in a residential setting. 

Extensive data on food consumption patterns are compiled by the USDA under the National 

Health and Nutrition Examination Survey, What We Eat in America, (NHANES/WWEIA) and 

are used in pesticide risk assessments for all registered food uses of a pesticide. These data are 

analyzed and categorized by subgroups based on age, season of the year, ethnic group, and 

region of the country. Additionally, OPP is able to assess dietary exposure to smaller, 

specialized subgroups and exposure assessments are performed when conditions or 

circumstances warrant. Whenever appropriate, non-dietary exposures based on home use of 

pesticide products and associated risks for adult applicators and for toddlers, youths, and adults 

entering or playing on treated areas post-application are evaluated. Further considerations are 

currently in development as OPP has committed resources and expertise to the development of 

specialized software and models that consider exposure to bystanders and farm workers as well 

as lifestyle and traditional dietary patterns among specific subgroups.

4.0 Hazard Characterization and Dose-Response Assessment

4.1 Toxicology Studies Available for Analysis

The toxicology database on 2,4-D is complete and sufficient for assessing the toxicity and 

characterizing the hazard of 2,4-D. The toxicology studies for 2,4-D are summarized in 

Appendix A. The database includes the following studies. 

Subchronic: 21-day dermal toxicity (rabbit), 90-day oral toxicity (rat), 13-week oral (diet) 

toxicity (dog), 13-week oral (capsule) toxicity (dog), 28-day inhalation toxicity (rat) 

Developmental toxicity: developmental toxicity (rat), developmental toxicity (rabbit)

Reproduction: 2-generation reproduction study (rat); an extended 1-generation 

reproductive toxicity study (rat)

Chronic: combined oral chronic toxicity/carcinogenicity (rat), carcinogenicity (mouse), 

chronic oral toxicity (dog)

Neurotoxicity: acute neurotoxicity (rat), subchronic neurotoxicity (rat), developmental 

neurotoxicity (rat)

Other: immunotoxicity study (rat), thyroid assessment (rat), mutagenicity battery, 

metabolism (rat)

The studies available for consideration of 2,4-D toxicity provide a comprehensive database, with

routes of administration that are consistent with potential exposure scenarios. Additionally, there 

are 90-day oral toxicity (dog and rat), 21-day dermal toxicity (rabbit), developmental toxicity (rat 
2,4-D Human Health Risk Assessment DP No. D389455

Page 13 of 96

and rabbit) studies available on the amine salts and esters of 2,4-D, which show a similar toxicity 

profile as that following exposure to 2,4-D.

4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkylchlorophenoxy herbicide. 2,4-D is well 

absorbed orally, undergoes limited metabolism, and is eliminated quickly from the body 

primarily unchanged in the urine by active saturable renal transport. The observed dosedependent, non-linear pharmacokinetics of 2,4-D is primarily due to the saturation of this renal 

secretory transport system. This saturation results in elevated plasma concentrations of 2,4-D that 

are associated with toxicity. The main target organ for 2,4-D is the kidney, where the highest 

tissue levels are found. There is a gender-based difference in the renal clearance of 2,4-D in adult 

rats; i.e., males show a greater ability to clear 2,4-D relative to females. Additionally, 

toxicokinetic studies conducted in pregnant rats show that 2,4-D is transferred through maternal 

milk to the pups. Due to a limited capacity to excrete organic acids, the dog is more sensitive to 

the effects of 2,4-D than the rat with respect to repeated dosing. Based on data obtained from the 

open literature, the calculation of relevant pharmacokinetic parameters for 2,4-D in different 

species shows that renal clearance, volume of distribution, and plasma half-life of 2,4-D correlate 

with body weight (allometric scaling) for the mouse, rat, pig, calf, and human but not the dog. 

The dog shows a lower than expected renal clearance and a longer than expected plasma half-life 

compared to the other species. The calculated renal clearance in dogs is about an order of 

magnitude lower than the values expected from allometric scaling for the other species. This is 

consistent with acute oral toxicity data, which show that the NOAEL in the dog is about an order 

of magnitude lower than rodent NOAELs.

4.2.1 Dermal Absorption

There is an extensive set of high quality human in vivo dermal absorption data available, and a 

dermal absorption factor of 10% has been used previously. However, quantification of dermal 

risk is not required, as discussed in Section 4.5.1 (Dose-Response Assessment).

4.3 Toxicological Effects

The toxicity profile of 2,4-D shows that the principal toxic effects are changes in the kidney 

[increased kidney weight, histopathological lesions], thyroid [decreased thyroxine, increased 

thyroid weight, hyperplasia and hypertrophy of follicular cells], liver [increased liver weight, 

increased ALT, and AST, histopathological lesions, including hypertrophy], adrenal [increased 

adrenal weight, histopathological lesions], eye [retinal degeneration, cataract formation, lens 

opacity], and ovaries/testes [decreased testes weight and ovarian weight, atrophy] in the rat 

following exposure to 2,4-D via the oral route at dose levels above the threshold of saturation of 

renal clearance. No systemic toxicity was observed in rabbits following repeated exposure via

the dermal route at dose levels up to the limit dose [1000 mg/kg/day]. In the dog, the kidney 

[increased kidney weight, elevated blood urea nitrogen and creatinine, decreased glucose, 

cellular alterations of the proximal tubule], thyroid, [decreased T4, increased thyroid weights],

and testis [decreased testes weight, testes degeneration, and hypospermatogenesis/juvenile testis] 

are target organs following exposure via the oral (diet and capsule) route at dose levels above the 
2,4-D Human Health Risk Assessment DP No. D389455

Page 14 of 96

threshold of saturation of renal clearance. Effects in dogs were observed at lower dose levels 

than those observed in rodents, and this is attributed to the dog’s limited capacity to eliminate 2, 

4-D and other organic acids. 2,4-D is not acutely (lethal) toxic via the oral, dermal, and 

inhalation routes, is not a dermal irritant or a dermal sensitizer, but it is a severe eye irritant.

Maternal toxicity (decreased body weight gain) and developmental toxicity (skeletal 

malformations) were observed in the rat developmental toxicity study at a dose level that exceeds 

the threshold of saturation of renal clearance. In the rabbit, clinical signs (ataxia, decreased 

motor activity, loss of righting reflex, cold extremities) and decreased body weight gain were 

observed in the maternal animal at the high dose, and there were 2 abortions at the high dose. In 

the rat 2-generation reproductive toxicity study, decreased maternal body weights and offspring 

deaths and skeletal variations were observed at the high dose level, which exceeds the threshold 

of saturation of renal clearance. 

Neurotoxicity, as evidenced by the increased incidence of in-coordination and slight gait 

abnormalities (forepaw flexing or knuckling), was observed following oral exposure to rats 

during the FOB assessment in the acute neurotoxicity study in rats. Relative forelimb grip 

strength was significantly increased in rats of both sexes at the high-dose level in the subchronic 

neurotoxicity study, although there was no treatment-related change in absolute grip strength. 

Developmental neurotoxicity and developmental immunotoxicity were not observed in the 

extended 1-generation reproduction toxicity study in rats, and the findings in the thyroid were 

considered adaptive.

2,4-D is classified as “not classifiable as to human carcinogenicity”, based upon bioassays in rats 

and mice that showed no statistically significant tumor response in either species. The Agency 

determined, based on several reviews of epidemiological studies, in addition to the animal 

studies, that the existing data did not support a conclusion that links human cancer to 2,4-D 

exposure.

4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

HED recommends that the 10X FQPA Safety Factor (for the protection of infants and children) 

be reduced to 1X. An FQPA Safety Factor of 1X is appropriate for the following reasons:

The toxicity database is complete and adequate to assess safety for infants and children. There is 

evidence of increased susceptibility in the rat developmental toxicity study and in the rat 2-

generation reproduction study. These studies have clearly defined NOAELs/LOAELs, and the 

points of departure used in the risk assessment are below where these findings occur and are 

protective. There are acute and subchronic neurotoxicity studies, a developmental neurotoxicity 

study, a detailed evaluation of thyroid function across life stages, and a developmental 

immunotoxicity study on 2,4-D. The exposure assessment will not underestimate children’s 

exposure to 2,4-D. Further details may be found in the following sections.

4.4.1 Completeness of the Toxicology Database
2,4-D Human Health Risk Assessment DP No. D389455

Page 15 of 96

The toxicology database for 2,4-D is complete. Acceptable rat and rabbit developmental toxicity 

studies, a rat 2-generation reproduction study, an extended 1-generation rat reproduction toxicity 

study (F1 offspring evaluated for potential effects on the nervous system, immune system, 

reproductive and endocrine systems, thyroid function, and other systemic toxicity parameters), 

and acute, subchronic, and developmental neurotoxicity studies in rats are available. 

4.4.2 Evidence of Neurotoxicity

Evidence of neurotoxicity was observed in the acute neurotoxicity study in rats, as evidenced by 

an increase in the incidence of in-coordination and slight gait abnormalities (forepaw flexing or 

knuckling) during the FOB assessment at the high dose in both sexes. In the subchronic 

neurotoxicity study, relative forelimb grip strength was significantly increased in rats of both 

sexes at the high-dose level, although there was no treatment-related change in absolute grip 

strength. Clinical signs of neurotoxicity (decreased motor activity, ataxia, loss of righting reflex, 

extremities cold to the touch) were observed in maternal rabbits in the developmental toxicity 

study. Developmental neurotoxicity was not observed in the developmental neurotoxicity study 

in rats. Neuropathological effects were not observed in any study.

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal

There is evidence of increased susceptibility following in utero exposure to 2,4-D in the rat 

developmental toxicity study and following in utero and/or pre-/post-natal exposure in the rat 2-

generation reproduction study. There is no evidence of increased susceptibility following in 

utero exposure to 2,4-D in the rabbit developmental toxicity study or following in utero and/or 

pre-/post-natal exposure in the rat extended 1-generation reproduction toxicity study.

2,4-D has been evaluated for potential developmental effects in the rat and rabbit. Maternal 

toxicity included decreased body weight gains in the rat study at the same dose level where 

developmental effects (occurrence of skeletal malformations) were observed. Maternal toxicity 

in the rabbit included decreased body weight gain, clinical signs of toxicity (decreased motor 

activity, ataxia, loss of righting reflex, extremities cold to the touch), and abortions, the latter 

being indicative of developmental toxicity. Decreased maternal body weight gains were observed 

in the rat 2-generation reproduction study at a dose that exceeded renal saturation and resulted in 

reduced viability of the F1 pups. There are clearly established NOAELs and LOAELs for the 

population of concern, there are no data gaps in the toxicology database, and the points of 

departure (POD) are protective of susceptibility.

4.4.4 Residual Uncertainty in the Exposure Database

There are no residual uncertainties in the exposure database. The dietary exposure estimates are 

unrefined and reflect primarily tolerance-level residue in food, 100% CT, and upper-bound 

drinking water estimates based on modeling. These assumptions and refinements are detailed in 

the Section 5.4.1. Additionally, HED does not believe that non-occupational exposure estimates 

are underestimated. The assumptions are detailed in Section 6.0. 

4.5 Toxicity Endpoint and Point of Departure Selections
2,4-D Human Health Risk Assessment DP No. D389455

Page 16 of 96

4.5.1 Dose-Response Assessment

A detailed description of the toxicity studies used for selecting toxicity endpoints and points of 

departure for various exposure scenarios is presented in the appendix. The available hazard 

database is adequate to characterize any potential for prenatal or postnatal risk for infants and 

children. 

An acute dietary endpoint for females 13+ was selected from the developmental toxicity study in 

rats with a NOAEL of 25 mg/kg/day. At the study LOAEL of 75 mg/kg/day, fetal skeletal 

malformations (14th rudimentary ribs) were observed. A 100X uncertainty factor was applied to 

account for inter- and intra-species variability resulting in an acute reference dose (RfD) of 0.25 

mg/kg/day. 

An acute dietary endpoint for the general population, including infants and children, was selected 

from the acute neurotoxicity study in rats with a NOAEL of 67 mg/kg. At the study LOAEL of 

225 mg/kg, an increased incidence of incoordination and slight gait abnormalities (forepaw 

flexing or knuckling) and decreased motor activity were observed. A 100X uncertainty factor 

was applied to account for inter- and intra-species variability resulting in an acute reference dose 

(RfD) of 0.67 mg/kg/day. 

The chronic dietary endpoint was selected from the extended one-generation reproduction 

toxicity study in rats with a NOAEL of 21 mg/kg/day. At the study LOAEL of 55.6/46.7 

mg/kg/day, kidney toxicity, manifested as increased kidney weights and increased incidence of 

degeneration of the proximal convoluted tubules, was observed and decreased body weight in 

pups was observed throughout lactation. A 100X uncertainty factor was applied to account for 

inter- and intra-species variability resulting in a chronic reference dose (cRfD) of 0.21 

mg/kg/day. 

Short-term and intermediate-term incidental oral endpoints for risk assessment were selected 

from the extended one-generation reproduction toxicity study in rats with a NOAEL of 21 

mg/kg/day. At the study LOAEL of 55.6/46.7 mg/kg/day, kidney toxicity, manifested as 

increased kidney weights and increased incidence of degeneration of the proximal convoluted 

tubules, was observed and decreased body weight in pups was observed throughout lactation. A 

100X uncertainty factor was applied to account for inter- and intra-species variability.

Short-term and intermediate-term inhalation endpoints for risk assessment were selected from 

the route-specific 28-day inhalation toxicity study in rats with a LOAEL of 0.05 mg/L/day. A 

NOAEL for portal-of-entry effects was not determined. At the study LOAEL of 0.05 mg/L/day,

squamous metaplasia and epithelial hyperplasia with increased mixed inflammatory cells within 

the larynx, which was not totally resolved following a 4-week recovery period, were observed. 

Human Equivalent Concentrations (HEC)/Human Equivalent Doses (HED) for residential and 

occupational scenarios were calculated. For details regarding the calculation of the HEC and 

HED, see Appendix A. A 3X uncertainty factor was applied to account for inter-species 

variability (to account for the PD differences), a 10X uncertainty factor was applied to account 

for intra-species variability, and a 10X UFLOAEL→NOAEL was applied to account for the lack of a 
2,4-D Human Health Risk Assessment DP No. D389455

Page 17 of 96

NOAEL. Although there was no assessment of the thyroid in the inhalation study, the rat 

extended 1-generation reproduction toxicity (oral) study performed an assessment of the thyroid 

for several age groups at dose levels up to/approaching renal saturation. The changes in thyroid 

hormones observed, along with thyroid histopathological findings, were considered treatmentrelated, although not adverse. The lack of an assessment of the thyroid in the inhalation study is 

considered inconsequential because the portal of entry endpoint is protective of potential thyroid 

effects expected to occur at higher concentrations; i.e., at doses that exceed the level of renal 

clearance. Portal-of-entry effects were observed at all dose levels, and an additional 10X 

uncertainty factor is applied to the LOAEL to obtain an extrapolated NOAEL used for the 

inhalation risk assessments. The use pattern indicates that dose levels required to exceed the 

renal clearance mechanism would not be attained following human inhalation exposure.

No quantification of dermal risk is required. Although the dermal toxicity study did not evaluate 

developmental endpoints, (1) there was no dermal or systemic toxicity observed following 

repeated dermal applications to rabbits at the Limit Dose (1000 mg/kg/day); (2) there was no 

quantitative susceptibility observed in the developmental or reproductive toxicity studies; (3) the 

use of a 10% human dermal absorption factor (DAF) with the oral developmental LOAEL of 90 

mg/kg/day established in the rabbit developmental toxicity study yields a dermal equivalent dose 

(DED) of 900 mg/kg/day, which is numerically similar to the high-end dermal NOAEL (1000 

mg/kg/day) in the dermal rabbit study; (4) the use of the 10% human DAF with the oral 

developmental LOAEL of 75 mg/kg/day established in the rat developmental study yields a DED 

of 750 mg/kg/day; (5) the developmental findings in the rat and rabbit occurred at oral dose 

levels exceeding renal clearance, and clear NOAELs were obtained (dermal equivalent doses of 

250 and 300 mg/kg/day); (6) although there was no assessment of the thyroid in the dermal

study, the rat extended 1-generation reproduction toxicity (oral) study performed an assessment 

of the thyroid for several age groups at dose levels up to/approaching renal saturation. The 

changes in thyroid hormones (↓ T3 and T4 with ↑TSH levels) observed, along with thyroid 

histopathological findings, were considered treatment-related, although not adverse (NOAEL for 

thyroid effects is ≈40 mg/kg/day; dermal equivalent dose of 400 mg/kg/day); and (7) the use 

pattern indicates that dose levels required to exceed the renal clearance mechanism would not be 

attained following human dermal exposure. 

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

Oral and inhalation endpoints are not the same and cannot be aggregated. 

4.5.3 Cancer Classification and Risk Assessment Recommendation

The Cancer Peer Review Committee (CPRC; TXR No. 0050017, dated January 29, 1997) 

classified 2,4-D as “not classifiable as to human carcinogenicity”, based upon bioassays in rats

and mice that showed no statistically significant tumor response in either species. The Agency 

determined, based on several reviews of epidemiological studies, in addition to the animal 

studies, that the existing data did not support a conclusion that links human cancer to 2,4-D 

exposure.
2,4-D Human Health Risk Assessment DP No. D389455

Page 18 of 96

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk 

Assessment

The points of departure, uncertainty factors, and toxicity endpoints are presented in the following 

table.

Table 4.5.4.1 Summary of Toxicological Doses and Endpoints for 2,4-D for Use in Dietary and Occupational

and Non-Occupational Human Health Risk Assessments

Exposure/ 

Scenario

Point of 

Departure

Uncertainty/FQ

PA Safety 

Factors

RfD, PAD, 

Level of 

Concern for 

Risk 

Assessment

Study and Toxicological Effects

Acute Dietary 

(Females 13-

49 years old)

Developmental 

NOAEL = 25 

mg/kg/day

UFA= 10x

UFH=10x

FQPA SF= 1x

aRfD=aPAD = 

0.25 

mg/kg/day

Developmental Toxicity Study – rat

MRID 00130407, 00130408 (1983)

Developmental LOAEL = 75 mg/kg/day 

based on fetal skeletal abnormalities (14th

rudimentary ribs)

Acute Dietary 

(General 

Population, 

including 

Infants and 

Children)

NOAEL = 67 

mg/kg/day

UFA= 10x

UFH=10x

FQPA SF= 1x

aRfD=aPAD = 

0.67 

mg/kg/day

Acute Neurotoxicity Study – rat

MRID 43115201 (1994)

LOAEL = 227 mg/kg/day based on slight 

gait abnormalities (forepaw flexing and 

knuckling) and increased incidence of 

incoordination.

Chronic 

Dietary (All 

Populations)

NOAEL= 21

mg/kg/day

UFA=10x

UFH=10x

FQPA SF = 1x

cRfD =cPAD= 

0.21

mg/kg/day

Extended 1-generation reproduction 

(Crl:CD(SD) rat)

MRID 47972101 (2010)

Parental LOAEL=800/600 ppm (males 

55.6 mkd; females 46.7 mkd), based on 

kidney toxicity manifested as increased 

kidney weights and increased incidence of 

degeneration of the proximal convoluted 

tubules and for offspring based on 

decreased body weight observed 

throughout lactation. 

Incidental 

Oral 

Short- and 

IntermediateTerm (1-30 

days and 1-6 

months)

NOAEL = 21 

mg/kg/day 

UFA= 10x

UFH=10x

FQPA SF= 1x

Residential 

LOC for MOE 

= 100

Extended 1-generation reproduction 

(Crl:CD(SD) rat)

MRID 47972101 (2010)

LOAEL=800/600 ppm (males 55.6 mkd; 

females 46.7 mkd), based on kidney 

toxicity manifested as increased kidney 

weights and increased incidence of 

degeneration of the proximal convoluted 

tubules, and for offspring based on 

decreased body weight observed 

throughout lactation. 

Dermal (All 

durations)

No potential hazard via the dermal route, based on the lack of systemic effects following repeat

dermal exposure of rabbits at dose levels up to 1000 mg/kg/day. Although developmental toxicity 

was not assessed in the dermal study, clear NOAELs (dermal equivalent doses of 250 and 300 

mg/kg/day) were determined; the developmental effects occurred at dose levels that exceed renal 

clearance mechanism (dermal equivalent doses of 750 and 900 mg/kg/day); dose levels required to 

exceed the renal clearance mechanism would not be attained following dermal exposure to humans.
2,4-D Human Health Risk Assessment DP No. D389455

Page 19 of 96

Table 4.5.4.1 Summary of Toxicological Doses and Endpoints for 2,4-D for Use in Dietary and Occupational

and Non-Occupational Human Health Risk Assessments

Exposure/ 

Scenario

Point of 

Departure

Uncertainty/FQ

PA Safety 

Factors

RfD, PAD, 

Level of 

Concern for 

Risk 

Assessment

Study and Toxicological Effects

Inhalation (all 

durations)

LOAEL = 0.05 

mL/kg/day

HEC=0.013 

mg/L/dayA

(residential)

HEC= 0.056 

mg/L/dayA

(occupational)

HED=1.9 

mg/kg/dayB

(residential)

HED= 2.68 

mg/kg/dayB

(occupational)

UFA = 3X

UFH = 10X

UF LOAEL→NOAEL

= 10X

Residential 

and 

Occupational 

LOC for MOE 

= 300

Subchronic inhalation toxicity study (SD 

CD rat)

MRID 47398701 (2008)

LOAEL = 0.05 mg/L/day, based on 

portal-of-entry effects

(squamous metaplasia and epithelial 

hyperplasia with increased mixed 

inflammatory cells within the larynx); not 

totally resolved following a 4-week 

recovery period.

Cancer (oral, 

dermal, 

inhalation)

Classification: Group D – not classifiable as to human carcinogenicity

Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and 

used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human 

exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = 

uncertainty factor. UFA = extrapolation from animal to human (interspecies). UFH = potential variation in 

sensitivity among members of the human population (intraspecies). UFL = use of a LOAEL to extrapolate a 

NOAEL. UFS = use of a short-term study for long-term risk assessment. UFDB = to account for the absence of key 

data (i.e., lack of a critical study). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c 

= chronic). RfD = reference dose. MOE = margin of exposure. LOC = level of concern. N/A = not applicable.

1 The HEC used for risk assessment is the most sensitive one derived from the nasal degeneration effects.

A Residential HEC (portal of entry endpoint) = 0.05 mg/L * (6/24; 0.25) * (5/7; 0.714) * Tracheobronchial RDDR (1.490) = 0. 

013 mg/L ; Occupational HEC (portal of entry endpoint) = 0.05 mg/L * (6/8; 0.75) * (5/5; 1) * Tracheobronchial RDDR (1.490) 

= 0. 056 mg/L

B Residential HED (portal of entry endpoint) = HEC * 6*24 = 1.9 mg/kg/day; Occupational HED (portal of entry endpoint) = 

HEC*6*8 = 2.68 mg/kg/day
2,4-D Human Health Risk Assessment DP No. D389455

Page 20 of 96

5.0 Dietary Exposure and Risk Assessment 

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

An acceptable metabolism study using AAD-12 soybean was submitted to elucidate the nature of 

the residue in plants. The predominant residues in soybean forage and hay were 2,4-D, 2,4-DCP, 

acetyl-glucose-DCP conjugate and a glucose conjugate of 2,4-DCP. In soybean seed the primary 

identified residues were 2,4-D, glucose conjugate of 2,4-DCP and 2,4-DCP disaccharide. The 

study indicates that the primary pathway of metabolism of 2,4-D in AAD-12 soybean proceeds 

rapidly via dissociation to the acid. After cleavage of the acid side chain, the resulting 

metabolite 2,4-DCP is rapidly conjugated with glucose. Very low levels of 2,4-DCP compared 

to conjugated 2,4-DCP indicate that conjugation is rapid and is a preferential route of 

metabolism. Metabolism proceeds through incorporation of the radiolabeled carbon into natural 

plant constituents. 

Two acceptable metabolism studies of 2,4-D in AAD-1 corn were submitted in support of this 

action. 2,4-D is more readily metabolized into 2,4-DCP in AAD-1 corn than in non-tolerant 

corn. In these studies, 2,4-D and the conjugates of 2,4-DCP were the major residue identified in 

forage and fodder. These plant metabolism studies conducted on AAD-1 corn also concluded 

that metabolism of 2,4-D proceeded to 2,4-DCP, glucose conjugates of 2,4-DCP, and to 

incorporation into natural plant constituents. EPA has determined the residue of concern for 

plant tolerance enforcement is parent 2,4-D only. The residues of concern for risk assessment of 

transgenic corn and soybean are 2,4-D and 2,4-DCP. 

The requirements for livestock metabolism are fulfilled. Adequate goat and hen metabolism 

studies are available, and the metabolism of 2,4-D is similar in both species. Based on the 

available data, on 9/3/03, the Agency determined that the residue of concern in livestock for the 

tolerance expression and for risk assessment is 2,4-D, free and conjugated, determined as the 

acid (W. Hazel and L. Taylor, 12/3/03, D293119, TXR No. 0052264). The existing livestock 

commodity tolerances are adequate to cover the use on feedstuffs of AAD-1 field corn. A 

livestock feeding/grazing restriction for the soybean use is proposed and is considered practical; 

therefore, the current tolerances for residues of 2,4-D on soybean feedstuffs are not being 

reevaluated at this time.

5.1.2 Summary of Environmental Degradation

According to the 2005 RED, as the major route of degradation is aerobic microbial metabolism, 

2,4-D is non-persistent (t1/2=6.2 days) in terrestrial (aerobic) environments, moderately persistent 

(t1/2=45 days) in aerobic aquatic environments, and highly persistent (t1/2= 231 days) in anaerobic 

terrestrial and aquatic environments. Because 2,4-D will be anionic under most environmental 

conditions, it is expected to be mobile (Koc=61.7) in soil and aquatic environments. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 21 of 96

In the six aquatic field dissipation studies, 2,4-D was the predominant residue. There are three 

major degradates identified in the submitted environmental fate studies for 2,4-D, 1,2,4-

benezenetriol, 2,4-dichlorophenol (2,4-DCP), and chlorohydroquinone (CHQ). Metabolite 

Assessment Review Committee (MARC) has determined that residues other than 2,4-D are not 

of risk concern due to low occurrence under environmental conditions, comparatively low 

toxicity, or a combination thereof (W. Hazel and L. Taylor, TXR No. 0052264, D293119, 

12/3/03). Therefore, EDWCs for human health are based on 2,4-D acid equivalent (a.e.) of 2,4-D 

choline.

5.1.3 Comparison of Metabolic Pathways

An acceptable 2,4-D metabolism study in rats is available (MRID 41737302). 2,4-D is well 

absorbed orally, undergoes limited metabolism, and is eliminated quickly from the body 

primarily unchanged in the urine. Parent 2,4-D was the major metabolite found in the urine, 

amounting to 72.9 - 90.5% of the dose.

Residues other than 2,4-D are not of risk concern in the environment as there is low occurrence 

and/or low toxicity. 

In non-transgenic crops, milk, tissue, and poultry, the primary residue is parent 2,4-D. 2,4-DCP 

is a minor metabolite in non-transgenic plants, milk, fat, and eggs. It was determined that 2,4-

DCP is not of concern at the levels expected in livestock tissues and considering the lower 

toxicity of 2,4-DCP compared to 2,4-D.

2,4-D is more readily metabolized into 2,4-DCP in transgenic corn and soybean than in nontransgenic corn and soybean. Although the metabolite 2,4-DCP found in transgenic corn and 

soybean was not a major metabolite in rat metabolism, since 2,4-DCP is less toxic than 2,4-D, 

comparing 2,4-DCP residues to 2,4-D endpoints would be protective for risk assessment 

purposes. 

5.1.4 Residues of Concern Summary and Rationale

HED’s Metabolism Assessment Review Committee (MARC) determined that the residue of 

toxicological concern to be included in non-transgenic crop and livestock tolerances and in 

dietary risk assessments (food and water) is 2,4-D, both free and conjugated, determined as the 

acid (Memo, T. Jimerson, 10/13/04). 

For 2,4-D-tolerant field corn and soybean, the metabolite 2,4-DCP was determined to also be 

included as a residue of concern for dietary risk assessment purposes as there are greater 

amounts of 2,4-DCP found in resistant crop compared to non-resistant crop. Since 2,4-DCP is 

less toxic than 2,4-D, comparing 2,4-DCP residues to 2,4-D endpoints would be protective for 

risk assessment purposes (D394981, A. LaMay, 10/27/11). As 2,4-D is still a major metabolite 

in transgenic corn and soybean, the residues of concern for tolerances will stay the same, the free 

and conjugated forms of parent compound. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 22 of 96

Table 5.1.4 Summary of Metabolites and Degradates to be included in the Risk 

Assessment and Tolerance Expression

Matrix Residues included in Risk 

Assessment

Residues included in 

Tolerance Expression

Plants

Primary Crop Parent (2,4-D) Parent (2,4-D)

Rotational Crop Parent (2,4-D) Parent (2,4-D)

Transgenic Corn and 

Soybean 2,4-D and 2,4-DCP Parent (2,4-D)

Livestock

Ruminant Parent (2,4-D) Parent (2,4-D)

Poultry Parent (2,4-D) Parent (2,4-D)

Drinking Water Parent (2,4-D) Not Applicable

2,4-D is 2,4-Dichlorophenoxyacetic acid, and 2,4-DCP is 2,4-dichlorophenol.

5.2 Food Residue Profile

Adequate residue data are available to support the use of 2,4-D choline on transgenic corn and 

soybean and for risk assessment purposes.

Magnitude of the residue data for transgenic field corn and soybean have been submitted to 

support the proposed 2,4-D choline use. The number of trials and locations were adequate as 

were application rates. Pre-harvest intervals (PHI) for corn reflect the proposed label. However, 

the label specifies a PHI of 30 days for soybean and the minimum PHI in the field trial study was 

51 days for soybean seed. An acceptable method was used for quantitation of 2,4-D and 2,4-

DCP. Applications were made as 2,4-D DMA; these data support of the use of 2,4-D choline on 

field corn and soybean as 2,4-D salts readily dissociate into 2,4-D parent and the cation. 

Based on the corn trials results, residues of 2,4-D and 2,4-DCP, respectively, may be up to 3.0 

and 4.0 ppm on early-harvest forage; 4.4 and 5.0 ppm on typical-harvest forage; and 8.9 and 5.8 

ppm on stover; residues on corn grain were non-detectable (<0.01ppm for each analyte). 

For soybean, residues of 2,4-DCP were considerably higher than 2,4-D. Residues of 2,4-D and 

2,4-DCP, respectively may be up to 4.0 ppm and 25 ppm on early-harvest forage, up to18 ppm 

and 40 ppm on typical-harvest forage, up to 22 ppm and 190 ppm on early-harvest hay, up to 60 

ppm and 133 ppm on typical-harvest hay. On soybean seed, residues of 2,4-D were nondetectable (<0.01ppm) and residues of 2,4-DCP were up to 0.054 ppm. 

The submitted soybean residue decline data indicate that residues of 2,4-D generally decreased in 

forage and hay with increasing PHI. Residues of 2,4-D in soybean seed were <LOQ and residue 

decline could not be assessed. Residues of 2,4-DCP in forage, hay, and seed generally decreased 

or did not increase with increasing PHI. 

Processing factors for 2,4-D and 2,4-DCP could not be calculated for corn flour, grits, meal, 

refined oil, and starch because residues were below the LOQ in/on all samples of field corn grain
2,4-D Human Health Risk Assessment DP No. D389455

Page 23 of 96

and the processed commodities. Residues of 2,4-D may concentrate up to 2.1x in aspirated grain 

fractions (AGF).

Processing factors for 2,4-D could not be calculated for soybean refined oil, meal, and hulls 

because residues were below the LOQ in/on all samples of soybean seed and processed 

commodities. The data indicated that residues of 2,4-D may concentrate in AGF. Residues of 

2,4-DCP may concentrate in AGF and meal but do not concentrate in oil and hulls. The 

observed concentration factors were less than the theoretical concentration factors.

Storage stability data are available for 2,4-D indicating that residues are stable under frozen 

storage conditions for up to 13 months in corn grain, forage, fodder, starch, flour and oil; Storage 

stability data are available demonstrating the stability of 2,4-DCP in corn stover, starch, and oil

stored frozen for 13 months.

Residues of 2,4-DCP do not appear to be stable in herbicide tolerant corn forage, grain, and 

flour, with significant decline noted after 13 months of frozen storage. However, the 

contribution to the dietary burden from corn is not significant when compared to the contribution 

from the currently registered uses on grass forages and hay; therefore, even if residues of 2,4-

DCP are corrected for significant loss during storage, they are not likely to increase the dietary 

burden. Furthermore, if residues of 2,4-DCP are corrected for recovery, the combined residues 

of 2,4-D and 2,4-DCP in field corn foodstuffs are not anticipated to be greater than the tolerance. 

Since tolerance level residues for corn commodities were included in the dietary risk assessment, 

that assessment is still adequate.

Storage stability data are available indicating residues of 2,4-D and 2,4-DCP are stable for up to 

27.8 months in soybean raw agricultural commodities (RACs; seed, hay, forage). The available 

storage stability data are adequate to support sample storage conditions and durations in the 

soybean field trials. Storage stability data are available to support the storage durations (up to 

10.3 months) for the processed commodities soybean hulls, meal, AGF, and refined oil.

Concerning corn, as the combined residues of 2,4-D and 2,4-DCP found in transgenic corn feed 

items are lower than those residues expected in other crop feed commodities, the additional of 

corn feedstuffs to the livestock diet would not increase the dietary burden. The existing livestock 

commodity tolerances are adequate to cover the use on feedstuffs of AAD-1 field corn. A 

livestock feeding/grazing restriction for the soybean use is proposed; therefore, the current 

tolerances for residues of 2,4-D on soybean feedstuffs are not being reevaluated at this time.

No residues of concern or 2,4-D metabolites were detected at 0.01 ppm in any rotated crop 

matrix from either 30- or 139-day plant back intervals (PBIs). No PBIs or rotational crop 

tolerances are required to support the proposed use. 

5.3 Water Residue Profile

The dietary analyses incorporated the drinking water estimates provided by Environmental Fate 

and Effects Division (EFED). The estimated drinking water concentrations (EDWCs) were 

estimated using the Pesticide Root Zone Model/Exposure Analysis Modeling System 
2,4-D Human Health Risk Assessment DP No. D389455

Page 24 of 96

(PRZM/EXAMS) models for surface water and Screening Concentration In GROund Water 

model (SCI-GROW) for ground water. For ground water, the maximum concentration detected 

from monitoring data was 15 μg/L. For surface water, the 1-in-10 year acute (peak) and annual 

average concentrations are 136 and 23 μg/L, respectively (D403774 and 404562, F. Khan, 

10/2/2012). EFED indicated that the estimated drinking water concentration for the acute 

assessment should be 136 ppb, the 1 in 10 year annual peak exposure based on the MS corn 

scenario. The chronic assessment should use the 1 in 10 year annual mean value of 23 ppb

(email communication between Faruque Khan/EFED and Alexandra LaMay/HED, 10/03/12).

Table 5.3 Summary of Surface Water and Groundwater Concentrations for 2,4-D

Scenario Surface Water Conc., ppb Groundwater Conc., ppb

Acute 136a15c

Chronic (non-cancer) 23b

a

From the Tier II PRZM-EXAMS - Index Reservoir model. 1 in 10 year peak exposure. Input parameters are 

based on MS corn scenario

b

From the Tier II PRZM-EXAMS – previously reported 1 in 10 year annual mean

c

From the United States Geological Survey (USGS) National Water-Quality Assessment (NAWQA) program 

monitoring data. Maximum concentration detected.

Source: F. Khan, 10/2/12, D403774 and 404562. Drinking Water Assessment for the Section 3 Proposed 

Registration of 2,4-D Choline (Ethanaminium) on Corn and Soybean 

5.4 Dietary Risk Assessment

Acute and chronic aggregate (food + dietary drinking water) exposure and risk assessments were 

conducted using the Dietary Exposure Evaluation Model software with the Food Commodity 

Intake Database (DEEM-FCID) Version 3.16. This software uses 2003-2008 food consumption 

data from the U.S. Department of Agriculture’s (USDA’s) National Health and Nutrition 

Examination Survey, What We Eat in America, (NHANES/WWEIA). 

5.4.1 Description of Residue Data Used in Dietary Assessment

Concerning transgenic field corn, as the combined residues of 2,4-D and 2,4-DCP found in 

transgenic field corn food items are less than the tolerances of parent only found in nontransgenic field corn, using field corn tolerance-level values for 2,4-D in the acute and chronic 

dietary analyses are protective of residues of 2,4-D and 2,4-DCP in transgenic field corn. 

Concerning transgenic soybean, the combined 2,4-D and 2,4-DCP residues were used for the 

acute and chronic dietary analyses as the combined residues found in tolerant soybean were 

greater than the established tolerance of parent only for soybean. Residue levels of parent 2,4-D 

in/on soybean were non-detectable, thus estimated 2,4-D residues (at 1⁄2 LOD of 0.003 ppm, or 

0.0015 ppm) were added to the 2,4-DCP highest average field trial residue (HAFT is 0.047 

ppm) to be used in the acute and chronic dietary analyses. Please refer to D400085 (D. Drew, 

7/11/13) for more information.
2,4-D Human Health Risk Assessment DP No. D389455

Page 25 of 96

A livestock feeding/grazing restriction for the soybean use is proposed; those are not considered 

feedstuffs associated with the proposed use. Therefore, the current tolerances for residues of 2,4-

D on soybean feedstuffs are not being reevaluated at this time. Concerning corn, as the 

combined residues of 2,4-D and 2,4-DCP found in transgenic corn feed items are lower than 

those residues expected in other crop feed commodities, the additional of corn feedstuffs to the 

livestock diet would not increase the dietary burden. No changes to the existing livestock 

commodity tolerances are required for the proposed new uses. 

Tolerance-level residues were used as exposure values for all commodities (existing and 

proposed uses) in the acute and chronic dietary exposure assessment with the exception of 

soybean, as described above. For soybeans, field trial residue data for transgenic soybean were

used in the assessment. It was assumed that 100% of all crops had been treated. Empirical and 

default processing factors were used as appropriate. 

For water residues, EFED indicated that the estimated drinking water concentration for the acute 

assessment should be 136 ppb, the 1 in 10 year annual peak exposure based on the MS corn 

scenario using Tier II PRZM-EXAMS. The chronic assessment should use the 1 in 10 year 

annual mean value of 23 ppb using Tier II PRZM-EXAMS (email communication between 

Faruque Khan/EFED and Alexandra LaMay/HED, 10/03/12).

5.4.2 Percent Crop Treated Used in Dietary Assessment

Percent crop treated (%CT) data were not applied to the acute and chronic assessments; 100% 

CT was assumed for all commodities, including field corn and soybean, in both analyses. 

5.4.3 Acute Dietary Risk Assessment

For acute exposure assessments, individual one-day food consumption data are used on an 

individual-by-individual basis. The reported consumption amounts of each food item can be 

multiplied by a residue point estimate and summed to obtain a total daily pesticide exposure for a 

deterministic exposure assessment, or “matched” in multiple random pairings with residue values 

and then summed in a probabilistic assessment. The resulting distribution of exposures is expressed 

as a percentage of the aPAD on both a user (i.e., only those who reported eating relevant 

commodities/food forms) and a per-capita (i.e., those who reported eating the relevant commodities 

as well as those who did not) basis. In accordance with HED policy, per capita exposure and risk are 

reported for analyses. 

The resulting acute food plus drinking water risk estimates are not of concern to HED (≤100% 

aPAD) at the 95th percentile of the exposure distribution for the general population and all 

population subgroups. The resulting acute risk estimate for children 1 to 2 years old, the subgroup 

with the greatest exposure, was 14% of the aPAD at the 95th percentile of the exposure. Refer to the 

summary table in 5.4.6. The acute dietary assessment is unrefined; to further refine the 2,4-D dietary 

exposure and risk estimates, %CT, anticipated residues, or monitoring data, if available, could be 

used.

5.4.4 Chronic Dietary Risk Assessment
2,4-D Human Health Risk Assessment DP No. D389455

Page 26 of 96

For chronic dietary exposure assessment, an estimate of the residue level in each food or foodform (e.g., orange or orange juice) on the food commodity residue list is multiplied by the 

average daily consumption estimate for that food/food form to produce a residue intake estimate. 

The resulting residue intake estimate for each food/food form is summed with the residue intake 

estimates for all other food/food forms on the commodity residue list to arrive at the total 

average estimated exposure. Exposure is expressed in mg/kg body weight/day and as a percent 

of the cPAD. This procedure is performed for each population subgroup.

The resulting chronic food plus drinking water risk estimates are not of concern to HED for the 

general population and all population subgroups. The most highly exposed population was children 

1 to 2 years old utilizing 15% of the cPAD. Refer to the summary table in 5.4.6. The chronic dietary 

assessment is unrefined; to further refine the 2,4-D dietary exposure and risk estimates, %CT, 

anticipated residues, or available monitoring data could be used.

5.4.5 Cancer Dietary Risk Assessment

A cancer dietary exposure and risk assessment was not conducted because 2,4-D is classified as 

“not classifiable as to human carcinogenicity”.

5.4.6 Summary Table

Table 5.4.6. Summary of Dietary (Food and Drinking Water) Exposure and Risk for 2,4-D

Population Subgroup

Acute Dietary

(95th Percentile) Chronic Dietary Cancer

Dietary 

Exposure 

(mg/kg/day)

% aPAD*

Dietary 

Exposure

(mg/kg/day)

% cPAD*

Dietary 

Exposure

(mg/kg/day)

Risk

General U.S. Population 0.032197 4.8 0.009882 4.7

N/A N/A

All Infants (< 1 year old) 0.040354 6.0 0.008879 4.2

Children 1-2 years old 0.095100 14 0.030838 15

Children 3-5 years old 0.077053 11 0.025920 12

Children 6-12 years old 0.044063 6.6 0.015028 7.2

Youth 13-19 years old 0.030918 4.6 0.009103 4.3

Adults 20-49 years old 0.025487 3.8 0.007842 3.7

Adults 50+ years old 0.021883 3.3 0.007282 3.5

Females 13-49 years old 0.025718 10 0.007453 3.5

*The values for the highest exposed population for each type of risk assessment are bolded.

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are registered residential uses of 2,4-D for use on ornamental turf, including parks, sports 

fields, and golf courses, as well as aquatic uses; these uses have been assessed previously and 

there are no new residential uses proposed. The existing residential uses that have been 

reassessed in this document to reflect updates to HED’s 2012 Residential SOPs3along with 

 

3 Available: http://www.epa.gov/pesticides/science/residential-exposure-sop.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 27 of 96

policy changes for body weight assumptions. The revision of residential exposures will impact 

the human health aggregate risk assessment for 2,4-D.

Please refer to D413002, A. LaMay, for details on the updated residential exposure assessment. 

6.1 Residential Handler Exposure

HED uses the term “handlers” to describe those individuals who are involved in the pesticide 

application process. HED believes that there are distinct tasks related to applications and that 

exposures can vary depending on the specifics of each task. Residential handlers are addressed 

somewhat differently by HED as homeowners are assumed to complete all elements of an 

application without use of any protective equipment.

The quantitative exposure/risk assessment developed for residential handlers is based on the 

following scenarios: 

Mixing/Loading/Applying Liquid/WP/DF to Lawns/Turf with Hose-End Sprayer

Mixing/Loading/Applying Liquid/WP in WSP to Lawns/Turf/Aquatic Sites with 

Manually-pressurized handwand

Mixing/Loading/Applying Ready-to-Use/WP in WSP to Lawns/Turf with Hose-End 

Sprayer

Mixing/Loading/Applying Liquid to Lawns/Turf/Aquatic Sites with Backpack

Mixing/Loading/Applying Liquid/WP/DF to Lawns/Turf with Manually-pressurized 

handwand or backpack

Mixing/Loading/Applying Granule to Lawns/Turf with Push-type spreader, Belly 

Grinder, Spoon, Shaker Can, Cup or Hand dispersal

Residential Handler Exposure Data and Assumptions

A series of assumptions and exposure factors served as the basis for completing the residential 

handler risk assessments. Each assumption and factor is detailed below.

Application Rate:

The maximum application rate for lawns/turf is 1.45 lb ae/acre for registered uses.

Unit Exposures and Area Treated or Amount Handled:

Unit exposure values and estimates for area treated or amount handled were taken from HED’s 

2012 Residential SOPs4. 

Exposure Duration: 

Residential handler exposure is expected to be short-term in duration. Intermediate-term 

exposures are not likely because of the intermittent nature of applications by homeowners.

Residential Handler Non-Cancer Exposure and Risk Estimate Equations

The algorithms used to estimate exposure and dose for residential handlers can be found in 

Appendix A and/or the 2012 Residential SOPs5.

 

4 Available: http://www.epa.gov/pesticides/science/residential-exposure-sop.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 28 of 96

Combining Exposures/Risk Estimates:

There is no potential hazard via the dermal route for 2,4-D. Only inhalation risk estimates were 

quantitatively assessed. 

Summary of Residential Handler Non-Cancer Exposure and Risk Estimates

The residential handler risk estimates are not of concern for 2,4-D (MOEs are greater than the 

level of concern of 300 for inhalation) for all scenarios.

Table 6.1. Residential Handler Non-cancer Exposure and Risk Estimates for 2,4-D.

Exposure Scenario Level of Concern Inhalation Unit 

Exposure (mg/lb ai)

Maximum 

Application 

Rate1

Area Treated or 

Amount 

Handled Daily2

Inhalation

Dose

(mg/kg/day)3

MOE4

(LOC = 300)

Mixer/Loader/Applicator

Lawns/Turf, Liquid/ 

WP/DF, Hose-end sprayer

Inhalation = 300

0.022 1.5 lb ae/acre 0.5 acres 0.00021 9,200

Lawns/Turf/ Aquatic Sites, 

Liquid/WP in WSP, 

Manually-pressurized 

handwand

0.018 0.06 lb 

ae/gallonA 5 gallons 0.000068 28,000

Lawns/Turf, Ready-touse/WP in WSP, Hose-end 

sprayer

0.034 1.5 lb ae/acre 0.5 acres 0.00032 6,000

Lawns/Turf/ Aquatic Sites, 

Liquid, Backpack 0.14 0.06 lb ae/gallonA 5 gallons 0.00053 3,600

Lawns/Turf, WP/DF, 

Manually-pressurized 

handwand or Backpack

1.1 0.06 lb 

ae/gallonA 5 gallons 0.0041 460

Lawns/Turf, Granule, Pushtype spreader 0.0026 1.5 lb ae/acre 0.5 acres 0.000024 78,000

Lawns/Turf, Granule, Belly 

Grinder 0.039 0.0000344 lb ai/ft2,B 1200 ft2

0.00002 94,000

Lawns/Turf, Granule, Spoon 0.087 0.0000344 lb 

ai/ft2,B 100 ft2

0.0000037 510,000

Lawns/Turf, Granule, Cup/ 

Shaker Can 0.013 0.0000344 lb ai/ft2,B 100 ft2

0.00000056 3,400,000

Lawns/Turf, Granule, Hand 

Dispersal 0.38 0.0000344 lb ai/ft2,B 100 ft2

0.000016 120,000

1 Based on registered or proposed label.

2 Based on HED’s 2012 Residential SOPs (http://www.epa.gov/pesticides/science/residential-exposure-sop.html).

3 Inhalation Dose = Inhalation Unit Exposure (mg/lb ai) × Application Rate (lb ai/acre or gal) × Area Treated or Amount 

Handled (A/day or gallons/day) ÷ BW (kg).

4 Inhalation MOE = Inhalation POD (mg/kg/day) ÷ Inhalation Dose (mg/kg/day).

A 1.5 lb ai per acre with minimum of 25 gallons used per acre = 0.06 lb ai/gal

B 1.5 lb ai/acre * 1 acre/43560 ft2= 0.0000344 lb ai/ft2

6.2 Post-Application Exposure

 

5 Available: http://www.epa.gov/pesticides/science/residential-exposure-sop.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 29 of 96

There is the potential for post-application exposure for individuals exposed as a result of being in 

an environment that has been previously treated with 2,4-D. The quantitative exposure/risk 

assessment for residential post-application exposures is based on the following scenarios: 

As a result of existing residential uses:

Incidental ingestion (i.e., hand-to-mouth, object-to-mouth, soil ingestion exposure) from 

contact with treated turf (children 1 < 2 years old only)

Episodic granular ingestion on treated turf (children 1 < 2 years old only)

Incidental ingestion of water during recreational swimming (both adults and children 3 < 

6 years old).

Assessment of post-application exposure to turf treated with liquid formulations is protective of 

exposure to solid formulations.

The lifestages selected for each post-application scenario are based on an analysis provided as an 

Appendix in the 2012 Residential SOPs6. These lifestages are not the only lifestages that could 

be potentially exposed for these post-application scenarios; however, the assessment of these 

lifestages is health protective for the exposures and risk estimates for any other potentially 

exposed lifestages.

Residential Post-application Exposure for Turf Use

Residential Post-application Exposure Data and Assumptions for Turf Use

A series of assumptions and exposure factors served as the basis for completing the residential 

post-application risk assessment. Each assumption and factor is detailed in the 2012 Residential 

SOPs6.

Chemical-specific TTR Data 

There are three transferable turf residue studies available for 2,4-D, MRIDs 45033101, 

44655703, and 44655702. These studies have been determined to be acceptable for risk 

assessment purposes. The highest predicted day 0 TTR value is 0.28 μg/cm2(from MRID 

44655703, TRT3, North Carolina, application rate of 1.79 lb ae/acre). For risk assessment 

purposes, this residue value is scaled to the 1.45 lb ae/acre application rate; the residue value for 

risk assessment is 0.23 μg/cm2on day 0.

Residential Post-application Non-Cancer Exposure and Risk Equations

The algorithms used to estimate residential post-application exposure and dose can be found in 

D413002 and/or the 2012 Residential SOPs6.

Combining Exposure and Risk Estimates

There is no potential hazard via the dermal route for 2,4-D. Only incidental oral risk estimates 

were quantitatively assessed. The incidental oral scenarios (i.e., hand-to-mouth and object-tomouth) should be considered inter-related and it is likely that they occur interspersed amongst 

 

6 Available: http://www.epa.gov/pesticides/science/residential-exposure-sop.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 30 of 96

each other across time. Combining these scenarios would be overly-conservative because of the 

conservative nature of each individual assessment. The granular ingestion scenario is not 

combined as this exposure would not occur as a result of routine behavior and is considered an 

episodic event related to poisoning. Therefore, no post-application exposure scenarios were 

combined for children 1 < 2 years old. 

Summary of Residential Post-application Non-Cancer Exposure and Risk Estimates for Turf Use

The residential post-application risk estimates are not of concern for 2,4-D (MOEs are greater 

than the level of concern of 100) for all incidental oral scenarios.

Table 6.2.1. Residential Post-application Non-cancer Exposure and Risk Estimates for 2,4-D.

Lifestage

Post-application Exposure 

Scenario Application 

Rate1

Dose 

(mg/kg/day)2 MOEs3 Combined Routes

Combined 

MOEs Use Site Route of Exposure

1 to <2 years Turf

Hand-to-Mouth

1.45 lb 

ae/acre

0.0315 670

NA NA

Object-to-Mouth 0.00096 22,000

Soil Ingestion 4.9x10-5430,000

Episodic Granular 

Ingestion

3% ai; 1.5 lb 

ae/acre 0.14 490A

1 Based on registered or proposed label.

2 Dose equations can be found in Appendix A.

3 MOE = POD (mg/kg/day) / Dose (mg/kg/day).

4 Combined MOE = NA.

A Ingestion of granules is considered episodic in nature; MOE was calculated using the acute dietary endpoint and POD

Residential Post-application Exposure for Aquatic Use

2,4-D can be used for aquatic weed control of surface and submerged weeds. According to the 

2005 RED for 2,4-D, the target concentration in the water column surrounding submerged weeds 

is 2 to 4 ppm. Although many treatments are applied to aquatic areas where recreational 

swimming is not likely to occur, some subsurface treatments are made at recreational lakes. As a 

result, individuals can be exposed to 2,4-D residues in water by entering these areas if they have 

been previously treated. A 24 hour swimming restriction was also added to the master label 

according to the RED. Of the possible post-application exposures, swimming in treated water is 

considered by HED to be worst case and is used as a surrogate for all other possible postapplication exposures. The extent of exposure during recreational swimming is assumed to be 

short-term in duration. Risks estimates were calculated for post-application incidental oral 

ingestion while swimming in treated lakes or ponds. Inhalation exposure is expected to be 

negligible for swimmers; therefore, a post-application inhalation assessment was not conducted. 

Furthermore, the inhalation assessment for residential handlers is expected to be protective of 

potential post-application exposure and risk.

Residential Post-application Exposure Data and Assumptions for Aquatic Use

A series of assumptions and exposure factors served as the basis for completing the residential 

post-application risk assessment for the swimmers scenario and can be found in D413002. 

Inhalation exposure from swimming is expected to be negligible, especially in compared to 

ingestion. Furthermore, 2,4-D will exist primarily in anion form in the environment; therefore, 

significant volatilization from water surfaces is not anticipated because anions are not expected 

to volatilize.
2,4-D Human Health Risk Assessment DP No. D389455

Page 31 of 96

The Agency considered residential post-application exposure for different segments of the 

population. The lifestages selected for each post-application scenario are based on an analysis 

provided as an Appendix in the 2012 Residential SOPs7. These lifestages are not the only 

lifestages that could be potentially exposed for these post-application scenarios; however, the 

assessment of these lifestages is health protective for the exposures and risk estimates for any 

other potentially exposed lifestages. 

The scenarios, routes of exposure and lifestages assessed include ingestion of water during 

recreational swimming (both adults and children 3 < 6 years old).

The swimmer calculations have been updated from the previous assessment to reflect the revised 

body weights for adults and children. Also the water ingestion rates and the time spent in the 

water have been revised to correspond with the 2012 Residential SOPs. A body weight of 80 kg 

is used for assessing incidental oral ingestion for adults since the toxicity endpoint selected is not 

based on a developmental study where developmental and/or fetal effects were observed. The 

level of concern for adults and children (3 to < 6 years old) for short-term incidental oral 

exposures is MOEs < 100.

Residential Post-application Non-Cancer Exposure and Risk Equations

The equations and inputs are generally derived from SWIMODEL 3.0, developed by EPA as a 

screening tool to conduct exposure assessments of pesticides found in swimming pools and spas. 

It uses well-accepted screening exposure assessment equations to calculate the high end exposure 

for swimmers expressed as a mass-based intake value (mg/event). The model focuses on 

potential chemical intakes only and does not take into account metabolism or excretion of the 

chemical of concern. Detailed information and the algorithms used to estimate residential postapplication exposure from uses of 2,4-D for aquatic application are presented in D413002.

Summary of Residential Post-application Exposure and Risk Estimates for Aquatic Use

Table 6.2.2 presents the post-application incidental oral ingestion MOE values calculated for 

adults and children 3 to <6 years old after aquatic applications of 2,4-D. 

Post-application risk estimates do not exceed HED’s level of concern for any of the scenarios 

assessed. 

Table 6.2.2. Residential Post-application Non-cancer Exposure and Risk Estimates for 2,4-

D (Swimmer Scenario)

Exposure Scenario Level of 

Concern

Maximum 

Concentration 

in Water 1

(mg/L)

Exposure Dose

(mg/kg/day) 2

Short-term 

MOE 3

Ingestion of water, 

Adult 100 4

0.0039 5,300

Ingestion of water,

Child 3<6 years 0.0306 690

1. Maximum concentration in water = 4 mg/L (4 ppm) based on maximum application rate for 

 aquatic weed control: 10.8 lb ae/acre-foot. It is possible that the concentration may exceed this value immediately after 

application before mixing. However it is conservative to assume exposure to this concentration for a short-term duration 

(1 to 30 days) as well as considering the 24 hour swimming restriction on the master label.

2. Exposure dose (mg/kg/day) = Refer to Appendix A.

 

7 Available: http://www.epa.gov/pesticides/science/residential-exposure-sop.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 32 of 96

3. MOE = NOAEL/Exposure dose. The level of concern for adults and children (3< 6 years old) is MOEs < 100.

6.3 Combined Residential Risk Estimates (Multiple Exposure Scenarios)

There are no combined residential exposure scenarios.

6.4 Residential Risk Estimates for Use in Aggregate Assessment

Table 6.4 reflects the residential risk estimates that are recommended for use in the aggregate 

assessment for 2,4-D.

Inhalation exposures are not included in the aggregate assessment since effects from the 

inhalation endpoint are not systemic

Post-application granular pesticide exposure following applications to lawns and turf are not 

included in the aggregate assessment as this exposure would not occur as a result of routine 

behavior and is considered an episodic event related to poisoning

The recommended residential exposure for use in the adult aggregate assessment reflects 

incidental oral exposure from post-application exposure swimmer scenario

The recommended residential exposure for use in the children 1<2 years old aggregate

assessment reflects hand-to-mouth exposures from post-application turf scenario (i.e., 

post-application exposure to turf applications)
2,4-D Human Health Risk Assessment DP No. D389455

Page 33 of 96

Table 6.4. Recommendations for the Short- and Intermediate-Term Residential Exposures for the [Chemical] Aggregate Assessment 1

Lifestage

Handler Exposure 

(mg/kg/day)

Residential

Handler Total 

Exposure 

(mg/kg/day)

Residential 

Handler 

Total MOE 

Post-application Exposure 

(mg/kg/day) 2

Residential Postapplication Total 

Exposure 

(mg/kg/day)

Residential Postapplication MOE 3

Dermal Inhalation Dermal Inhalation Oral

Short-Term

Adult N/A N/A N/A N/A N/A N/A 0.0039 0.0039 5,300

Child N/A N/A N/A N/A N/A N/A 0.0315 0.0315 670

1 Bolded risk estimates should contribute to the residential exposure portion of the aggregate assessment. 

2

Post-application exposure represents high-end incidental oral exposure for the relevant exposure duration

3 Total MOE = 1/ (1/Incidental oral MOE). Incidental oral NOAEL = 21 mg/kg/day.
2,4-D Human Health Risk Assessment DP No. D389455

Page 34 of 96

6.5 Residential Bystander Post-application Inhalation Exposure (Volatilization)

The potential exposure to bystanders from vapor phase 2,4-D residues emitted from treated fields 

has been evaluated for the proposed use of 2,4-D choline on herbicide-tolerant corn and soybean. 

Bystander exposure from 2,4-D emitted from treated fields depends on two main factors: 1) the 

rate at which these chemicals come off a treated field (described as the off-gassing, emission or 

flux) and 2) how those vapors are dispersed in the air over and around the treated field. 

Volatilization can occur during the application process or thereafter. It can result from aerosols 

evaporating during application, while deposited sprays are still drying (e.g., possibly via codistillation), or after as dried deposited residues volatilize. The volatilization assessment uses an 

analysis after sprays have dried.

Approaches EPA has used previously to assess inhalation exposures to fumigant pesticides8 were 

used for the assessment and is also consistent with the recommendations of the December 2009 

Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP)9 meeting 

on the scientific issues associated with field volatilization of conversional (semi-volatile) 

pesticides.

Flux Data

Flux data for 2,4-D were submitted and reviewed by the Agency10. The study measured the flux 

rates of 2,4-D ethylhexyl ester (EHE), 2,4-D dimethylamine salt (DMA salt) and the 2,4-D 

choline salt. According to the registrant, 2,4-D choline has a reduced potential for volatility. For 

this assessment, the data from the 2,4-D choline applications only were used as this action 

specifically seeks registration for 2,4-D choline product use in conjunction with the use of 

soybean and corn with resistance traits. 

Volatilization Modeling and Risk Assessment

Volatilization modeling for a single day was completed using Probabilistic Exposure and Risk 

model for FUMigants (PERFUM). There are a variety of factors that potentially affect the 

emission rates of 2,4-D and subsequent offsite transport including: field condition (i.e., bare soil, 

growing or mature crop canopy), field parameters (i.e., soil type, moisture, etc.), formulation 

type, meteorological conditions, and application scenario (i.e., rate, method). To the extent 

possible, based on the limited information available and a lack of intentional statistical design to 

quantitatively evaluate such factors, the impact of these variables was considered. Flux 

estimates from all monitored trials, a number of field sizes, and various meteorological data were 

used with PERFUM to estimate risk based on the 2,4-D field volatility study data described 

above. The PERFUM modelling results are based on Bradenton, FL; Yakima, WA; Flint, MI; 

and Ventura, CA weather datasets which have been used in the past for other volatilization 

analyses and represent a range of conditions including those which have consistently provided 

the highest risk estimates. The analysis included a 40, 80, and 120 acre field using the flux data 

 

8 U.S. EPA 2004d. FIFRA Science Advisory Panel Meeting Minutes - Fumigant Bystander Exposure Model 

Review: Probabilistic Exposure and Risk Model for Fumigants (PERFUM) Using Iodomethane as a Case Study. 

Available at http://www.epa.gov/scipoly/sap/meetings/2004/august1/august2425minutes.pdf

9 U.S. EPA 2009. FIFRA Science Advisory Panel Meeting Minutes - Scientific Issues Associated with Field 

Volatilization of Conventional Pesticides. Available at 

http://www.epa.gov/scipoly/sap/meetings/2009/december/120309meetingminutes.pdf

10 MRID 48862902. Field Volatility of Different 2,4-D Forms. June 15, 2012. Dow AgroSciences LLC. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 35 of 96

submitted for 2,4-D choline and each source of weather data. The short-term residential 

inhalation endpoint was used in the volatilization assessment; it is a conservative assumption to 

compare the day 1 volatilization exposure to a short-term HEC. Furthermore, a 6-hour exposure 

averaging period was used in the model; it is a conservative assumption to compare the 6 hour 

average exposure from the model to the HEC calculated for 24 hours of exposure especially 

since the 6 hour exposure period used as the basis for the comparison represents the peak 

emissions period after application.

Volatilization Risk Estimates

The field volatility study suggests that volatilization of 2,4-D from treated crops does occur and 

could result in bystander exposure to vapor phase 2,4-D. Results of PERFUM modeling, 

however, indicate that airborne concentrations, even at the edge of the treated fields, are not of 

concern. The maximum proposed application rate of 1 lb a.e./A was assessed. There were no 

whole field and maximum field buffers estimated using PERFUM for any field size (i.e., risks 

were acceptable at all percentiles of exposure at the edge of a treated field). 

6.6 Spray Drift

Spray drift is always a potential source of exposure to residents nearby to spraying operations. 

Off-target movement of pesticides can occur via many types of pathways and it is governed by a 

variety of factors. Sprays that are released and do not deposit in the application area end up offtarget and can lead to exposures to those it may directly contact. They can also deposit on 

surfaces where contact with residues can eventually lead to indirect exposures (e.g., children 

playing on lawns where residues have deposited next to treated fields). The potential risk 

estimates from these residues can be calculated using drift modeling coupled with methods 

employed for residential risk assessments for turf products.

The approach to be used for quantitatively incorporating spray drift into risk assessment is based 

on a premise of compliant applications which, by definition, should not result in direct exposures 

to individuals because of existing label language and other regulatory requirements intended to 

prevent them.11 Direct exposures would include inhalation of the spray plume or being sprayed 

directly. Rather, the exposures addressed here are thought to occur indirectly through contact 

with impacted areas, such as residential lawns, when compliant applications are conducted. 

Given this premise, exposures for children (1 to 2 years old) and adults who have contact with 

turf where residues are assumed to have deposited via spray drift thus resulting in an indirect 

exposure are the focus of this analysis analogous to how exposures to turf products are 

considered in risk assessment. 

Several 2,4-D products have existing labels for use on turf, thus it was considered whether the 

risk assessment for that use may be considered protective of any type of exposure that would be 

associated with spray drift. If the maximum application rate on crops adjusted by the amount of 

drift expected is less than or equal to existing turf application rates, the existing turf assessment 

is considered protective of spray drift exposure. The proposed maximum single application rate 

of 2,4-D choline on herbicide-tolerant corn and soybean is 1 lb ae/acre. This is less than the 

registered application rate on turf of 1.5 lb ae/acre, which has been previously assessed and 

 

11 This approach is consistent with the requirements of the EPA’s Worker Protection Standard.
2,4-D Human Health Risk Assessment DP No. D389455

Page 36 of 96

which was updated based on the revised SOPs for Residential Exposure Assessment (i.e., see 

above in Section 5.2). Thus, even if 100% of the application rate on herbicide-tolerant corn and 

soybean is deposited on an adjacent lawn, calculated risk estimates from drift would not be of 

concern. This again is because all existing registered uses on lawns have been previously 

assessed and no risks of concern were identified. 

The registered residential uses on turf result in exposure greater than potential exposure from 

spray drift; therefore, no new residential assessment needs to be completed. 

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate pesticide exposures and risks 

from three major sources: food, drinking water, and residential exposures. In an aggregate 

assessment, exposures from relevant sources are added together and compared to quantitative 

estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When 

aggregating exposures and risks from various sources, HED considers both the route and 

duration of exposure.

7.1 Acute Aggregate Risk

The acute aggregate risk assessment includes only food and water exposure. The acute food plus 

drinking water risk estimates are not of concern to HED (≤100% aPAD) at the 95th percentile of 

the exposure distribution for the general population and all population subgroups. Refer to 

section 5.4.3 for a detailed discussion of the acute dietary assessment.

7.2 Short-Term Aggregate Risk

The short-term aggregate risk assessment includes food, water, and residential exposure. The 

resulting short-term aggregate risks are not of concern to HED (MOEs > LOC of 100) for adults 

and children.

Table 7.2 Short-Term and/or Intermediate Term Aggregate Risk Calculations

Population

Short- or Intermediate-Term Scenario

NOAEL 

mg/kg/day LOC1

Max 

Allowable 

Exposure2

mg/kg/day

Average 

Food and 

Water 

Exposure 

mg/kg/day

Residential 

Exposure 

mg/kg/day3

Total 

Exposure 

mg/kg/day4

Aggregate 

MOE (food, 

water, and 

residential)5

Adult 21 100 0.21 0.007842 0.0039 0.011742 1,800

Child 21 100 0.21 0.030838 0.0315 0.062338 340

1 LOC = inter- and intra- species uncertainty factors totaling 100

2 Maximum Allowable Exposure (mg/kg/day) = NOAEL/LOC

3 Residential Exposure = Oral exposure. (Table 6.4).

4 Total Exposure = Avg Food & Water Exposure + Residential Exposure)

5 Aggregate MOE = [N

7.3 Intermediate-Term Aggregate Risk
2,4-D Human Health Risk Assessment DP No. D389455

Page 37 of 96

Intermediate-term residential exposures are not likely because of the intermittent application of 

2,4-D by homeowners; therefore, the intermediate-term aggregate risk assessment includes only 

food and drinking water exposure, and are equivalent to the chronic food plus drinking water 

exposure. The chronic food plus drinking water risk estimates are not of concern to HED for the 

general population and all population subgroups. Refer to section 5.4.4 for a detailed discussion 

of the chronic dietary assessment.

7.4 Chronic Aggregate Risk

The chronic aggregate risk assessment includes only food and water exposure. The chronic food 

plus drinking water risk estimates are not of concern to HED for the general population and all 

population subgroups. Refer to section 5.4.4 for a detailed discussion of the chronic dietary 

assessment.

7.5 Cancer Aggregate Risk

2,4-D has been classified as a Category D chemical, i.e., not classifiable as to human 

carcinogenicity. A quantitative cancer risk assessment is not required. 

8.0 Cumulative Exposure/Risk Characterization

2,4-D is a member of the alkylphenoxy herbicide class of pesticides. This class also includes 

MCPA, 2,4-DB, and 2,4-DP. A cumulative risk assessment has not been performed as part of 

this human health risk assessment because the Agency has not made a determination as to which 

compounds to which humans may be exposed, if any, have a common mechanism of toxicity.

Unlike other pesticides for which EPA has followed a cumulative risk approach based on a 

common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as 

to 2,4-D and any other substances. For the purposes of this tolerance action, therefore, EPA has 

not assumed that 2,4-D has a common mechanism of toxicity with other substances.

For information regarding EPA’s efforts to determine which chemicals have a common

mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy 

statements released by EPA’s Office of Pesticide Programs concerning common mechanism 

determinations and procedures for cumulating effects from substances found to have a common 

mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

9.1 Short-/Intermediate-Term Handler Risk

HED uses the term handlers to describe those individuals who are involved in the pesticide 

application process. HED believes that there are distinct job functions or tasks related to 

applications and exposures can vary depending on the specifics of each task. Job requirements 

(amount of chemical used in each application), the kinds of equipment used, the target being 

treated, and the level of protection used by a handler can cause exposure levels to differ in a 

manner specific to each application event. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 38 of 96

Based on the anticipated use patterns and current labeling, types of equipment and techniques 

that can potentially be used, occupational handler exposure is expected from the proposed uses. 

The quantitative exposure/risk assessment developed for occupational handlers is based on the 

following scenarios: 

Mixing/Loading Liquid for Groundboom Application

Applying Spray by Groundboom Equipment

Occupational Handler Exposure Data and Assumptions

A series of assumptions and exposure factors served as the basis for completing the occupational 

handler risk assessments. Each assumption and factor is detailed below on an individual basis.

Application Rate: 

The proposed maximum single application rate on herbicide-tolerant corn and soybean is 1 lb 

ae/acre.

Unit Exposures: It is the policy of HED to use the best available data to assess handler exposure. 

Sources of generic handler data, used as surrogate data in the absence of chemical-specific data, 

include PHED 1.1, the AHETF database, the Outdoor Residential Exposure Task Force 

(ORETF) database, or other registrant-submitted occupational exposure studies. Some of these 

data are proprietary (e.g., AHETF data), and subject to the data protection provisions of FIFRA. 

The standard values recommended for use in predicting handler exposure that are used in this 

assessment, known as “unit exposures”, are outlined in the “Occupational Pesticide Handler Unit 

Exposure Surrogate Reference Table12”, which, along with additional information on HED 

policy on use of surrogate data, including descriptions of the various sources, can be found at the 

Agency website13.

Area Treated or Amount Handled: 

Area treated or amount handled includes:

Groundboom equipment: 200 acres per day for high-acreage field crops 

Exposure Duration: 

HED classifies exposures from 1 to 30 days as short-term and exposures 30 days to six months 

as intermediate-term. Exposure duration is determined by multiple factors, including the 

exposed population, the use site, the pest pressure triggering the use of the pesticide, and the 

cultural practices surrounding that use site. For most agricultural uses, it is reasonable to believe 

that occupational handlers will not apply the same chemical every day for more than a onemonth time frame; however, there may be a large agribusiness and/or commercial applicators 

who may apply a product over a period of weeks (e.g., completing multiple applications for 

multiple clients within a region). 

For 2,4-D, based on the proposed use, short- and intermediate-term exposures are anticipated for 

the following reasons: (1) the product can be applied multiple times per year (2) the product can 

 

12 Available: http://www.epa.gov/opp00001/science/handler-exposure-table.pdf

13 Available: http://www.epa.gov/pesticides/science/handler-exposure-data.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 39 of 96

be applied to multiple application sites and (3) there may be a large agribusiness and/or 

commercial applicators who may apply a product over a period of weeks.

Mitigation/Personal Protective Equipment: Estimates of inhalation exposure are calculated for 

various levels of PPE. Results are presented for “baseline,” defined as a single layer of clothing 

consisting of a long sleeved shirt, long pants, shoes plus socks, no protective gloves, and no 

respirator. The 2,4-D product labels direct mixers, loaders, applicators and other handlers to 

wear long-sleeved shirt and long pants, shoes plus socks, chemical-resistant gloves, and 

protective eyewear; a chemical-resistant apron is required when mixing or loading, cleaning up 

spills or equipment, or otherwise exposed to the concentrate.

Occupational Handler Non-Cancer Exposure and Risk Estimate Equations

The algorithms used to estimate non-cancer exposure and dose for occupational handlers can be 

found in D413002.

Combining Exposures/Risk Estimates:

There is no potential hazard via the dermal route for 2,4-D; therefore, only occupational handler 

inhalation exposures are quantitatively assessed.

Summary of Occupational Handler Non-Cancer Exposure and Risk Estimates

Occupational handler risk estimates are not of concern (i.e., MOEs > LOC of 300) for all 

scenarios for use of 2,4-D choline on herbicide-tolerant corn and soybean. At baseline PPE (i.e., 

no respirator), the occupational handler inhalation MOE is 4,900 for mixer/loaders and 3,200 for 

applicators using groundboom equipment. 

Table 9.1. Occupational Handler Non-Cancer Exposure and Risk Estimates for 2,4-D.

Exposure Scenario Crop or 

Target

Inhalation Unit 

Exposure (μg/lb 

ai)1

Maximum

Application 

Rate2

Area 

Treated 

Daily3

Inhalation

Mitigation Level: 

No respirator

Dose 

(mg/kg/day)4 MOE5

Mixer/Loader

Mixer/Loader, 

Liquid, Groundboom

High Acreage 

Crop 0.219 1 lb ae/acre 200 acres 0.000548 4,900

Applicator

Applicator, Spray, 

Groundboom

High Acreage 

Crop 0.34 1 lb ae/acre 200 acres 0.00085 3,200

1 Based on the “Occupational Pesticide Handler Unit Exposure Surrogate Reference Table” (March 2013); Level of mitigation: Baseline.

2 Based on proposed labels.

3 Exposure Science Advisory Council Policy #9.1.

4 Inhalation Dose = Dermal Unit Exposure (μg/lb ai) × Conversion Factor (0.001 mg/μg) × Application Rate (lb ai/acre or gal) × Area Treated or 

Amount Handled Daily (A or gal/day) ÷ BW (kg).

5 Inhalation MOE = Inhalation NOAEL (mg/kg/day) ÷ Inhalation Dose (mg/kg/day).

9.2 Short-/Intermediate Term Post-Application Risk

HED uses the term post-application to describe exposures that occur when individuals are 

present in an environment that has been previously treated with a pesticide (also referred to as reentry exposure). Such exposures may occur when workers enter previously treated areas to 

perform job functions, including activities related to crop production, such as scouting for pests 
2,4-D Human Health Risk Assessment DP No. D389455

Page 40 of 96

or harvesting. Post-application exposure levels vary over time and depend on such things as the 

type of activity, the nature of the crop or target that was treated, the type of pesticide application, 

and the chemical’s degradation properties. In addition, the timing of pesticide applications, 

relative to harvest activities, can greatly reduce the potential for post-application exposure.

9.2.1 Dermal Post-application Risk

Occupational Post-application Dermal Exposure

There is no potential hazard via the dermal route for 2,4-D; therefore, a quantitative occupational 

post-application dermal risk assessment was not completed.

Restricted Entry Interval

The REI specified on the proposed label is based on the acute toxicity of 2,4-D. 2,4-D is 

classified as Toxicity Category III via the dermal route, Toxicity Category IV for skin irritation 

potential, and Toxicity Category I for eye irritation. It is not a skin sensitizer. Under 40 CFR 

156.208 (c) (2) (iii), ai’s classified as Acute Toxicity Category I for acute dermal, eye irritation 

or primary skin irritation are assigned a 48-hour REI. Therefore, the [156 subpart K] Worker 

Protection Statement interim REI of 48 hours is adequate to protect agricultural workers from 

post-application exposures to 2,4-D. 

9.2.2 Inhalation Post-application Risk

Based on the Agency's current practices, a quantitative occupational post-application inhalation 

exposure assessment was not performed for 2,4-D at this time primarily because of the low acute 

inhalation toxicity (Toxicity Category III) and vapor pressure (1.4 x 10-7 mm Hg at 25°C for 2,4-

D acid). However, there are multiple potential sources of post-application inhalation exposure to 

individuals performing post-application activities in previously treated fields. These potential 

sources include volatilization of pesticides and resuspension of dusts and/or particulates that 

contain pesticides. The Agency is in the process of evaluating available post-application 

inhalation exposure data generated by the ARTF and may, as appropriate, develop policies and 

procedures, to identify the need for and, subsequently, the way to incorporate occupational postapplication inhalation exposure into the Agency's risk assessments. If new policies or 

procedures are put into place, the Agency may revisit the need for a quantitative occupational 

post-application inhalation exposure assessment for 2,4-D.

Although a quantitative occupational post-application inhalation exposure assessment was not 

performed, an inhalation exposure assessment was performed for occupational/commercial

handlers. Handler exposure resulting from application of pesticides outdoors is anticipated to 

result in higher exposure than post-application exposure. Therefore, it is expected that these 

handler inhalation exposure estimates would be protective of most occupational post-application 

inhalation exposure scenarios. Furthermore, a quantitative volatilization inhalation exposure 

assessment was assessed for bystanders and indicates no risk of concern for bystanders.

10.0 References

Dole, T. 05/12/05, D316597. 2,4-D. HED’s Revised Human Health Risk Assessment for the 

Reregistration Eligibility Decision (RED) Revised to Reflect Public Comments.
2,4-D Human Health Risk Assessment DP No. D389455

Page 41 of 96

Drew, D. 7/11/13, D400085. 2,4-D. Summary of Analytical Chemistry and Residue Data for 

Use of 2,4-D Choline in/on Herbicide Tolerant Soybeans Containing the Aryloxyalkanoate 

Dioxygenase-12 (AAD-12) Gene.

Goodlow, T. 1/14/09. D339654. 2,4-D: Residue Analytical Method for Livestock Commodities.

Jimerson, T. 10/13/04, D309452. 2,4-D: Health Effects Division (HED) Metabolism Assessment 

Review Committee (MARC) Decision Document-Revised. Chemical I.D. No. 030001. Case 

No. 0073. Meeting date: 9/3/03.

Khan, F. 10/2/12, D403774 and 404562. Drinking Water Assessment for the Section 3 Proposed 

Registration of 2,4-D Choline (Ethanaminium) on Corn and Soybean 

LaMay, A. 10/27/11, D384441. 2,4-D. Summary of Analytical Chemistry and Residue Data for 

Use of 2,4-D Choline in/on Herbicide Tolerant Field Corn Containing the Aryloxyalkanoate 

Dioxygenase-1 (ADD-1) Gene.

LaMay, A. 10/27/11, D394981, 2,4-D: Petition for the Establishment of a New Formulation of 

2,4-D Choline on Herbicide Tolerant Field Corn Containing the Aryloxyalkanoate Dioxygenase1 (ADD-1) Gene. 

LaMay, A. 2/22/12, D398209, 2,4-D: Amended Petition for the Establishment of a New 

Formulation of 2,4-D Choline on Herbicide Tolerant Field Corn Containing the 

Aryloxyalkanoate Dioxygenase-1 (ADD-1) Gene. 

LaMay, A. 8/8/13, D413002, 2,4-D. Occupational and Residential Exposure Assessment for a 

Proposed Use of 2,4-D Choline on Herbicide-Tolerant Corn and Soybean. 

LaMay, A. 8/8/13, D413003, 2,4-D. Acute and Chronic Aggregate Dietary (Food and Drinking 

Water) Exposure and Risk Assessment for the Section 3 Registration Action on HerbicideTolerant Field Corn and Soybean. 

LaMay, A. 8/8/13, D410196, 2,4-D. Data Provided to Support the Stability of 2,4-D and 2,4-

DCP for Herbicide-Tolerant Corn Commodities Held in Frozen Storage.
2,4-D Human Health Risk Assessment DP No. D389455

Page 42 of 96

Appendix A. Toxicology Profile and Executive Summaries

A.1 Toxicology Data Requirements

The toxicology data requirements (40 CFR 158.340) for the food uses of 2, 4-D are presented 

below. Use of the new guideline numbers does not imply that the new (1998) guideline protocols 

were used.

Study

Technical

Required Satisfied

870.1100 Acute Oral Toxicity.......................................................

870.1200 Acute Dermal Toxicity ..................................................

870.1300 Acute Inhalation Toxicity..............................................

870.2400 Primary Eye Irritation....................................................

870.2500 Primary Dermal Irritation ..............................................

870.2600 Dermal Sensitization......................................................

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.3100 Oral Subchronic (rodent)...............................................

870.3150 Oral Subchronic (nonrodent) .........................................

870.3200 21-Day Dermal ..............................................................

870.3250 90-Day Dermal ..............................................................

870.3465 90-Day Inhalation..........................................................

yes

yes

yes

no

yes

yes

yes

yes

no

yes

870.3700a Developmental Toxicity (rodent)...................................

870.3700b Developmental Toxicity (nonrodent).............................

870.3800 Reproduction .................................................................

yes

yes

yes

yes

yes

yes

870.4100a Chronic Toxicity (rodent) ..............................................

870.4100b Chronic Toxicity (nonrodent)........................................

870.4200a Oncogenicity (rat)..........................................................

870.4200b Oncogenicity (mouse)....................................................

870.4300 Chronic/Oncogenicity....................................................

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.5100 Mutagenicity—Gene Mutation – bacterial ....................

870.5375 Mutagenicity—Structural Chromosomal Aberrations...

870.5550 Mutagenicity—Other Genotoxic Effects.......................

yes

yes

yes

yes

yes

yes

870.6100a Acute Delayed Neurotoxicity (hen)...............................

870.6100b 90-Day Neurotoxicity (hen)...........................................

870.6200a Acute Neurotoxicity Screening Battery (rat) .................

870.6200b 90-Day Neurotoxicity Screening Battery (rat)...............

870.6300 Developmental Neurotoxicity........................................

no

no

yes

yes

yes

-

-

yes

yes

yes

870.7485 General Metabolism ......................................................

870.7600 Dermal Penetration........................................................

870.7800 Immunotoxicity .............................................................

yes

no

yes

yes

yes

yes

Special Studies Comparative thyroid yes yes
2,4-D Human Health Risk Assessment DP No. D389455

Page 43 of 96

A.2 Toxicity Profiles

Table A.2.1 Acute Toxicity Profile – 2,4-D 

Guideline 

No. Study Type MRID Guideline No. Guideline No.

870.1100 Acute oral [rat] 00101605 LD50 = 699 mg/kg III

870.1200 Acute dermal [rabbit] 00101596 LD50 = >2000 mg/kg III

870.1300 Acute inhalation [rat] 00161660 LC50 = > 1.79 mg/L III

870.2400 Acute eye irritation [rabbit] 41125302 Severe eye irritant I

870.2500 Acute dermal irritation [rabbit] 42232701 not an irritant IV

870.2600 Skin sensitization [guinea pig] 00161659 not a dermal sensitizer N/A

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.3100 90-Day oral toxicity 

(Fischer 344 rats)

MRID 41991501 (1991)

Acceptable/Guideline

0, 1, 15, 100, 300

mg/kg/day

NOAEL = 15 mg/kg/day

LOAEL = 100 mg/kg/day, based on decreases 

body weight/gain, alterations in hematology

and clinical chemistry parameters, and cataract

formation in females.

At HDT, effects listed above occurred more

frequently and/or to a greater extent, as well as 

histopathological lesions in eyes, liver, testes, 

adrenals, kidneys, thymus, bone marrow,

spleen, thyroids, lungs.

870.3150 Subchronic oral

(capsule) toxicity -

dogs

MRID 41737301 (1990)

Acceptable/Guideline

0, 0.3, 1.0, 3.0, and 10

mg/kg/day]

NOAEL = 1 mg/kg/day

LOAEL = 3 mg/kg/day, based on decreased 

bodyweight/body-weight gain and food

consumption (males), alterations in clinical

chemistry parameters [increased BUN (both

sexes), creatinine (males)], and decreased testis

weight in males. At the highest dose tested,

mortality (2 males, 1 female sacrificed

moribund), hematology alterations, and 

microscopic lesions of the kidneys and testes 

[hypospermatogenesis] were observed.

870.3150 Subchronic oral

(diet) toxicity (dog)

MRID 42780001 (1993)

Acceptable/Guideline

0, 0.5, 1.0, 3.75, and 7.5

mg/kg/day 

NOAEL = 1 mg/kg/day

LOAEL = 3.75 mg/kg/day, based on decreased 

body-weight gain (both sexes) and food 

consumption (males), as well as alterations in 

clinical chemistry parameters[increased BUN,

creatinine, and alanine aminotransferase] in

both sexes, and decreased testes weight and

slightly higherincidence of

hypospermatogenesis/juvenile testis and

inactive/juvenile prostate were observed. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 44 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.3200 21-Day dermal 

toxicity (rabbit)

MRID 41735301,

41735304 (1990)

Acceptable/Guideline

0, 10, 100, 1000

mg/kg/day

NOAEL = 1000 mg/kg/day

LOAEL > 1000 mg/kg/day, no adverse effects

observed at the limit dose

870.3465 90-Day inhalation 

toxicity (Sprague 

Dawley CD®)

6 hr/day, 5 

days/week for 28 

days

MRID 47398701 (2008)

Acceptable/Guideline

0, 0.05, 0.1, 0.3, 1.0 mg/L

Systemic NOAEL = 0.30 mg/L/day

Systemic LOAEL = 1.0 mg/L/day based on 

increased alkaline phosphatase and aspartate 

aminotransferase levels in females and 

decreased spleen weights in females. 

NOAEL (portal-of-entry effects) = not 

determined. 

LOAEL (portal-of-entry effects) = 0.05 

mg/L/day, based on squamous metaplasia and 

epithelial hyperplasia with increased mixed 

inflammatory cells within the larynx; not totally 

resolved following a 4-week recovery period. 

870.3700a Developmental 

toxicity

Fischer 344 rats

(GD 6-15)

MRID 00130407,

00130408 (1983)

Acceptable/Guideline

0, 8, 25, and 75

mg/kg/day

Maternal NOAEL = 25 mg/kg/day

Maternal LOAEL = 75 mg/kg/day, based on

decreased body weight gains. Survival was not 

affected by treatment.

Developmental NOAEL = 25 mg/kg/day

Developmental LOAEL = 75 mg/kg/day,

based on skeletal abnormalities/

malformations.

870.3700b Prenatal 

developmental in 

(New Zealand white 

rabbit)

GD 6-18

MRID 41747601 [1990]

Acceptable/Guideline

0, 10, 30, and 90

mg/kg/day

Maternal NOAEL = 30 mg/kg/day

Maternal LOAEL = 90 mg/kg/day, based on 

clinical signs [ataxia, decreased motor activity,

loss of righting reflex, cold extremities], 

abortion (2), decreased body-weight gains. 

Survival was not affected by treatment.

Developmental NOAEL = 30 mg/kg/day

Developmental LOAEL = 90 mg/kg/day, based

on abortions.
2,4-D Human Health Risk Assessment DP No. D389455

Page 45 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.3800 Reproduction and 

fertility effects

(Fischer 344 rat)

MRID 00150557 and 

00163996 (1985)

acceptable/guideline*

*new 870.3800 required 

(see MRID 47972101 

below)

Parental NOAEL = 5 mg/kg/day.

Parental LOAEL = 20 mg/kg/day, based on 

decreased female body weight/body weight

gain (F1) and male renal tubule alteration

(F0 and F1). At HDT, excess toxicity and too 

few pups for second litter (dose group 

terminated).

Reproductive NOAEL = 20 mg/kg/day. 

Reproductive LOAEL = 80 mg/kg/day, based 

on increased gestation length. However, since 

an increase of less than a day is not considered 

adverse, reproductive toxicity was not observed 

in this study. The dose of 80 mg/kg/day was not 

assessed in the second generation due to the 

lack of sufficient F1 offspring to mate 

(excessive pup mortality). Dose is above the 

threshold of saturation of renal clearance.

Offspring NOAEL = 5 mg/kg/day

Offspring LOAEL = 20 mg/kg/day based on 

pup deaths.
2,4-D Human Health Risk Assessment DP No. D389455

Page 46 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.3800

OECD 443 

and 416

Reproduction and 

fertility effects

(Crl:CD(SD) rat)

Extended One 

Generation 

Reproductive 

Toxicity (EOGRT) 

study

47972101 (2010)

acceptable/non-guideline*

(satisfies OECD 

guideline)

*satisfies the requirement

for reproduction/ fertility 

effects and DNT studies

Parental (male) systemic NOAEL = 300 ppm 

(16.6 mg/kg/day).

Parental (male) systemic LOAEL = 800 ppm 

(45.3 mg/kg/day), based on nephrotoxicity 

manifested as increased kidney weights, and 

degenerative lesions in the proximal convoluted 

tubules in the main study P1 rats.

Parental (female) systemic NOAEL = 600 

ppm (40.2 mg/kg/day). No toxicologically 

relevant effects were identified in P1 females or 

in the GD 17 satellite female groups at the 

highest dose tested.

Thyroid toxicity NOAEL = 800/600 ppm 

(45.3 mg/kg/day in males and /40.2 mg/kg/day 

in females), the highest dose tested. The 

thyroid effects noted in the database were 

considered to be adaptive. 

Offspring (F1 adults) NOAEL = 300 ppm 

(20.9/ mg/kg/day in males and 23.3 mg/kg/day 

in females). 

Offspring (F1 adults) LOAEL = 800/600 ppm 

(55.6 mg/kg/day in males and 46.7 mg/kg/day 

in females), based on kidney toxicity 

manifested as increased kidney weights and 

increased incidence of degeneration of the 

proximal convoluted tubules. 

F1 offspring NOAEL = 300 ppm. The dose on 

a mg/kg/day basis for the PND 22 F1 offspring 

was not calculated.

F1 offspring (PND 22) LOAEL = 800/600 

ppm, based on decreased body weight observed 

throughout lactation. 

DNT offspring NOAEL = 800 ppm/600 ppm 

(81.7 mg/kg/day in males, 59.2 mg/kg/day in 

females), the highest dose tested [lack of 

evidence of DNT (FOB parameters, motor 

activity, and acoustic startle response)]. 

DIT offspring NOAEL = 800/600 ppm (71.8 

mg/kg/day in males and 55.3 mg/kg/day in 

females), the highest dose tested [lack of 

evidence of DIT [SRBC antibody-forming cell 

assay (PND 66-70) and Natural Killer Cell 

assay (PND 87-93)]. 

Reproductive NOAEL = 800/600 ppm (45.3 

mg/kg/day in males, 40.2 mg/kg/day in 

females), the highest dose tested [lack of effect 

on estrous cyclicity, (P1 females, satellite GD 

17 dams, Set 3 F1 offspring) or reproductive 

indices (mating, fertility, time to mating, 

gestation length, pre-and post-implantation 

loss, number of corpora lutea (satellite GD 17 

dams), sperm parameters, ovarian follicle 

counts, and reproductive organ histopathology). 
2,4-D Human Health Risk Assessment DP No. D389455

Page 47 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.4100a Chronic toxicity

(Fischer 344 rat)

MRID 43293901 (1993)

MRID 43612001 (1995)

MRID 44284501 (1997)

0, 5, 75, or 150 mg/kg/day

Acceptable/Guideline

NOAEL = 5 mg/kg/day. 

LOAEL = 75 mg/kg/day, based on decreased

body-weight gain (females) and food

consumption (females), alterations in

hematology [decreased RBC (females), HGB 

(females), platelets (both sexes)] and clinical

chemistry parameters [increased creatinine

(both sexes), alanine and aspartate

aminotransferase (males), alkaline

phosphatase (both sexes), decreased T4 (both 

sexes), glucose (females), cholesterol (both

sexes), and triglycerides(females)], increased

thyroid weights (both sexes at study

termination), decreased testes and ovarian 

weights, and microscopic lesions in the lungs

(females). At the high-dose level, there were

microscopic lesions in the eyes, liver, adipose

tissue, and lungs.

There was no treatment-related increase in

the incidence of any tumor.

870.4100b Chronic toxicity 

(Beagle dog)

MRID 43049001 (1993)

Acceptable/Guideline

0, 1, 5, or 7.5/10 

mg/kg/day

NOAEL = 1 mg/kg/day

LOAEL = 5 mg/kg/day, based on decreased

body-weight gain (both sexes) and food

consumption (females), as well as alterations

in clinical chemistry parameters[increased 

BUN, creatinine, and alanine

aminotransferase, decreased glucose] in both

sexes, decreasedbrain weight in females, and 

histopathological lesions in the liver and

kidneys. At the highest dose tested, 

aspermatogenesis and degeneration of the testes 

was observed in one male and decreased brain 

weight was observed in both sexes.

870.4200 Carcinogenicity

(Fischer 344 rat)

MRID 43612001 (1995)

MRID 44284501 (1997)

0, 5, 75, or 150 mg/kg/day

Acceptable/Guideline

See under 870.4100a above

870.4300 Carcinogenicity

(B6C3F1 CRL BR 

mouse)

MRID 40061801 (1993)

Acceptable/Non-guideline

0, 1, 15, or 45 mg/kg/day

NOAEL = 1 mg/kg/day

LOAEL = 15 mg/kg/day, based on treatmentrelated increase in kidney weights in both

sexes and microscopic renal lesions in males.

There was no treatment-related increase in

the incidence of any tumor type.

Doses not adequate to assess carcinogenic 

potential.
2,4-D Human Health Risk Assessment DP No. D389455

Page 48 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.4300 Carcinogenicity

(B6C3F1 CRL BR 

mouse)

MRID 43879801 (1995)

MRID 43597201(1995)

Acceptable/Guideline

0, 5, 62/150, or 120/300 

mg/kg/day (male/female)

NOAEL = 5 mg/kg/day

LOAEL =62/150 mg/kg/day, based on based

on an increased absolute and/or relative

kidney weights and an increased incidence

of renal microscopic lesions.

There was no treatment-related increase in

the incidence of any tumor type.

870.5100 Salmonella 

typhimurium

TA98, TA100, 

TA1535, Ta1537, 

Ta1538

MRID 41409801(1990)

Acceptable/guideline

100-1000 μg/plate w/ S9;

66.7-6670 μg/plate w/out

S9

No evidence of bacterial mutation w/ and

w/out S9

870.5395 in vivo mouse 

micronucleus assay

ICR mouse (bone 

marrow)

MRID 41409804,

41870101 (1990)

Acceptable/guideline

40-400 μg/kg

Negative.

870.5450 Unscheduled DNA

synthesis assay

MRID 41409807 (1990)

Acceptable/guideline

No evidence of induction of unscheduled

DNA synthesis

Literature 

studies 

cited by 

NRDC

Mutagenicity/

Genotoxicity

(in vivo and in 

vitro)

Office of Pesticide 

Programs, EPA, 

Reevaluation of the 

Genetic Toxicology 

Profile of 2, 4-D. 

(December 12, 2011).

FRN April 18, 2012 denial of NRDC 

petition (mutagenicity summary).

870.6200a Acute neurotoxicity 

screening battery

(Fischer 344 rat)

MRID 43115201 (1994)

Acceptable/guideline

0, 15, 75, or 250 

mg/kg/day 

Achieved: 0, 13, 67, or 

227 mg/kg/day

NOAEL = 67 mg/kg.

LOAEL = 227 mg/kg/day, based on an

increased incidence ofincoordination and

slight gait abnormalities (described as

forepaw flexing or knuckling) and decreased

total motor activity.

870.6200b Subchronic 

neurotoxicity 

screening battery

(Fischer 344 rat)

MRID 43293901 (1994) 

Acceptable/guideline

0, 5, 75, or 150 mg/kg/day 

Males 0, 4.6, 71.2, or 

141.1 mg/kg/day

Females 0, 4.6, 68, 138.9 

mg/kg/day

NOAEL = 71/68 mg/kg/day. 

LOAEL = 141/139 mg/kg/day, based on

increased relative forelimb grip strength and 

increased incidence of bilateral retinal 

degeneration.

870.6300 Developmental 

neurotoxicity

(Crl:CD(SD) rat)

MRID 47972101 (2010)

See under EOGRT

DNT offspring NOAEL = 800 ppm/600 ppm 

(81.7 mg/kg/day in males, 59.2 mg/kg/day in 

females), the highest dose tested [lack of 

evidence of DNT (FOB parameters, motor 

activity, and acoustic startle response)]. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 49 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.7485 Metabolism and 

pharmacokinetics

(Fischer 344 rat)

MRID 41737302 (1990) 2,4-D (sodium salt) is well absorbed orally, 

undergoes limited metabolism, and is 

eliminated quickly from the body primarily 

unchanged in the urine; was nearly completely 

eliminated via the urine following single low 

(>95%; ≈1 mkd) and repeat low (>90%; 100 

mkd) doses; majority eliminated within 12 

hours; highest levels found in kidneys; 

following single high dose (≈100 mkd), ≈90% 

was eliminated in the urine, with 40%-46% 

being eliminated within the first 12 hours; 

highest levels in perirenal fat.

870.7485 Metabolism and 

pharmacokinetics

(Crl:CD(SD) rat)

MRID 47417902 (2008) Compared to the lowest dose (100 ppm), there 

was an 11-fold, 31-fold, and 60-fold difference 

in the AUC24h at 400 ppm, 600 ppm and 800 

ppm, demonstrating nonlinear kinetics.

870.7485 Metabolism and 

pharmacokinetics

(Crl:CD(SD) rat)

MRID 47417901 (2008) Study provides plasma levels of 2,4-D for both 

maternal rats and pups of both sexes, as well as 

levels of 2,4-D in maternal milk, demonstrating 

that the pups would be exposed to 2,4-D during 

lactation. Kidney findings confirm the genderbased difference in renal clearance of 2,4-D in 

adult rats.
2,4-D Human Health Risk Assessment DP No. D389455

Page 50 of 96

Table A.2.2. Subchronic, Chronic and Other Toxicity Profile – 2,4-D

Guideline 

No. 

Study Type MRID No. (year)/

Classification /Doses

Results

870.7600 Dermal penetration 

(human, male)

Feldman. R. J. and

Maibach,

H. I. (1974).

Percutaneous Penetration

of Some Pesticides and 

Herbicides in Man. 

Toxicology and Applied 

Pharmacology. 28: 126-

132].

Ross, R.H.; Driver, J.H.; 

Harris, S.A.; Maibach, 

H.I. (2005). Dermal 

absorption of 2,4-D: a 

review of species 

differences. Regulatory 

Toxicology and 

Pharmacology 41: 82-91.

Moody, R.P.; Wester, 

R.C.; Melendres, J.L.; 

Maibach, H.I. . Journal of 

Toxicology and 

Environmental Health

36(3):241-50.,1992. 

MRID 48772102.

Harris, S.A.; Solomon, 

K.R. 1992. Journal of 

Toxicology and 

Environmental Health 36, 

233-240. MRID 

48772104.

Maibach, H.I.; Feldmann, 

R.J., 1974. MRID 

46859102.

Wester, R.C.; Melendres, 

J.; Sedik, L.; Maibach, H.; 

Riviere, J.E., 1998. 

Toxicology and Applied 

Pharmacology 151, 159-

165. MRID 48772101

dermal application was 5.8% ± 2.4%; 10% 

DAF used in previous risk assessments

870.7800 Immunotoxicity

SRBC antibodyforming cell assay 

and Natural Killer 

Cell assay

(Crl:CD(SD) rat)

MRID 47972101 (2010)

See under EOGRT

DIT offspring NOAEL = 800/600 ppm (71.8 

mg/kg/day in males and 55.3 mg/kg/day in 

females), the highest dose tested [lack of 

evidence of DIT [SRBC antibody-forming cell 

assay (PND 66-70) and Natural Killer Cell 

assay (PND 87-93)]. 

A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Reference Dose (aRfD) - Females age 13-49
2,4-D Human Health Risk Assessment DP No. D389455

Page 51 of 96

Study Selected: Developmental Toxicity Study - rat 

MRID No.: 00130407/00130408

Executive Summary: See Appendix A, Guideline § 870.3700a

Dose and Endpoint for Risk Assessment: NOAEL = 25 mg/kg/day based on fetal skeletal 

malformations (14th rudimentary ribs) at LOAEL = 75 mg/kg/day, which exceeds the threshold 

of saturation of renal clearance.

Comments about Study/Endpoint/Uncertainty Factors: This endpoint is appropriate for the 

population of concern (females of child bearing age) and is considered to be a single dose effect. 

It was concluded that the skeletal findings are real and treatment-related. Although the findings 

occur only at a dose level that exceeds the renal clearance mechanism (75 mg/kg/day), regulating 

below the level where this occurs is protective. There are clearly established NOAELs and 

LOAELs for the population of concern, there are no data gaps in the toxicology database, and the 

point of departure (POD) is protective of susceptibility.

Uncertainty Factor (UF): 100X [10X interspecies extrapolation (UFA), 10X intraspecies 

variability (UFH), FQPA SF 1X].

A.3.2 Acute Reference Dose (aRfD) - General Population

Study Selected: Acute Neurotoxicity Study - rat 

MRID No.: 43115201

Executive Summary: See Appendix A, Guideline § 870.6200

Dose and Endpoint for Risk Assessment: NOAEL = 67 mg/kg/day, based on an increased

incidence of incoordination and slight gait abnormalities(forepaw flexing or knuckling) and

decreased total motor activity at the LOAEL = 227 mg/kg/day.

Comments about Study/Endpoint/Uncertainty Factors: The endpoint is appropriate for the 

population of concern (general population) and is considered to be a single dose effect. 

Uncertainty Factor (UF): 100X [10X interspecies extrapolation (UFA), 10X intraspecies 

variability (UFH), FQPA SF 1X].

A.3.3 Chronic Reference Dose (cRfD) `

Study Selected: Extended 1-Generation Reproduction Toxicity Study - rat

MRID No.: 47972101 

Executive Summary: See Appendix A, Guideline [§ 870.3800; OECD 443 and 416]

Dose and Endpoint for Risk Assessment: parental NOAEL = 21 mg/kg/day (300 ppm), based 

on kidney toxicity manifested as increased kidney weights and increased incidence of 

degeneration of the proximal convoluted tubules at LOAEL = 800/600 ppm (males 55.6 

mg/kg/day; females 46.7 mg/kg/day).

Comments about Study/Endpoint/Uncertainty Factors: The EOGRT study is considered 

appropriate for dose and endpoint selection for the chronic dietary exposure assessment. This is a 

very robust long-term study with a decent dose spread, which evaluates a whole host of 

endpoints, including the target organ (kidney). The chronic rat study, which was selected 

previously for the chronic dietary endpoint, was not selected since it has a very large dose spread 

and the high dose (75 mg/kg/day) exceeds the level of renal clearance in the rat (≈50 mg/kg/day). 

Other studies in the database with lower NOAELs; e.g., two mouse carcinogenicity studies, also 
2,4-D Human Health Risk Assessment DP No. D389455

Page 52 of 96

have similar dose-spacing issues and were not selected for the same reason. The NOAEL of the 

EOGRT study is protective of the LOAELs from the other chronic studies.

Uncertainty Factor (UF): 100X [10X interspecies extrapolation (UFA), 10X intraspecies 

variability (UFH), FQPA SF 1X].

A.3.4 Incidental Oral Exposure (Short- and Intermediate-Term)

Study Selected: Extended 1-Generation Reproduction Toxicity Study - rat

MRID No.: 47972101 

Executive Summary: See Appendix A, Guideline [§ 870.3800; OECD 443/416]

Dose and Endpoint for Risk Assessment: parental NOAEL = 21 mg/kg/day, based on 

decreased body weight observed throughout lactation in F1 offspring (PND 22) at LOAEL = 47

mg/kg/day (800/600 ppm).

Comments about Study/Endpoint/Uncertainty Factors: The EOGRT study is considered 

appropriate for dose and endpoint selection for the incidental oral exposure assessments. This 

study was selected instead of the rat developmental toxicity and rat subchronic oral toxicity 

studies used previously since there is more confidence in the recent EOGRT study, which is a 

more robust study evaluating multiple parameters. Doses for the EOGRT study were selected 

based on pharmacokinetics (PK) data from adult rats and offspring. This study is appropriate for 

both short- and intermediate-term since the study assessed several durations of exposure and life 

stages [postnatal day (PND) 4, PND 22, and PND 60).

Uncertainty Factor (UF): 100X [10X interspecies extrapolation (UFA), 10X intraspecies 

variability (UFH), FQPA SF 1X].

A.3.5 Dermal Exposure (Short- and Intermediate-Term)

A quantitative dermal assessment is not required for the short-term and intermediate-term dermal 

scenarios. Since the dermal toxicity study did not evaluate developmental endpoints, and to 

ensure that developmental effects would not be expected in the dermal toxicity study, the 

following hazard and exposure characteristics were considered: 1) There was no quantitative 

susceptibility observed in the developmental or reproductive toxicity studies; developmental 

toxicity was observed at the same dose as maternal toxicity; 2) there was no dermal or systemic 

toxicity observed following repeated dermal applications to rabbits at the Limit Dose (1000 

mg/kg/day) in a 21-day dermal toxicity study; 3) the use of a 10% human dermal absorption 

factor (DAF) with the oral developmental LOAEL of 90 mg/kg/day established in the rabbit 

developmental toxicity study yields a dermal equivalent dose (DED) of 900 mg/kg/day, which is 

numerically similar to the high-end dermal NOAEL (1000 mg/kg/day) in the dermal rabbit 

study; 4) similarly, use of the 10% human DAF with the oral developmental LOAEL of 75 

mg/kg/day established in the rat developmental study yields a DED of 750 mg/kg/day; 5) the 

developmental findings in the rat and rabbit occurred at oral dose levels exceeding renal 

clearance, and clear NOAELs were obtained (dermal equivalent doses of 250 and 300 

mg/kg/day); and 6) the use pattern indicates that dose levels required to exceed the renal 

clearance mechanism would not be attained following human dermal exposure. 

Additionally, although there was no thyroid hormone assessment and the thyroid was not 

weighed in the dermal study, the rat extended 1-generation reproduction toxicity (oral) study 
2,4-D Human Health Risk Assessment DP No. D389455

Page 53 of 96

performed an assessment of the thyroid for several age groups (F1 offspring on PND 4, PND 22, 

and PND 62-64, and the P1 gestation day 17 females at dose levels up to/approaching renal 

saturation (males 45/females 40 mg/kg/day). The changes in thyroid hormones (↓ T3 and T4 with 

↑TSH levels) observed, along with thyroid histopathological findings, were considered 

treatment-related, although not adverse (NOAEL for thyroid effects is ≈40 mg/kg/day; dermal 

equivalent dose of 400 mg/kg/day). 

A.3.6 Inhalation Exposure (All Durations) 

Study Selected: 28-Day Inhalation Toxicity Study - rat

MRID No.: 47398701 

Executive Summary: See Appendix A, Guideline [§ 870.3465]

Dose and Endpoint for Risk Assessment: No NOAEL for portal of entry effects was 

determined, and the LOAEL was used as the point of departure. The LOAEL for portal of entry 

effects is 0.05 mg/L (lowest dose tested), based on squamous metaplasia and epithelial 

hyperplasia with increased mixed inflammatory cells within the larynx, which was not totally 

resolved following a 4-week recovery period. Excessive salivation, labored breathing, and 

chromodacryorrhea were observed at the high-dose level following the 12th exposure and 

continued during the remainder of the exposures.

Comments about Study/Endpoint/Uncertainty Factor: This route specific study is appropriate 

for the short-term and intermediate-term inhalation assessments. The portal of entry effects were 

squamous metaplasia and epithelial hyperplasia with increased mixed inflammatory cells within 

the larynx. These effects occurred at all dose levels (NOAEL was not identified). The incidence 

and severity of these effects were increased in a dose-related manner, and the effects persisted 

following the 4-week recovery period, although the incidence and severity were reduced. A 

human-equivalent concentration (HEC) was derived from this study based upon the portal of 

entry effects (residential: 0.013 mg/L; occupational: 0.056 mg/L). A human-equivalent dose 

(HED) was also calculated (residential: 1.9 mg/kg/day; occupational: 2.68 mg/kg/day). For 

details regarding the calculation of the HEC and HED, see Appendix B. Although there was no 

thyroid hormone assessment and the thyroid was not weighed in the inhalation study, the 

Crl:CD(SD) rat extended 1-generation reproduction toxicity (oral) study (EOGRT) performed an 

assessment of the thyroid for several age groups (F1 offspring on PND 4, PND 22, and PND 62-

64, and the P1 gestation day 17 (GD 17) females at dose levels up to/approaching renal 

saturation (males 45/females 40 mg/kg/day). The changes in thyroid hormones (↓ T3 and T4 with 

↑TSH levels) observed, along with thyroid histopathological findings, were considered 

treatment-related, although not adverse (NOAEL for thyroid effects is ≈40 mg/kg/day). The lack 

of an assessment of the thyroid in the inhalation study is considered inconsequential because the 

portal of entry endpoint is protective of potential thyroid effects expected to occur at higher 

concentrations; i.e., at doses that exceed the level of renal clearance. Portal-of-entry effects were 

observed at all dose levels, and an additional 10X uncertainty factor is applied to the LOAEL to 

obtain an extrapolated NOAEL used for the inhalation risk assessments. 

Uncertainty Factor (UF): LOC=300 (UFA = 3X, UFH = 10X, UF LOAEL→NOAEL= 10X). For the 

inhalation exposure assessments for short- and intermediate-term durations, a total uncertainty 

factor of 300X is appropriate [3X for interspecies extrapolation (reduced from 10X because RfC 

methodology was used, which takes into consideration the pharmacokinetic differences between 
2,4-D Human Health Risk Assessment DP No. D389455

Page 54 of 96

animals and humans), 10X for intraspecies variation, and a 10X FQPA database uncertainty 

factor (for extrapolation from a LOAEL to a NOAEL)].

A.4. HEC/HED Calculations

Methodologies for HEC Calculations

Calculations used to estimate the inhalation risk to humans from aerosols are dependent not on 

the regional gas dose ratio (RGDR) as for gases, but on the regional deposited dose ratio 

(RDDR). Inhalation studies using aerosols characterize particulate exposure by defining the 

particulate diameter [mass median aerodynamic diameter (MMAD)] and the geometric standard 

deviation (σg), which is then used to determine the RDDR. The RDDR is a multiplicative factor 

used to adjust an observed inhalation particulate exposure concentration of an animal (A) to the 

predicted inhalation particulate exposure concentration for a human (H) that would be associated 

with the same dose delivered to the affected region (r) or target tissue.

RDDRr = (RDDr/Normalizing Factor)A / (RDDr/Normalizing Factor)H 

As with calculations for gases, the affected regions and potential target tissues are the three 

respiratory regions (extrathoracic, tracheobronchial, and pulmonary). The RDDR is easily 

calculated by using a software program designed specifically for computing the RDDR from the 

MMAD and σg defined from an aerosol inhalation study. The values for the species-specific 

parameters used to calculate the RDDR are provided in the EPA document “Methods for 

Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry.” 

The magnitude of the UFs applied is dependent on the methodology used to calculate risk. The 

RfC methodology takes into consideration the PK differences, but not the PD differences. 

Consequently, the UF for interspecies extrapolation may be reduced to 3X (to account for the PD 

differences) while the UF for intraspecies variation is retained at 10X. Thus, the UF when using 

the RfC methodology is customarily 30X.

Regional Deposited Dose Ratio (RDDR) 

MMAD = 1.7

Sigma g = 1.98

 Body Minute 

SPECIES weight (g) Ventilation (VE, ml) 

-----------------------------------------------------------------------------

Rat 236 171.5 

Human 80000 13800.0 

-----------------------------------------------------------------------------

 

Tracheobronchial

SA (cm^2) deposited fraction 

--------------------------------------------------------------------------------

Rat 22.5 0.063 

Human 3200.0 0.075 
2,4-D Human Health Risk Assessment DP No. D389455

Page 55 of 96

--------------------------------------------------------------------------------

RATIO 0.007 0.843

RDDR = (VE/SA)animal/(VE/SA)human * deposited fractionanimal/deposited fractionhuman

= (171.7/22.5)animal/(13800/3200)human * 0.063/0.075

= 1.490

HEC Calculations for Short- and Intermediate-term Occupational Exposure:

Assume worker exposure for 8 hrs/day and 5 days/week and residential exposure for 24 hrs/day 

and 7 days/week:

HEC = NOAELstudy * (daily duration of exposureanimal/daily duration of exposurehuman) * (days/week of 

exposureanimal/days/week of exposurehuman) * RDDR

Occuaptional HEC = 0.05 mg/L * (6/8) * (5/5) * 1.490 = 0.056 mg/L.

Residential HEC = 0.05 mg/L* (6/24)*(5/7)* 1.490 = 0.013 mg/L.

Table A.4.. HEC Array for Occupational/Residential Risk Assessments.

Relevant Study LOAEL

(mg/L)

NOAEL

(mg/L) Da Dh Wa Wh RDDR HEC (mg/L) inter intra

UF

(LOAEL to 

NOAEL)

Short-& Intermediate-Term Exposure

Occupational

Inhalation – Rat

(MRID 47398701)

28-day

TB 0.05

Not 

attained 6 8 5 5 1.490 0.056 3 10 10

Residential

Inhalation – Rat

(MRID 47398701)

28-day

TB 0.05 Not 

attained 6 24 5 7 1.490 0.013 3 10 10

Long-Term Exposure: Not appropriate for 2,4-D.

Key for Array Tables

LOAEL: Lowest observed adverse effect level

NOAEL: No observed adverse effect level

Da: Daily animal exposure (hrs/day)

Dh: Anticipated daily human exposure (hrs/day)

Wa: Weekly animal exposure (days/week)

Wh: Anticipated weekly human exposure (days/week)

RRDR: Regional Deposited Dose Ratio

HEC: Human Equivalent Concentration

inter: interspecies extrapolation UF

intra: intraspecies variation UF

UF: Other UF(s)

TB: Tracheobronchial region

Route-to-Route Extrapolation

More information and details in: Memo, “Route-to-Route Extrapolations” J. Whalen and H. Pettigrew, 10/10/98.
2,4-D Human Health Risk Assessment DP No. D389455

Page 56 of 96

HED’s route-to-route extrapolation converts human and animal values from mg/L concentrations 

to mg/kg oral equivalent doses. The equation uses a single conversion factor to account for 

default body weights and respiratory volumes. An activity factor is used to account for increased 

exposure resulting from increased respiration. 

Using the HEC calculated (based upon squamous metaplasia, epithelial hyperplasia with mixed 

inflammatory cells within larnyx), a conversion of the inhalation concentration to a dose (mg/L 

to mg/kg/day) was conducted as follows: 

Human-Equivalent Dose (HED, mg/kg/day) = Dose (systemic HEC value, mg/L) x A x CF (L/hr/kg) x D (hours) x 

AF = mg/kg

 

Where: 

A = absorption: ratio of deposition and absorption in respiratory tract compared to 

absorption by the oral route.

CF = conversion Factor; a L/hr/kg factor which accounts for respiratory volume and body 

weight for a given species and strain (Table 1 of J. Whalen and H. Pettigrew, 10/10/98).

D = duration; duration of daily animal or human exposure (hours).

AF = activity Factor; animal default is 1.

Therefore, the occupational human equivalent dose for 2,4-D is calculated as follows:

Occupational HED: (0.056 mg/L) x 1 x 6 x 8 x 1 = 2.68 mg/kg/day 

Residential HED: (0.013 mg/L) x 1 x 6 x 24 x 1 = 1.9 mg/kg/day

Based on the current 2,4-D label, HED believes exposures can be short- (1-30 days) or 

intermediate- (1 to 6 months) term in duration. Long-term exposures are not anticipated for 2,4-

D based on proposed labeled uses. For the short- and intermediate-term scenarios, inhalation 

data from the 28-day inhalation rodent study was most appropriate for determining HECs. In the 

RfC methodology, different HECs may be calculated for the same experimental NOAEL due to 

the following:

1. Different algorithms are used to derive HECs for systemic versus port-of-entry effects. 

Typically, HECs are calculated separately for systemic versus port-of-entry effect. For 2,4-

D, port-of-entry effects was observed and, therefore, only port-of-entry HECs were derived. 

2. Time adjustments are traditionally needed for non-occupational (bystander) versus 

occupational exposure scenarios. Traditionally, HECs for non-occupational exposure are 

based on the number of hours an individual may be at home. Therefore, the most 

conservative estimate of hours spent at home would be 24 hours/day and 7 days/week. In 

comparison, the average workweek for an occupational worker is 8 hours/day and 5 

days/week. The HEC array table reflects the time adjustment in the calculations.

A.5 Executive Summaries

A.5.1 Subchronic Toxicity

870.3100 90-Day Oral Toxicity - Rat
2,4-D Human Health Risk Assessment DP No. D389455

Page 57 of 96

In a subchronic oral toxicity study [MRID 41991501], 10 Fischer 344 rats/sex/group 

were administered 2,4-dichlorophenoxyacetic acid [96.1% a.i.] via the diet for 13 weeks 

at concentrations of 0, 1 mg/kg/day, 15 mg/kg/day, 100 mg/kg/day, and 300 mg/kg/day.

There were no treatment-related deaths. Clinical signs of toxicity occurred mainly in the 

high-dose females and included hunched posture [1 high-dose female during weeks 13-

14], depressed activity [all high-dose females during first week], and few/no feces [4 

high-dose males and all high-dose females during week 1; 5 high-dose females during 

week 12; 1 high-dose female during week 13]. There was a dose-related increase in the 

incidence of pale/opaque eyes in both sexes [1/10 controls, 2/10 at 1, 15, and 100 

mg/kg/day and 4/10 at 300 mg/kg/day in males; 1/10 controls, 2/10 at 1 mg/kg/day, 1/10 

at 15 mg/kg/day, 4/10 at 100 mg/kg/day, and 8/10 at 300 mg/kg/day in females]. 

Decreased body weights were observed throughout the study at the high-dose level [both 

sexes], with the magnitude of the deficit increasing with time [males 85%/77% and 

females 79%/72% of control at weeks 6/13, respectively], and decreased body-weight 

gains [weeks 0-6: males 72%/females 50% of control; overall: males 63%/females 43% 

of control] were observed in both sexes at the highest dose level throughout the study. At 

the next highest dose level, decreased body weight [93% of control at 13 weeks; both 

sexes] and body-weight gain [overall males 91%/females 89% of control] were observed.

A corresponding decrease in food consumption was observed in both sexes at the two 

highest dose levels. 

Complete cataract formation was observed in 7 high-dose [300 mg/kg/day] females and 

in one female at the next highest [100 mg/kg/day] dose, and posterior subcapsular 

cataract was observed in 5 high-dose females. Treatment-related alterations in 

hematology [statistically significant decreases in RBC, HGB, HCT, platelet count, 

absolute and corrected leukocyte counts, and lymphocyte counts] were observed at the 6-

and/or 13-week intervals at the high-dose level [both sexes], and decreased platelet 

counts were observed in both sexes at the next highest dose level at week 13. Alterations 

in clinical chemistry [decreased thyroxine and triiodothyronine levels] were observed at 

100 and 300 mg/kg/day at both intervals in one or both sexes. 

Changes in absolute and/or relative organ weights [adrenals, brain, thymus, heart, 

kidneys, testes with epididymides (males), ovaries (females), pituitary, liver (increased),

thyroids/parathyroids (increased) were observed primarily at the high-dose level [both 

sexes], and many of these changes may be attributable to decreased body weight. Gross 

findings, mainly in the high-dose group, included small testes and epididymis and opaque 

eyes [females]. Treatment-related histopathological changes were observed primarily in 

the high-dose group and included centrilobular hepatocellular hypertrophy [liver], 

bilateral retinal degeneration and cataract formation [females], atrophy of the testes 

[males], hypertrophy of the zona glomerulosa [adrenal cortex, both sexes] and follicular 

cells [thyroid, females], atrophy of the thymus [both sexes] and spleen [both sexes], 

congestion and edema of the bone marrow [both sexes], brush border loss in proximal 

tubular cells [kidney, both sexes]. Many of the lesions correlated well with the alterations 
2,4-D Human Health Risk Assessment DP No. D389455

Page 58 of 96

observed in hematology and clinical chemistry parameters and/or organ-weight data of 

the high-dose groups. 

The NOAEL is 15 mg/kg/day, based on decreased body weight/body-weight gain, 

alterations in some hematology [decreased platelets (both sexes)] and clinical 

chemistry [decreased T3 (females) and T4 (both sexes)] parameters, and cataract 

formation in females at the LOAEL of 100 mg/kg/day.

This subchronic oral toxicity study is classified Acceptable/Guideline, and the study 

satisfies the guideline requirement [OPPTS 870.3100; §82-1] for a subchronic oral 

toxicity study in the rodent.

870.3150 90-Day Oral Toxicity - Dog

(1) In a subchronic oral toxicity study [MRID 41737301], 5 beagle dogs/sex/group were 

administered 2,4-dichlorophenoxyacetic acid [96.1%] via capsules for 13 weeks at 

concentrations of 0, 0.3 mg/kg/day, 1.0 mg/kg/day, 3.0 mg/kg/day, and 10 mg/kg/day.

Two males [weeks 4, 13] and one female [week 9] at the high-dose level were sacrificed 

moribund. Treatment-related clinical signs [thin, languid appearance, anorexia, tremors] 

were observed at the high-dose level in the three dogs sacrificed in extremis. Decreased 

body weights were observed in males at the two highest dose levels [83% and 80% of 

control at 3 and 10 mg/kg/day, respectively] and in females at the high-dose level [86% 

of control] at week 13. Body-weight gains were decreased throughout the study in males 

at the two highest dose levels [weeks 0-13: 56% and 44%, respectively]. The mid- and 

high-dose females displayed decreased body-weight gains also [weeks 0-13: 84% and 

52% of control, respectively]. Decreased food consumption was observed in both sexes at 

the highest dose level and in males at the next highest dose level throughout the study. 

Decreased RBC, hemoglobin, hematocrit, and platelet count values were observed in both 

sexes at the highest dose level, although statistical significance was not always attained 

[n=4-5]. Treatment-related alterations in clinical chemistry parameters were observed 

mainly at the two-highest dose levels in both sexes at various times during the dosing 

period [increased blood urea nitrogen (both sexes), creatinine (both sexes), alanine 

aminotransferase (high-dose females; males not statistically significant); decreased T4 

(both sexes at high-dose; not statistically significant), and glucose (high dose, both 

sexes]. High-dose females displayed slightly increased kidney and thyroid weights 

[absolute, relative to body and brain weights] compared to the control group. Decreased 

testis weight was observed at the two-highest dose levels, and two of the 3 surviving 

high-dose males and one of the 2 high-dose males sacrificed moribund displayed 

hypospermatogenesis and giant cell formation. Microscopic lesions [cellular alterations, 

proximal tubule] were observed in the kidney of both sexes at the high-dose level [3 of 5 

males at 3 mg/kg/day, 3/3 surviving males at 10 mg/kg/day, 1 of 4 surviving females at 

10 mg/kg/day, and all dogs sacrificed moribund].
2,4-D Human Health Risk Assessment DP No. D389455

Page 59 of 96

The NOAEL is 1.0 mg/kg/day. The LOAEL of 3.0 mg/kg/day is based on decreased 

body weight/body-weight gain and food consumption (males), alterations in clinical 

chemistry parameters [increased BUN (both sexes), creatinine (both sexes)], and 

decreased testis weight (males).

This subchronic oral toxicity study is classified Acceptable/Guideline, and it satisfies the 

guideline requirement [OPPTS 870. 3150; §82-1(b)] for a subchronic non-rodent oral 

toxicity study.

(2) In a subchronic oral toxicity study [MRID 42780001], 4 beagle dogs/sex/group were 

administered 2,4-dichlorophenoxyacetic acid [96.7%] via the diet for 13 weeks at 

concentrations of 0, 0.5 mg/kg/day, 1.0 mg/kg/day, 3.75 mg/kg/day, and 7.5 mg/kg/day. 

The dose levels were selected based on the results of a 4-week range-finding study 

[MRID 42780004]in which decreased body weight, food consumption, alterations in 

clinical chemistry parameters, and distended gall bladders were observed at 10 

mg/kg/day.

There were no deaths, and no treatment-related clinical signs were observed. Decreased 

body weights were observed in males at the highest dose level [85% of control at week 

14] and in females at the lowest and highest dose levels [91% of control] at week 14. 

Body-weight gains were decreased throughout the study in both sexes at the two highest 

dose levels but there was no dose-response [overall gain: males 52% and 55% of the 

control value/females 53% and 58% of the control value at 3.75 and 7.5 mg/kg/day, 

respectively]. Females at the two lowest dose levels also displayed decreased bodyweight gains [overall gain: 74% and 79% of control at 0.5 and 1.0 mg/kg/day, 

respectively] compared to the control. Decreased food consumption was observed in 

males at the two highest dose levels and in all female groups compared to their respective 

controls, but there was no consistent dose response. 

Ophthalmology findings were comparable among the groups for both sexes. There were 

no apparent treatment-related alterations in hematology among the groups for either sex, 

with the exception of decreased platelet counts. Decreased platelet counts [73% and 87% 

of control] were observed in males at the high-dose level at both [week 4 and week 13, 

respectively] time points. Elevations in blood urea nitrogen [two highest dose levels, both 

sexes], creatinine [three highest dose levels, both sexes], and alanine aminotransferase 

[two highest dose levels, both sexes] and decreased glucose levels [two highest dose 

levels, females] were observed at week 4 and/or week 13; however, a dose response was 

not always apparent. Although no corroborative histopathological changes were observed 

in the liver or kidneys, similar changes in several of these clinical chemistry parameters 

have been observed in other studies on both 2,4-D and the 2,4-D salts and esters in rats 

and in an earlier subchronic toxicity study in dogs with 2,4-D [MRID 41737301]. In this 

latter study, microscopic lesions were observed in the kidneys of both sexes at 10 

mg/kg/day. 

Males at the two highest dose levels displayed a dose-related decrease in testes weight 

[absolute, relative to body, and relative to brain], although statistical significance was not 
2,4-D Human Health Risk Assessment DP No. D389455

Page 60 of 96

attained [n=4]. Decreased adrenal weight [absolute and relative to brain] was observed in 

both sexes at the highest dose level, decreased liver weight [absolute and relative to 

brain] was observed in both sexes at the highest dose level, and decreased heart weight 

[absolute and relative to brain] was observed in both sexes at the two highest dose levels, 

but there was no dose response. Microscopically, hypospermatogenesis/ juvenile testis 

was observed in 1 control, 1 mid-high [3.75 mg/kg/day], and 2 high-dose [7.5 mg/kg/day] 

males, and inactive, juvenile prostate was observed in one male in the control, 0.5 

mg/kg/day, 1 mg/kg/day, and 3.75 mg/kg/day dose groups and in all 4 high-dose males. 

The NOAEL is 1.0 mg/kg/day. The LOAEL of 3.75 mg/kg/day is based on decreased 

body-weight gain (both sexes) and food consumption (males), as well as alterations 

in clinical chemistry parameters [increased BUN, creatinine, and alanine 

aminotransferase] in both sexes, and decreased testes weight.

This subchronic oral toxicity study is classified Acceptable/Guideline, and it satisfies the 

guideline requirement [OPPTS 870. 3150; §82-1(b)] for a subchronic non-rodent oral 

toxicity study.

870.3200 21-Day Dermal Toxicity – Rabbit

In a 21-day dermal toxicity study [MRID 41735304], 5 Hra: (NZW) SPF 

rabbits/sex/group were administered the 2,4-dichlorophenoxyacetic acid [96.1%] via

dermal application [neat material (powder) spread evenly on 4" x 4" gauze pad and 

moistened with 0.5mL distilled water [6 hours/day, 7 days/week for 21 days at 

concentrations of 0 [distilled water], 10, 100, and 1000 mg/kg/day.

Treatment had no adverse effect on survival, clinical signs, mean body weight, 

hematology, clinical chemistry, urinalysis, gross pathology, or histopathology. At the 

high-dose level, increased kidney weights [absolute and relative] were observed in both 

sexes, but statistical significance was attained in the females only, and there were no 

corroborating changes in clinical chemistry parameters or histopathology in the kidneys. 

2,4-D was only slightly irritating to the skin of the rabbits, inducing very slight erythema 

and epidermal scaling, with the females displaying a higher incidence than males.

The NOAEL for dermal and systemic toxicity is 1000 mg/kg/day, the highest dose 

tested.

This 21-day dermal toxicity study is classified Acceptable/Guideline and satisfies the 

guideline requirement [§82-2; 870.3200] for a 21-day dermal toxicity study.

870.3465 28-Day Inhalation – Rat

In a subchronic inhalation toxicity study (MRID 47398701), 2,4-D (99% a.i., Lot # 2006 

24833 8006-USA) was administered to 10 or 20 Sprague-Dawley CD® 

rats/sex/concentration by dynamic [nose only] exposure at concentrations of 0, 0.05, 0.10, 
2,4-D Human Health Risk Assessment DP No. D389455

Page 61 of 96

0.30 or 1.00 mg/L for 6 hours per day, 5 days/week for 28 days (for a total of at least 20 

exposures).

Following nose-only inhalation exposure, 2, 4-D was associated with portal-of-entry 

effects that consisted of squamous metaplasia and epithelial hyperplasia with increased 

mixed inflammatory cells within the larynx. The incidence and severity of the effects 

were increased in a dose-related manner, and the effects persisted following the 4-week 

recovery period, although the incidence and severity were reduced. Clinical signs 

associated with exposure at the high dose included excessive salivation (day 13 and 

subsequently), labored breathing (day 13 and subsequently), and chromodacryorrhea (day 

12 and intermittently thereafter). A slight decrease in body weight was observed in the 

high-dose females by day 14 and continued throughout the remainder of the dosing 

(↓10%) and the recovery periods (↓12%). Body-weight gains were reduced in the highdose female group throughout the study and recovery period and were accompanied by a 

reduction in food intake. Although a treatment-related reduction in reticulocyte counts 

was observed at the mid-high and high-dose levels in both sexes at terminal sacrifice, the 

toxicological significance is not known. Alkaline phosphatase values were increased in 

the mid-high and high-dose females and aspartate aminotransferase values were increased 

slightly in the high-dose females, but no correlating microscopic pathology findings were 

observed. Lung weights were unaffected by treatment. Females at the high-dose level 

displayed slight reductions in spleen and thymus weights. Organ weights were 

comparable among the male groups. 

The systemic toxicity LOAEL is 1.0 mg/L/day, based on excessive salivation, labored 

breathing, and chromodacryorrhea, decreased body weight in females and 

decreased spleen weights in females. The NOAEL is 0.30 mg/L/day.

A NOAEL for portal-of-entry effects was not determined. The LOAEL for portalof-entry effects (squamous metaplasia and epithelial hyperplasia with increased mixed 

inflammatory cells within the larynx; not totally resolved following a 4-week recovery 

period) is 0.05 mg/L, the lowest dose tested. 

This subchronic inhalation toxicity study in the rat is Acceptable/guideline, and it 

satisfies the guideline requirement for a subchronic inhalation study (OPPTS 870.3465; 

OECD 413) in the rat. 

A.5.2 Prenatal Developmental Toxicity

870.3700a Prenatal Developmental Toxicity Study - Rat

In a developmental toxicity study [MRID 00130407], pregnant Fischer 344 rats 

[35/group] were administered 2,4-dichlorophenoxyacetic acid [97.5%] via gavage at dose 

levels of 0 [corn oil], 8 mg/kg/day, 25 mg/kg/day, and 75 mg/kg/day from gestation day 

[GD] 6 through gestation day 15.
2,4-D Human Health Risk Assessment DP No. D389455

Page 62 of 96

There were no treatment-related deaths. Two (one control and one low-dose) dams 

delivered prematurely on gestation day 19 and, in both instances, the offspring produced 

were of similar size and development as those from full-term delivery. Clinical signs 

were comparable among the groups. Body weights were comparable among the groups 

throughout the study, but dams at the high-dose level displayed a decrease in bodyweight gain during the dosing period [79% of control for GD 6-15; 57% of control for 

GD 6-10], although statistical significance was not attained. The corrected body-weight 

gain was comparable among the groups. Food consumption data were not reported.

There was a slight decrease in pregnancy rate with increasing dose [85%, 85%, 80% and 

77%]. The numbers of corpora lutea, implantations, and live fetuses were comparable 

among the groups, and there were no dead fetuses. The numbers of resorptions, as well as 

pre- and post-implantation losses, were not adversely affected by treatment. The number 

of the dams with 100% resorptions was 2, 0, 1, and 1 [control, low-, mid-, and high-dose 

groups, respectively]. One control, 2 low-, 4 mid-, and 2 high-dose dams had late 

resorptions. Fetal body weight and crown-rump length were comparable among the 

groups, as was the sex ratio. 

There were no statistically-significant or treatment-related differences in the incidence of 

fetal external or visceral malformations. There was an increased incidence of skeletal 

malformations/variations at the high-dose level that included the presence of 7th cervical 

ribs [4 fetuses in 3 litters vs none in the control], presence of 14th rudimentary ribs [4 

fetuses in 3 litters vs none in the control]; malaligned sternebrae [15 fetuses in 10 litters 

vs 7 fetuses in 7 litters of the control]; reduced ossification of the vertebral arches [6 

fetuses in 5 litters vs2 fetuses in 1 litter of the control]; and unossified sternebrae # 5 or 

#6 [73 fetuses in 22 litters (3.32 fetuses/litter) vs 62 fetuses in 24 litters (2.58 

fetuses/litter)]. Although none of the increases attained statistical significance, they were 

attributed to treatment since some of the findings [maligned sternebrae, 14th rudimentary 

ribs, and reduced ossification of vertebral arches] were also observed in the F1b pups of 

dams fed 2,4-D at 80 mg/kg/day [actual dose 75 mg/kg/day] in the 2-generation 

reproduction study in the same strain of rat. Additionally, skeletal findings [2nd wavy ribs, 

lumbar ribs] and missing sternebrae were observed in another developmental toxicity 

study using a different strain of rat [Sprague-Dawley] at a comparable dose of 87.5 

mg/kg/day [2,4-D]. 

The maternal toxicity NOAEL is 25 mg/kg/day, and the maternal toxicity LOAEL is 

75 mg/kg/day, based on decreased body-weight gain. 

The NOAEL for developmental toxicity is 25 mg/kg/day, and the developmental 

toxicity LOAEL is 75 mg/kg/day, based on increased incidence of skeletal 

abnormalities. 

This developmental toxicity study is classified Acceptable/guideline, and it satisfies the 

guideline requirement [OPPTS 870.3700; §83-3(a)] for a developmental toxicity study in 

the rodent. NOTE: In 1996, it was determined that the original DER [Document No. 

003887] was inadequate, and the study was re-evaluated [Document No. 011934]. The 
2,4-D Human Health Risk Assessment DP No. D389455

Page 63 of 96

NOEL/LOEL were the same in both reviews. The current EXECUTIVE SUMMARY 

does not alter the no-effect and effect levels but reflects current terminology for the noobserved-adverse-effect level [NOAEL] and lowest-observed-adverse-effect level 

[LOAEL]. Additionally, the presence of 7th cervical ribs and 14th rudimentary ribs are 

considered skeletal malformations; previously listed as skeletal variations.

870.3700b Prenatal Developmental Toxicity Study - Rabbit

In a developmental toxicity study [MRID 41747601], artificially-inseminated female 

New Zealand White rabbits [20/group] were administered 2,4-Dichlorophenoxyacetic 

acid [96.1%] at dose levels of 0 [aqueous 0.5% methylcellulose], 10 mg/kg/day, 30 

mg/kg/day, and 90 mg/kg/day from gestation day 6 through gestation day 18. NOTE: All 

dose concentrations were corrected for the 96.1% purity of the test material.

There were no treatment-related deaths. Two does at 90 mg/kg/day aborted [days 21 and 

24]. Treatment-related clinical signs of toxicity were observed in the two does that 

aborted [days 21 and 24, after 13 doses each] and included ataxia in both [days 16-19 and 

after day 13], and decreased motor activity, loss of righting reflex, extremities that were 

cold to the touch, and dried feces in doe that aborted on day 21. Body weights were 

comparable among the groups throughout the study, but body-weight gains were 

decreased at the high-dose level [↓27%] during the dosing period [days 6-19; statistical 

significance was not attained]. During days 7-8, the low- and mid-dose groups showed no 

body-weight gain, and the high-dose group displayed a negative body-weight gain [-0.01 

grams] compared to the control [+0.01 gram]. During days 15-19, the high-dose group 

displayed no body-weight gain, and corrected body-weight gain was decreased at the 

high-dose level [↓23%; statistical significance was not attained] also. Food consumption 

was comparable among the groups. 

Pregnancy rates were comparable among the groups. Comparable numbers of corpora 

lutea, implantations, and live fetuses were observed among the groups, and there were no 

dead fetuses. One control doe had 100% resorptions. The number of resorption, pre- and 

post-implantation losses, and gravid uterine weights were comparable among the groups. 

Mean fetal body weight was comparable among the groups. At the high-dose level, there 

was a significant increase in the percent of live male fetuses [71.2%] compared to the 

control [52.8%] and other dose groups [low: 54.4%; mid: 59.4%]. At the high-dose level, 

the fetal incidence [3 fetuses of one litter; p<0.01] of hindlimbs turned inward was 

increased compared to the control (0) and other treatment groups (0), and the same 

fetuses displayed domed head [hydrocephaly]. This finding is not considered treatmentrelated. There were no apparent differences in the incidence of external, visceral, or 

skeletal variations, anomalies, retardations, or malformations among the groups.

The maternal toxicity NOAEL is 30 mg/kg/day, based on abortions, decreased bodyweight gain, and clinical signs of toxicity [decreased motor activity, ataxia, loss of 

righting reflex, extremities cold to the touch] at the maternal toxicity LOAEL of 90 

mg/kg/day. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 64 of 96

The developmental toxicity NOAEL is 30 mg/kg/day, based on abortions at the 

developmental toxicity LOAEL of 90 mg/kg/day.

This developmental toxicity study is classified Acceptable/Guideline, and it satisfies the 

guideline requirement [§83-3(b)/OPPTS 870.3700] for a developmental toxicity study in 

rabbits.

A.5.3 Reproductive Toxicity

870.3800 Reproduction and Fertility Effects - Rat

(1) In an extended dietary one-generation reproductive toxicity study (MRID 47972101), 

2,4-dichloro phenoxyacetic acid (2,4-D; 97.85%-98.6% a.i.; lot # 2006 2433 8006-USA)

was administered to 27 Crl:CD(SD) young adult rats/sex/dose via the diet at dose levels 

of 0, 100, 300, or 600 (females)/800 (males) ppm [equivalent to 0, ≈5, 15, or 30 

(females)/40 (males) mg/kg/day] for approximately four weeks prior to mating and 

continuing through mating (up to 2 weeks), gestation, and lactation. P1 males were 

exposed for a minimum of 11 weeks including 7 weeks from the initiation of the mating 

phase. P1 females were exposed until lactation day 22 (LD22). A satellite group of P1 

females (12/dose) were subject to the same exposures as the P1 females on the main 

study (exposure for 4 weeks during the pre-mating, up to 2 weeks during the mating 

period and during gestation until termination on gestation day 17 (GD 17). Satellite males 

were not exposed to dietary 2, 4-D except during co-housing with satellite females during

the mating period. 

P1 Generation: A comprehensive evaluation of P1 male and P1 female reproductive 

system was conducted, including an evaluation of gonadal function, the estrous cycle, 

sperm parameters, mating performance, conception, gestation, parturition and lactation, 

as well as survival, growth and development of the offspring. Selected systemic toxicity 

parameters were also evaluated in the P1 males and P1 females. 

Satellite GD 17 Females: A satellite group of P1 females (12/dose) was included for

assessments of selected systemic toxicity parameters, clinical chemistry/hematology, 

thyroid hormone levels, thyroid weights, plasma 2, 4-D levels, histopathology, and 

selected reproductive parameters during gestation (corpora lutea and implantation 

numbers). 

F1 Generation: F1 offspring were evaluated for potential effects on the nervous system, 

immune system, reproductive and endocrine systems, thyroid function, and other 

systemic toxicity parameters. 2, 4-D plasma levels were also assessed in the F1 offspring.

In-life parameters in all F1 offspring included clinical observations, body weights, feed 

consumption, anogenital distance, nipple retention and puberty onset. Selected F1 

offspring were divided into three different groups (Sets 1, 2, and 3) at weaning (postnatal 

day 21; PND 21). Each set of F1 offspring was maintained on the test diet until PND 60 

(Set 1b F1 offspring), ≈PND 70 (Sets 1a and 2a F1 offspring), or ≈PND 90-139 (Sets 2b 

and 3 F1 offspring). 
2,4-D Human Health Risk Assessment DP No. D389455

Page 65 of 96

Set 1a (10/sex/dose): assessment of general systemic and thyroid toxicity, which included 

clinical chemistry/hematology parameters, thyroid hormone assessment, and urinalysis 

(males only). Post-mortem evaluations in Set 1a (PND70) included gross pathology, 

organ weights and histopathology on a wide range of tissues, including thyroids.

Set 1b (10/sex/dose): developmental neurotoxicity (DNT) assessment, which included 

functional observational battery (FOB), motor activity and acoustic startle response 

(ASR). On PND 60, Set 1b animals were perfused for central nervous system (CNS) and 

peripheral nerve neuropathology evaluation and brain morphometry. A special stain 

(Luxol Fast Blue) was used to evaluate brain myelination. 

Set 2a (10sex/dose): assessment of potential developmental immunotoxicity (DIT): 

examination of humoral immune function using the sheep red blood cell (SRBC) 

antibody-forming cell (AFC) assay on PND 70-74.

Set 2b (10/sex/dose): assessment of potential developmental immunotoxicity (DIT): 

examination of innate cellular immunity using the natural killer cell (NK) assay on PND 

87-93.

Set 3 (23-27/sex/dose): assessment of reproductive/endocrine toxicity, which included 

estrous cycle evaluation and post-mortem evaluations that focused on reproductive 

organs, sperm assessment, and ovarian follicle counts on PND 139. TK analyses were 

conducted on Set 3 males and females on PND 63 and 84 to determine plasma 2, 4-D 

levels.

In addition, selected pups culled on PND 4 were used to assess thyroid hormone levels. 

Additional data were gathered from F1 offspring not assigned to Sets 1-3. On PND 22, 

unselected weanlings were either perfused for examination of neuropathology 

(12/sex/dose) or euthanized for assessment of systemic toxicity, which included thyroid 

hormone assessment, organ weights, and post-mortem examinations (gross pathology and 

histopathology) in 10/sex/dose. 

Reproductive and selected data from the F1 generation were used to assess whether a 

second generation would be produced. None of the criteria were met (Table 1), and a 

second generation was not assessed in this study. 

P1 Adult Rats: There were no treatment-related deaths or clinical signs of toxicity in 

either sex of P1 adults. Body weights and body-weight gains were comparable among the 

groups during the pre-mating and mating phases (both sexes) and during gestation and 

the latter part of lactation (dams). Prior to dietary adjustment of 2, 4-D concentration 

during the second week of lactation, the 600 ppm dams displayed a decrease in body 

weight (LD 7; ↓5%) and body-weight gain (LD 1-4; ↓64%), which is consistent with 

reduced food intake during the first week of lactation. The reduction in food intake can be 

attributed to the increase in the actual dose (≈65 mg/kg/day) above the targeted level (30 

mg/kg/day) during this time. After dietary adjustment, food intake for the 600 ppm dams 

was above control levels. 

There were no apparent treatment-related effects on hematology, differential white blood 

cell counts, and prothrombin time, and clinical chemistry and urinalysis parameters were 

comparable among the groups (both sexes). P1 males displayed increased kidney weights 
2,4-D Human Health Risk Assessment DP No. D389455

Page 66 of 96

(absolute and relative) at 800 ppm, which were accompanied by histopathological 

findings (degenerative lesion in the proximal convoluted tubules in the outer zone of the 

medulla) and are consistent with previous findings that the kidney is a target organ. There 

were no treatment-related findings in the P1 female kidney. Decreased reproductive and 

accessory sex gland weights were observed at 300 ppm and/or 800 ppm. These changes, 

however, are related to the concurrent control being outside of the laboratory historical 

control range. P1 females at 600 ppm displayed increased uterine weights (↑17%, both 

absolute and relative), although statistical significance was not attained. There were no 

alterations in estrous cycle pattern in the 600 ppm P1 females compared to the control, 

and no significant difference in mean estrous cycle length in P1 females at any dose level 

compared to the control. There were no significant, treatment-related effects on sperm 

motility or progressive motility, no differences in testicular spermatid and epididymal 

sperm counts, and no differences in the proportion of abnormal sperm. Male and female 

mating, conception, fertility, and gestation indices were comparable among the groups, 

and post-implantation loss was comparable among the groups. Both the time to mating 

and gestation length were comparable among the groups. 

GD 17 Satellite Females: All P1 satellite females survived to scheduled sacrifice, and 

body weights were comparable among the groups. Hematology and clinical chemistry 

parameters were comparable among the groups. Reproductive indices and the numbers of 

corpora lutea and implantations were comparable among the groups. There was a slight 

increase in resorptions at 600 ppm (0.9 vs 1.5), although there was wide variability 

(standard deviations exceed the means). There was a slight increase in post-implantation 

loss at 600 ppm (9.2 vs 5.5). It should be noted that this observation was not corroborated 

since post-implantation losses in the P1 adults of the definitive study were comparable 

amongst all dose groups. Both the 100 ppm and 600 ppm females displayed an increase 

in thyroid weight (↑9%), but there was no dose-response. There were no statistically 

significant, treatment-related differences in serum T3, T4, or TSH in the GD 17 satellite 

females. Although the 600 ppm GD 17satellite females displayed the predicted pattern of 

thyroid hormone changes (↓ T3 and ↓ T4 with ↑TSH levels) that suggest 2, 4-D exposure 

may adversely affect thyroid function at doses above the renal saturation clearance, the 

thyroid effects noted below renal saturation are not considered sufficiently robust to be 

adverse. 

F1 Offspring: There were no treatment-related effects on the numbers of live or dead F1 

pups born/litter or on pup survival or sex ratio. Slightly lower body weights were 

observed in the 600 ppm pups during early lactation, which coincided with the dams 

decreased food intake LD 1-4 and LD 4-7). Pup body weight (600 ppm) remained lower 

in the 600 ppm pups (↓6%) during PND 14-21. There was no significant, treatmentrelated difference in absolute or relative anogenital distance in either sex and no 

differences in nipple/areolae retention between control and high-dose groups in either 

sex. F1 males at 800 ppm displayed a 1.6 days delay in preputial separation (well within 

normal variability), which was accompanied by a very slight reduction in body weight 

compared to the control (↓2.1 grams; 99% of control). The age at vaginal opening was 

comparable among the groups of F1 females.
2,4-D Human Health Risk Assessment DP No. D389455

Page 67 of 96

F1 Offspring Thyroid Assessments: PND 4 - There were no statistically-significant 

differences in serum T3, T4, or TSH in PND 4 culled pups. T4 was reduced to a similar 

extent in both sexes at the 300 ppm (↓14%-15%) and 600 ppm/800 ppm (↓12%-14%) 

dose levels, and female PND 4 pups showed an increase in TSH (↑19%) at 600 ppm. F1 

PND 22 Weanlings - F1 PND 22 males displayed a statistically-significant reduction 

(↓28%) in T4 at 800 ppm, and F1 PND 22 females displayed a non-statistically 

significant reduction (↓20%) in T4 at 600 ppm. T3 was reduced in the males at 300 ppm 

(↓19%) and 800 ppm (↓13%), but there was no dose response. F1 PND 62-64 - Both 

sexes displayed increased TSH at 300 ppm (↑26%) and at 800 ppm (males ↑23%))/600 

ppm (females ↑24%)), although the increase in males was not dose-related and none of 

the differences in thyroid hormone levels were statistically significant. T4 was decreased 

at 800 ppm in males (↓13%). Though these findings suggest that 2, 4-D exposure may 

adversely affect thyroid function at doses above the renal saturation clearance, the thyroid 

effects noted below renal saturation are not considered sufficiently robust to be adverse.

F1 Unselected Offspring (PND 22 weanlings): There were no effects on survival of the 

unselected weanlings used for systemic toxicity (non-perfused). All treated males 

displayed a decrease in body weight (↓9%-10%) compared to the control males. 

Decreased adrenal weights were observed in males at 800 ppm (absolute ↓37% and 

relative ↓29%). The decreases in kidney (↓15%), liver (↓18%), testes (↓15%), and thyroid 

(↓14%) weights observed in males at 800 ppm were slightly greater than the body-weight 

deficit of 10%. Organ weights were comparable among the groups of females. There 

were no significant differences in perfused absolute brain weights, cerebral lengths and 

widths or cerebellar lengths and widths in perfused F1 PND 22 weanlings of either sex. 

There were no neuropathological observations attributed to treatment in the perfused F1 

PND 22 weanlings, and no treatment-related changes in myelin in either males at 800 

ppm or females at 600 ppm.

F1 Offspring Set 1a (PND 70): All Set 1a pups survived to scheduled sacrifice. Males at 

800 ppm displayed decreased body weight (↓11%-17%) and body-weight gains (↓11%-

25%) throughout the study period, with the magnitude of the reduction lessening with 

time of exposure. Females displayed comparable body weight/gain among the groups. 

Platelet counts were reduced in the 800 ppm males but not in the females at any dose 

level. Both sexes displayed a slight increase in ALT (↑18%/25%) and an increase in 

triglyceride (↑31%/43%) levels. Although some of the decreases in organ weights 

observed in the 800 ppm males may be attributed to the 10% decrease in body weight at 

termination, the decreases in liver (↓16%), pituitary (↓14%), and adrenal glands (↓12%) 

might be related to treatment. Increased uterine weights (↑31% absolute and ↑32% 

relative) were observed at 600 ppm. Although statistical significance was not attained, the 

finding is considered treatment-related since a similar increase was observed at 600 ppm 

in the P1 and Set 3 F1 females. Increased ovarian weight (↑9%) was observed in the 600 

ppm F1 Set 1a females, although statistical significance was not attained. Increased 

kidney weights (↑9% absolute and ↑11% relative) were observed in the females at 300 

ppm and 600 ppm, although there was no dose-response and kidney weights were 

comparable among the male groups. Decreased thymus weights (↓12% absolute and 

↓10% relative) were observed in females at 600 ppm and in Set 3 females at 600 ppm 
2,4-D Human Health Risk Assessment DP No. D389455

Page 68 of 96

(↓14% absolute and ↓13% relative). An increased incidence of degeneration of the 

proximal convoluted tubule in the kidney was observed in males at 300 ppm and 800 

ppm and in females at 600 ppm. Regarding the terminal stage of estrous, 2 of 10 females 

at 300 ppm and 3 of 10 females at 600 ppm displayed proestrus, whereas none of the 10 

females in the control and 100 ppm groups displayed proestrus.

F1 Offspring Set 1b (PND 54-56): There were no significant differences in body 

weight/gain in either sex. There was an increase in the level of urination in all treated 

male groups compared to the control group, but there was no dose response. There was a 

10% reduction in hind limb grip strength at 800 ppm in males and at 600 ppm in females. 

Males at 800 ppm displayed a decrease in total motor activity (↓10%), whereas females at 

600 ppm showed an increase (↑12%). During the first half of the session, all male groups 

displayed a similar motor activity level (were within 6%), whereas the 800 ppm males 

showed a progressive lessening of activity with increased time; i. e., the 800 ppm males 

displayed decreased activity compared to the control (↓11%, ↓16%, ↓30%, and ↓34% in 

Epochs 5, 6, 7, and 8, respectively). Males at 800 ppm displayed a different acoustic 

startle response (ASR) initially compared to the control males. There was no apparent 

difference in ASR in females. There were no significant differences in perfused absolute 

brain weights, cerebral lengths and widths, or cerebellar lengths and widths in either sex 

(PND 60). There were no treatment-related (1) microscopic changes in the central or 

peripheral nervous system in the perfused offspring; (2) changes in myelin; or (3) 

changes in microscopic measurements of structures in the cerebral cortex, cerebellum, 

thalamus, or hippocampus.

F1 Offspring Set 2a (PND 67-73): Developmental Immunotoxicity (Primary Immune 

Response to Sheep Red Blood Cells): There were no deaths. Slight decreases in body 

weights and body-weight gains were observed in males at 800 ppm (↓6%-10% and 

↓15%) and females at 600 ppm (↓8%-9% and ↓10%). Terminal body weights were 

comparable among the male and female groups. Both absolute (↓10%) and relative (↓8%) 

thymus weight decreases were observed in the males at 800 ppm and in the females at 

600 ppm [absolute (↓13%) and relative (↓10%)]. Males at 300 ppm showed a 17% 

decrease in thymus weight but no dose response. Spleen weights were slightly lower in 

females at 600 ppm [absolute (↓13%) and relative (↓14%)]. There was no significant 

difference in response for AFC/spleen and AFC/106 splenocytes among the male groups. 

Females at 600 ppm displayed a non-significant decrease of 54% for AFC/spleen and 

27% for the AFC/106 splenocytes. 

F1 Offspring Set 2b (PND 67-73): Developmental Immunotoxicity (Natural Killer 

Cell Activity): There were no deaths, and body weights/gains showed a similar slight 

reduction in males at 800 ppm as observed in the other offspring groups. Female body 

weights/gains were comparable among the groups. Terminal body weights (PND 87-93) 

were comparable among the groups (both sexes). There were no significant treatmentrelated effects on absolute or relative spleen or testes weights in males, and no significant 

treatment-related effects on spleen weights in females (only organs weighed). There were 

no significant, treatment-related differences in the percent target cell cytotoxicity at any 
2,4-D Human Health Risk Assessment DP No. D389455

Page 69 of 96

dose level compared to control (both sexes), and 2, 4-D did not alter the cytotoxic ability 

of splenic NK-cells in male or female rats at any dose level. 

F1 Offspring Set 3 (PND 90 or 139): Reproductive Toxicity: There were no treatmentrelated deaths or clinical signs of toxicity. Terminal body weights were comparable 

among the groups (both sexes). No significant differences were observed in mean estrous 

cycle length at any dose level compared to the control. There were no significant, 

treatment-related effects on the numbers of small follicles, growing follicles, or total 

follicles. There were no significant, treatment-related effects on sperm motility or 

progressive motility, no differences in testicular spermatid and epididymal sperm counts, 

and no differences in the proportion of abnormal sperm between the control and 800 ppm 

males. Absolute (↓9%) and relative (↓8%) pituitary gland weights were significantly 

lower in the 800 ppm males and absolute (↓9%) and relative (↓10%) pituitary gland 

weights were non-significantly lower in the 600 ppm females. There was no associated 

histopathology in the pituitary glands. Uterine weights were increased at 300 ppm (↑10% 

absolute and ↑10% relative) and 600 ppm (↑10% absolute and ↑11% relative) compared 

to the controls. Thymus weights were decreased (↓14% absolute and ↓13% relative) in 

females at 600 ppm, although statistical significance was not attained. No 

histopathological changes were observed in the pituitary or thymus in either sex. A 

degenerative lesion was observed in the kidney (proximal convoluted tubule) in both 

sexes at 300 ppm and at 600 ppm/800 ppm. Ovarian follicle counts were comparable 

between the control and 600 ppm females (PND 139). 

The parental systemic LOAEL is 800 ppm (45.3 mg/kg/day in males), based on 

nephrotoxicity manifested as increased kidney weights, and degenerative lesions in 

the proximal convoluted tubules in the main study P1 rats. The parental systemic 

NOAEL is 300 ppm (16.6 mg/kg/day in males). No toxicologically relevant effects 

were identified in P1 females or in the GD 17 satellite female groups at the highest 

dose tested (600 ppm; 40.2 mg/kg/day).

The thyroid toxicity NOAEL is established at 800/600 ppm (45.3 mg/kg/day in males 

and /40.2 mg/kg/day in females), the highest dose tested. The thyroid effects noted 

in the database were considered to be adaptive. 

The offspring (F1 adults) LOAEL is 800/600 ppm (55.6 mg/kg/day in males and 46.7 

mg/kg/day in females), based on kidney toxicity manifested as increased kidney 

weights and increased incidence of degeneration of the proximal convoluted tubules. 

The offspring NOAEL is 300 ppm (20.9/ mg/kg/day in males and 23.3 mg/kg/day in 

females). 

The F1 offspring (PND 22) LOAEL is 800/600 ppm, based on decreased body weight 

observed throughout lactation. The offspring NOAEL is 300 ppm. The dose on a 

mg/kg/day basis for the PND 22 F1 offspring was not calculated. 

The DNT offspring (PND 21-60) LOAEL is >800/600 ppm (81.7 mg/kg/day in males, 

59.2 mg/kg/day in females), based on the lack of evidence of DNT (FOB parameters, 
2,4-D Human Health Risk Assessment DP No. D389455

Page 70 of 96

motor activity, and acoustic startle response). The DNT offspring NOAEL is 800 

ppm/600 ppm (81.7 mg/kg/day in males, 59.2 mg/kg/day in females). 

The DIT offspring (PND 139) LOAEL is >800/600 ppm (71.8 mg/kg/day in males, 

55.3 mg/kg/day in females), based on the lack of evidence of DIT [SRBC antibodyforming cell assay (PND 66-70) and Natural Killer Cell assay (PND 87-93)]. The 

DIT offspring NOAEL is 800/600 ppm (71.8 mg/kg/day in males and 55.3 mg/kg/day 

in females), the highest dose tested. 

The reproductive LOAEL is > 800/600 ppm (45.3 mg/kg bw/day in males, 40.2 mg/kg 

bw/day in females), based on the lack of effect on estrous cyclicity, (P1 females, satellite 

GD 17 dams, Set 3 F1 offspring) or reproductive indices (mating, fertility, time to 

mating, gestation length, pre-and post-implantation loss, number of corpora lutea 

(satellite GD 17 dams), sperm parameters, ovarian follicle counts, and reproductive organ 

histopathology). The reproductive NOAEL is 800/600 ppm (45.3 mg/kg bw/day in 

males, 40.2 mg/kg bw/day in females), the highest dose tested. 

This study is classified Acceptable/non-guideline. The study does not satisfy a guideline 

requirement for 2, 4-D. It satisfies the data call-in requirements for 2, 4-D for OCSPP 

870.3800 (Reproduction and Fertility Effects), OCSPP 870.6300 (Developmental 

Neurotoxicity), OCSPP 870.7800 (Immunotoxicity). The study is in accordance with the 

OECD extended one-generation reproductive toxicity study guideline (OECD 443, 416; 

November, 2010). 

COMMENT: The major change is a change in the offspring (F1 adults) LOAEL from 

300 ppm to 600 ppm, making the new NOAEL 300 ppm. Both the PMRA and EPA do 

not consider the kidney effects occurring at PND 70 and PND 139 to be adverse at the 

300 ppm dose level in either sex. Although there was some degeneration in the males at 

300 ppm on PND 70, this did not correlate with increased kidney weight and was not 

really different from controls on PND 139. One would expect the degeneration to be 

worse after a prolonged exposure. There is clear correlation in both sexes at 800/600 

ppm and at both time points. 

A few typos were identified subsequent to the finalization of the DER, which include: 

(1) in the paragraph for P1 adult rats on page 3, it should have indicated that no 

alterations were observed in estrous cycle pattern in the P1 females rather than F1 

females; (2) page 50, first line should read from PND 21-69 (not PND 21-56). 

(2) In a 2-generation reproduction study (MRID 00150557 and MRID 00163996), 30 F0 

Fischer 344 rats/sex/dose were administered 2,4-D (97.5% a.i.) via the diet for 105 days 

prior to mating and through gestation and lactation of two litters and for 30 days after 

weaning of the last litter at target dose levels of 0, 5, 20, and 80 mg/kg/day. Rats were 

mated, one male with one female. The resulting F1a litters were weaned at day 28 post 

partum. After a two-week rest period, the Fo parental rats were re-bred using different 

male/female combinations to produce the F1b litters, from which 30 males/30 

females/group were selected to become the F1 parents. The F1 generation (30 
2,4-D Human Health Risk Assessment DP No. D389455

Page 71 of 96

rats/sex/group) was administered the test material at target dose levels of 0, 5, or 20 

mg/kg/day [high dose level dropped due to excess toxicity; there were an insufficient 

number of F1b pups] in utero and continuously via the milk or feed for 126 days 

postnatally and prior to mating and through gestation and lactation to two litters (F2a and 

F2b) and for 30 days after weaning the last litter.

There were no apparent treatment-related deaths, and clinical signs were comparable 

among the groups throughout the study. During the pre-mating dosing period, body 

weights of the F0 parental animals were slightly lower (males ↓3%-5% by week 6; 

females ↓4%-5% by week 13)) at the high-dose levels for both sexes. Body weight gains 

of the F0 high-dose males were decreased initially (weeks 2-3; ↓14%) and overall (weeks 

0-13 and weeks 0-40; ↓7%), as were these of the high-dose females [weeks 0-1 (↓21%); 

weeks 0-13 (↓8%); weeks 0-40 (↓6%). 

The high-dose F0 dams displayed a significantly lower body weight throughout gestation 

[F1a litter (↓5%-6%)] and by gestation day 20 during F1a pregnancy (↓10%). The highdose F0 dams displayed a significant reduction in body weight gains during both 

gestation periods, with the greater deficit being observed during the second gestation 

period [F1a liters: days 0-7 (↓33%*); days 13-20 ↓5%); days 0-20 (↓13%); F1b litters: 

days 0-7 (↓30%*); days 13-20 (↓41%**); days 0-20 (↓33%**)]. The high-dose F0 dams 

displayed decreased body weight on day 7 of lactation (both litters: ↓7%-8%), but body 

weights were significantly increased compared to the controls at day 28 of lactation 

(F1a↑8%/F1b ↑11%). Body weight gains were significantly reduced during lactation days 

1-7 for both litters (F1a ↓60%; F1b ↓94%). Overall, however, the high-dose dams 

displayed positive body weight gains during lactation days 1-28 compared to negative 

body weight gains in the control and other treatment groups.

Food consumption (g/rat/day) during the pre-mating period was slightly lower (↓5%-6%) 

in the high-dose females during a few weeks, but on a g/kg/day basis, both sexes at the 

high dose displayed a slight increase (↑4%) in food consumption compared to the control. 

During the first week of the 2-week rest period following the weaning of the first letter, 

the F0 dams displayed a significant decrease in food consumption (↑16%-17%). Food 

consumption was decreased at the high-dose level during both gestation periods (F1a 

during first 2 weeks (↑7%-9%); F1b during the third week (↑18%). A significant decrease 

in food consumption was observed throughout lactation (both litters) at the high dose 

(F1a litter (↓42%); F1b litter (↓17%-29%). At necropsy, no treatment-related adverse 

effects were observed at any dose level, although the F0 females displayed increased 

kidney weights at all dose levels, but there was no dose response. 

There were no apparent, treatment-related, adverse effects on body weights or body 

weight gains of the F1 parental animals during the pre-mating dosing period at the two 

remaining dose levels, although the mid-dose males (20 mg/kg/day) displayed an initial 

decrease in body weight gain [weeks 35-36 (↓9%**) and weeks 36-37 (↓11%**)]. At 20 

mg/kg/day, there were no significant differences in body weights in the F1 dams during 

gestation (F2a litters ↓1%-5%; F2b litters ↓4%-5%) or body weight gains [F2a litters 

↓15% (days 7-13); F2b litters ↓17% (days 0-7); ↓14% (days 13-20); ↓10% (days 0-20)], 
2,4-D Human Health Risk Assessment DP No. D389455

Page 72 of 96

and comparable body weights/gains were observed during lactation (both litters). Food 

consumption was comparable among the groups (both sexes) throughout the study. At 

necropsy, no treatment-related adverse effects were observed at either dose level, 

although the F1 males and females displayed slightly increased kidney weights at the 20 

mg/kg/day dose level, and the females at this dose displayed a slight increase in liver 

weight. 

F0 Generation: No apparent adverse effect was observed on fertility. Pre-coital intervals 

were comparable among the groups. The duration of gestation was significantly increased 

in the high-dose (80 mg/kg/day) F0 females producing the F1b pups (22.5 days vs 21.9 

days), although the delay was less than a day. The gestation survival index was 

comparable among the groups for the F1a pups but significantly decreased for the F1b 

litters (31.7% vs 97.8%). There was a significant decrease in the number of female 

fetuses at the high dose (39% vs 54%). The number of F1b pups born dead/dying by day 

1 (110) was significantly increased at the high-dose level compared to the control (5). 

F1a litter size was slightly lower at the high-dose level compared to the control (9.0 vs 

10.1), but F1b litter size was significantly lower than the control (5.1** vs 9.5). The F1a 

pup viability was comparable throughout weaning, but F1b pup viability was 

significantly lower throughout the weaning period. There was a significant decrease in 

F1b pup survival to lactation day 4 at the high-dose level (86%) compared to the control 

(100%) and other dose groups (≥98%), as well as survival to lactation day 28 (71% vs 

99%-100% in control and other dose groups). Decreased pup body weight (F1a males 

↓11%/F1a females ↓10% on day 1; F1a males ↓25%/F1a females ↓19% on day 28/F1b 

males ↓22%/F1b females ↓15% on day 1; F1b males ↓27%/F1b females ↓24% on day 28) 

and decreased body weight gains (F1a males ↓32%/F1a females ↓30% on days 1-4; F1a 

males ↓25%/F1a females ↓20% on days 4-28/F1b males ↓74%/F1b females ↓57% on 

days 1-4; F1b males ↓24%/F1b females ↓22% on days 4-28) were observed at the high 

dose level, with the F1b litters displaying the greater effect. At the mid-dose level, there 

was a slight decrease in body weight (F1a males ↓7%/females ↓6%); F1b males 

↓16%/females ↓13% on day 28) and body weight gains (F1a males ↓8%/females ↓7%; 

F1b males ↓17%/females ↓15% during days 4-28), with the F1b litters displaying the 

greater effect.

Skeletal anomalies and reduced ossification were observed in the high-dose F1b pups (80 

mg/kg/day) that were dead at birth (only dose level examined).

F1 Generation. No apparent adverse effect was observed on fertility at either dose level. 

Pre-coital intervals and gestation lengths were comparable among the groups. The 

gestation survival index and viability index were comparable among the groups for both 

the F2a and F2b litters. Litter size, pup body weights, and sex ratio were comparable 

among the groups in both the F2a nad F2b litters.

Degenerative changes in the tubules of the cortical region (high-dose F0 males) and outer 

medullary regions (mid- and high-dose F0 males, mid-dose F1 males) of the kidneys 

were found in a subsequent histopathological examination. The original reviewer noted 

that these effects on the kidney were not found originally but during a subsequent re-
2,4-D Human Health Risk Assessment DP No. D389455

Page 73 of 96

examination of the tissues, casting doubt on the quality of the histopathological 

examination of the reproductive organs. However, the RfD/QA Peer Review Committee 

determined that, based on the lack of effects on reproductive organs in the subchronic and 

chronic studies at similar or higher dose levels, reevaluation of these tissues (testes and 

ovaries) is not necessary (HED Document No. 011908, dated 5/9/96).

The NOAEL for parental toxicity is 5 mg/kg/day (target dose; actual dose range of 

3.8-13.5 mg/kg/day) and the parental LOAEL is 20 mg/kg/day (target dose; actual 

dose range of 14-48 mg/kg/day), based on decreased female body weight/body 

weight gain (F1) and male renal tubule alteration (F0 and F1).

The NOAEL for reproductive toxicity is 20 mg/kg/day (target dose; actual dose 

range of 18-35 mg/kg/day). Previously, the LOAEL for reproductive toxicity was set at 

80 mg/kg/day (target dose; actual dose range of 69-114 mg/kg/day), based on an increase 

in gestation length. However, the increase (<1 day) is not considered adverse. The dose 

of 80 mg/kg/day was not assessed in the second generation due to the lack of sufficient 

F1 offspring to mate (excessive pup mortality). The dose is above the threshold of 

saturation of renal clearance. Reproductive toxicity was not observed in this study.

The NOAEL for offspring toxicity is 5 mg/kg/day (target dose; actual dose range of 

7.2-13.5 mg/kg/day) and the LOAEL for offspring toxicity is 20 mg/kg/day (target 

dose; actual dose range of 26-48 mg/kg/day), based on decreased pup body weight 

(F1b). At 80 mg/kg/day (target dose; actual dose range of 76.1-133 mg/kg/day), there 

was an increase in pup deaths.

This 2-generation reproduction study is classified Acceptable/guideline. This study 

satisfies the guideline requirement (OCSPP 870.3800) for a 2-generation reproduction 

study in the rat.

A.5.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity – Rat

In a combined chronic toxicity/carcinogenicity study [MRID 43612001], 50 Fischer 344 

rats/sex/group were administered 2,4-dichlorophenoxyacetic acid [96.4%] via the diet for 

up to 24 months at concentrations of 0, 5 mg/kg/day, 75 mg/kg/day, and 150 mg/kg/day. 

The achieved doses were 4.77, 73.15, and 144.98 mg/kg/day [males] and 4.89, 73.11, and 

143.52 mg/kg/day [females], respectively. Additionally, 10 rats/sex/group were sacrificed 

at 12 months [interim sacrifice]. NOTE: The interim sacrifice data were reported in 

MRID 43293901, HED Document No. 011614, along with the chronic neurotoxicity 

screening battery substudy.

There were no treatment-related deaths or clinical signs of toxicity. Decreased body 

weight was observed throughout the study at the high-dose level in both sexes [males 

92%-96%/females 74%-90% of control] and at the mid-dose level in females [86%-90% 

of control]. At week 13, decreased body weight was very slight in the high-dose males 
2,4-D Human Health Risk Assessment DP No. D389455

Page 74 of 96

[96% of control] and somewhat greater in the high-dose females [90% of control]. At 

study termination, both sexes displayed decreased body weight at the high-dose level 

[males 92%/females 74% of control] with the females displaying a greater effect than 

males. The mid-dose females also displayed a decrease in body weight at study 

termination [86% of control]. Body-weight gains were decreased throughout the study in 

females [3-month interval (86% and 74% of control); 6-month interval (88% and 71% of 

control), and overall (77% and 52% of control)] at the mid- and high-dose levels, 

respectively. Similarly, high-dose males displayed decreased body-weight gains 

throughout the study [83%-87% of control]. Consistent with the decreased body-weight 

gains was a decrease in food consumption, which was observed at the mid-dose level in 

females [-3.9%] and in both sexes at the high-dose level [males (-4.7%)/females (-

11.6%)].

Ophthalmology findings at study termination consisted of increased incidences of 

constricted blood vessels, fundus and hyper-reflective, fundus in the high-dose males and 

an increased incidence of lens opacity in the high-dose females compared to the control 

and lower dose groups. Decreased RBC, HCT, and HGB values were observed at various 

time points in the mid- and high-dose females, and platelet counts were decreased at 

various time points in both sexes at the mid- and high-dose levels. Elevations in 

creatinine were observed in both sexes at the mid- and high-dose levels throughout the 

study, except at termination when comparable levels were observed among the male 

groups. Increased aspartate aminotransferase [mid- and high-dose males], alanine 

aminotransferase [mid- and high-dose males], and alkaline phosphatase [mid- and highdose, both sexes], and decreased glucose levels [mid-dose females, high-dose both 

sexes], cholesterol [mid- and high-dose, both sexes], and triglycerides [mid-dose females 

and high-dose both sexes] were observed throughout the study, although a dose response 

was not always apparent. There was a dose-related decrease in T4 values throughout the 

study in both sexes at the mid- and high-dose levels, and the females displayed the 

greater effect except at study termination.

Thyroid weights were increased in the mid-dose females and in both sexes at the highdose level at the 12-month interim sacrifice. At study termination, thyroid weights were 

increased in both sexes at the mid- and high-dose levels, although the mid-dose males did 

not attain statistical significance and the increase at the mid-dose level [both sexes] was 

greater than at the high-dose level. Decreased testes weights were observed at the highdose level at both the interim and terminal sacrifices and at the mid-dose level at study

termination, although statistical significance was not attained at the mid dose. Decreased 

ovarian weights were observed at the high-dose level at both sacrifice times and in the 

mid-dose females at study termination. The decreases in testes and ovarian weights are 

consistent findings in other studies on 2,4-D and its salts/esters. Kidney weights were 

increased only at the interim sacrifice in males at the mid- and high-dose levels [doserelated].

Gross pathology findings included decreased fat in high-dose females at both sacrifice 

times, multifocal pale foci in the lungs [interim: 1 mid-dose, 10 high-dose females; 

terminal: 4 high-dose males, one control, 4 mid-, 40 high-dose females], and lens opacity 
2,4-D Human Health Risk Assessment DP No. D389455

Page 75 of 96

in high-dose females at termination. Microscopically, there were increased incidences of 

lesions in the bone marrow [decreased hematopoiesis in high-dose females at the interim 

sacrifice], eyes [retina degeneration in 1 male and 9 females at high dose], kidney 

[proximal tubule degeneration in mid- and high-dose males and females], liver [altered 

tinctorial properties in mid-dose females and both sexes at high dose], lungs [multifocal 

alveolar histiocytosis in mid-dose females and both sexes at high dose], adipose tissue 

[atrophy in mid-dose female, both sexes at high dose], testes [atrophy at high dose], and 

thyroid [hyperplasia-high-dose males; hypertrophy and epithelial cells-high-dose 

females] at the interim sacrifice. At study termination, there were increased incidences of 

cataracts and retina degeneration of the eyes in both sexes at the high-dose level, and the 

severity of the retina degeneration was increased also. In the liver, there was an increased 

incidence of increased size of the hepatocytes with altered tinctorial properties in both 

sexes at the high-dose level. In the lungs, both the incidence and severity of 

inflammation were increased at the high-dose level in both sexes, and the incidence of 

atrophy of the adipose tissue was increased at the high-dose level in both sexes at 

termination. Tumor incidence was not affected by treatment.

The NOAEL is 5 mg/kg/day. The LOAEL of 75 mg/kg/day is based on decreased 

body-weight gain (females) and food consumption (females), alterations in 

hematology [decreased RBC (females), HGB (females), platelets (both sexes)] and 

clinical chemistry parameters [increased creatinine (both sexes), alanine and 

aspartate aminotransferases (males), alkaline phosphatase (both sexes), decreased 

T4 (both sexes), glucose (females), cholesterol (both sexes), and triglycerides

(females)], increased thyroid weights (both sexes at study termination), decreased 

testes and ovarian weights, and microscopic lesions in the lungs (females). At the 

high-dose level, there were microscopic lesions in the eyes, liver, adipose tissue, and 

lungs. There was no treatment-related increase in the incidence of any tumor.

This chronic toxicity/carcinogenicity study is classified ACCEPTABLE/Guideline, and it 

satisfies the guideline requirement [OPPTS 870. 4300; §83-5] for a chronic 

toxicity/carcinogenicity study in the rat.

NOTE: The current study was performed to address whether the finding of an increased 

incidence of astrocytomas of the brain found in the 1986 rat study [MRID 00160876; 

TXR# 0005234] was attributable to 2,4-D. The HED Carcinogenicity Peer Review 

Committee [CPRC; TXR No. 0050017] concluded that the doses used in the 1986 rat 

study were not adequate to assess the carcinogenic potential of 2,4-D. In the 1986 study, 

there was a significant trend for astrocytomas in male rats but no pair-wise significance. 

The incidence in both the treated and control males exceeded the historical control 

incidence for this tumor. Additionally, the CPRC concluded that the high-dose level in 

the 1986 study was not adequate. The current repeat chronic toxicity/carcinogenicity 

study in rats was performed at higher dose levels. There was one astrocytoma in the brain 

of males at the high dose vs none in the control. There was one tumor each in the control 

and high-dose female group. However, all of the brains of the rats in the low- and middose groups were not examined. The HED Carcinogenicity Peer Review Committee 

[CPRC; TXR No. 0050017] requested that the slides of the low- and mid-dose brains of 
2,4-D Human Health Risk Assessment DP No. D389455

Page 76 of 96

the males be evaluated. These additional data were submitted [MRID 44284501], and no 

additional tumors were observed. The additional data do not alter the findings of the 

study.

870.4100b Chronic Toxicity - Dog

In a chronic oral toxicity study [MRID 43049001], 5 beagle dogs/sex/group were 

administered 2,4-dichlorophenoxyacetic acid [96.7%] via the diet for 52 weeks at 

concentrations of 0, 1 mg/kg/day, 5 mg/kg/day, and 7.5 mg/kg/day. The target high-dose 

level of 10 mg/kg/day was reduced to 7.5 mg/kg/day during week 8, due to loss of body 

weight/failure to gain weight early in the study.

There were no treatment-related deaths or clinical signs. One female at 5 mg/kg/day was 

sacrificed on Day 130 in a moribund condition; however, death was attributed to an 

abdominal inflammatory condition. Decreased body weight was observed throughout the 

study at the high-dose level in both sexes, with females displaying the greater effect. By 

week 3, high-dose females displayed a 13% deficit in body weight compared to the 

control females. At the high-dose level at 8 weeks when the dose level was lowered, both 

sexes at the high-dose level displayed decreased body weight compared to the controls 

[males 90%/females 82% of control]. At week 13, the high-dose males were 94% of 

control and the high-dose females were 82% of control. At study termination, both sexes 

displayed decreased body weight at the mid- [males 89%/females 87% of control] and 

high-[males 87%/females 75% of control] dose levels compared to the controls. The lowdose females also displayed a decrease [87% of control] in body weight at study 

termination. Body-weight gain was decreased throughout the study in females at all dose 

levels [weeks 1-13 (75%, 70%, and 35% of control); 1-52 (73%, 64%, and 36% of 

control) at the low-, mid-, and high-dose levels, respectively]. Similarly, males displayed 

decreased body-weight gains throughout the study (except the low-dose group during 

weeks 1-13) compared to the control, although there was no dose-response [weeks 1-13 

(100%, 70%, 74% of control); 1-52 (78%, 63%, 67% of control]. Food consumption was 

decreased throughout most of the study, mainly at the high-dose level [both sexes], but 

statistical significance was not attained.

Ophthalmology findings were comparable among the groups for both sexes. There were

no apparent treatment-related alterations in hematology or urinalysis among the groups 

for either sex. Elevations in blood urea nitrogen, creatinine, and alanine aminotransferase, 

and decreased glucose levels [mid-dose males, high-dose both sexes] were observed 

throughout the study [mid- and high-dose levels, both sexes], although a dose response 

was not always apparent, especially in the males. Alkaline phosphatase was decreased in 

both sexes at the high-dose level at week 4. Similar alterations in these clinical chemistry 

parameters have been observed in subchronic studies in rats and dogs on 2,4-D, and its 

amine salts and esters.

There was a dose-related decrease in absolute brain weight in females, with statistical 

significance being attained at the mid- and high-dose levels. Males at the high dose also 

displayed decreased brain weight, but statistical significance was not attained. In general, 
2,4-D Human Health Risk Assessment DP No. D389455

Page 77 of 96

the organ-weight effects observed can be attributed to the lower body weight, although 

the decreased testes weights at the high-dose level and decreased ovarian weights at the 

mid- and high-dose levels are consistent findings in other studies on 2,4-D and its 

salts/esters. Microscopically, liver [perivascular, chronic active inflammation (males 1/5, 

1/5, 3/5, 4/5; females 0/5, 0/4, 4/5, 3/5 with increasing dose) and sinusoidal lining cell 

pigment (females; 1/5, 2/4, 5/5, 4/5 with increasing dose)] and kidney [minimal increase 

in the frequency and average severity of pigment in the tubular epithelium (males 2/5, 

4/5, 5/5, 5/5; females 1/5, 1/4, 5/5, 5/5 with increasing dose)] lesions were observed at the 

mid- and high-dose levels in both sexes. Aspermatogenesis and degeneration were 

observed in one male at the high-dose level compared to none in the other groups. 

The NOAEL is 1 mg/kg/day. The LOAEL of 5 mg/kg/day is based on decreased 

body-weight gain (both sexes) and food consumption (females), as well as alterations 

in clinical chemistry parameters [increased BUN, creatinine, and alanine 

aminotransferase, decreased glucose] in both sexes, decreased brain weight in 

females, and histopathological lesions in the liver and kidneys.

This chronic oral toxicity study is classified Acceptable/Guideline, and it satisfies the 

guideline requirement [OPPTS 870. 4100; §83-1] for a chronic, non-rodent, oral toxicity 

study.

A.5.5 Carcinogenicity

870.4200a Carcinogenicity Study - rat

In a chronic toxicity/carcinogenicity study with a chronic neurotoxicity screening 

battery substudy [MRID 43293901], 50 Fischer 344 rats/sex/group [main study]; 

15/sex/group [substudy] were administered 2,4-dichlorophenoxyacetic acid [96.4%] via

the diet for up to 24 months at concentrations of 0 [basal diet], 5 mg/kg/day, 75 

mg/kg/day, and 150 mg/kg/day [achieved doses were 4.6, 71.2, and 141.1 mg/kg/day 

[males] and 4.6, 68.0, and 138.9 mg/kg/day [females], respectively. Additionally, 10 

rats/sex/group were sacrificed at 12 months [interim sacrifice]. NOTE: This DER presents 

the results of the interim [one-year] sacrifice and neurotoxicity substudy.

There were no treatment-related deaths or clinical signs of toxicity. Decreased body 

weight was observed throughout the first year of the study at the high-dose level in both 

sexes [males 91%/females 88% of control at the interim sacrifice] and at the mid-dose 

level in females [94% of control at the interim sacrifice]. At 90 days, the decrease in body 

weight was very slight in the high-dose males [96% of control] and somewhat greater in 

the mid- [95% of control] and high-dose [90% of control] females. Body-weight gains 

were decreased throughout the first year at the mid- and high-dose levels in both sexes, 

although statistical significance was not always attained in males at the mid-dose level 

[2-week interval (males: mid-dose 90%/high-dose 84%; females: mid-dose 81%/highdose 63% of control); 3-month interval (males: mid-dose 98%/high-dose 88%; females: 

mid-dose 87%/high-dose 75% of control); and 1-year interval (males: mid-dose 

95%/high-dose 82%; females: mid-dose 89%/high-dose 73% of control). Consistent with 
2,4-D Human Health Risk Assessment DP No. D389455

Page 78 of 96

the decreased body-weight gains was a slight decrease in food consumption, which was 

observed in both sexes at the high-dose level.

Ophthalmology findings were comparable among the groups at the interim sacrifice. 

Decreased RBC [mid- and high-dose females (6 & 12 months) and high-dose males (12 

months)], HCT [mid- and high-dose females and high-dose males (6 & 12 months)], 

HGB [high-dose females (6 months)], WBC 9high-dose females (6 months)], and 

platelet counts [mid- and high-dose females and high-dose males (6 & 12 months) were 

observed. A dose-related increase in aspartate aminotransferase, alanine 

aminotransferase, and alkaline phosphatase was observed in the mid- and high-dose 

males at 6 months but not at 12 months. The mid- and high-dose females displayed a 

dose-related increase in alkaline phosphatase values at both the 6 and 12-month intervals. 

Cholesterol levels were decreased in the mid- (males at 12 months only) and high-dose 

rats of both sexes at both time intervals. T4 values were decreased at both time points in 

both sexes at the mid- and high-dose levels, although the mid-dose males at 6 months did 

not attain statistical significance. The only urinalysis finding was a decrease in specific 

gravity , which was observed in both sexes and time points at the mid- and high-dose 

levels.

Thyroid weights [absolute and relative] were statistically-significantly increased in the 

mid-dose females [ 20% greater than control] and in both sexes at the high-dose level 

[ 20% greater than control] at the 12-month interim sacrifice. Decreased testes weights 

[absolute and relative to brain] were observed at the high-dose level at 12 months [ 15% 

lower than control]. Kidney weights [absolute and relative] were increased at the 12-

months sacrifice in males at the mid- [7%-12% greater than control] and high-dose [8%-

16% greater than control] levels [dose-related].

Gross pathology findings included decreased fat in high-dose females [4/10] and 

multifocal pale foci in the lungs [1/10 mid-dose, 10/10 high-dose females]. 

Microscopically, there were increased incidences of lesions in the bone marrow 

[decreased hematopoiesis in high-dose females], eyes [bilateral retina degeneration in 1 

male and 10 females at high dose], kidney [proximal tubule degeneration in mid- and 

high-dose males and females], liver [altered tinctorial properties in mid-dose females and 

both sexes at high dose], lungs [multifocal , subacute to chronic inflammation (mid-dose 

females, both sexes at high dose), alveolar histiocytosis in females at high dose], adipose 

tissue [atrophy in mid- and high-dose females], testes [atrophy at high dose], and thyroid 

[decreased secretory material, epithelial cells-high-dose females].

The NOAEL is 5 mg/kg/day. The LOAEL of 75 mg/kg/day is based on decreased 

body weight (females)/body-weight gain (both sexes), alterations in hematology 

[decreased RBC, HCT, and platelets (females)], clinical chemistry parameters 

[increased alanine and aspartate aminotransferases (males), alkaline phosphatase 

(both sexes), decreased cholesterol (both sexes), and decreased T4 (both sexes)], and 

urinalysis [decreased specific gravity (both sexes)], increased kidney weights 

(males), increased incidence of degeneration of the descending proximal tubules 

(both sexes), hepatocellular hypertrophy with altered tinctorial properties (females), 
2,4-D Human Health Risk Assessment DP No. D389455

Page 79 of 96

lung inflammation (females), adipose tissue atrophy (females). At the high-dose 

level, there also were microscopic lesions in the eyes (both sexes), liver (males), testes 

(males), thyroid (females), and lungs (males).

Neurotoxicity Study

No treatment-related, hand-held, FOB observations were noted at any of the evaluation 

periods. Relative forelimb grip strength was significantly increased in both sexes at the 

high-dose level, but there was no treatment-related change in absolute grip strength. 

There was no treatment-related effect on motor activity. In agreement with the chronic 

toxicity portion of the study, an increased incidence of bilateral retinal degeneration was 

observed in the high-dose females. 

The NOAEL for neurotoxicity is 75 mg/kg/day, based on increased relative forelimb 

grip strength and increased incidence of bilateral retinal degeneration at the 

LOAEL of 150 mg/kg/day. 

This chronic toxicity/neurotoxicity study in the rat is classified Acceptable/guideline, 

and it satisfies the guideline requirement [OPPTS 870. 6200; §82-7] for a subchronic 

neurotoxicity study in the rat.

870.4200b Carcinogenicity (feeding) - Mouse

In a carcinogenicity study [MRID 43879801 and 43597201], 50 B6C3F1 CRL BR 

mice/sex/group were administered 2,4-dichlorophenoxyacetic acid [96.4%] via the diet 

for 104 weeks at concentrations of 0, 5 mg/kg/day [both sexes], 62 [males]/150 [females] 

mg/kg/day, and 120 [males]/300 [females] mg/kg/day. Additionally, 10 mice/sex/group 

were sacrificed at 52 weeks [interim sacrifice]. 

There were no treatment-related deaths or clinical signs of toxicity in either sex. Body 

weight, body-weight gain, and food consumption were comparable among the male 

groups throughout the study. Females at the high-dose level displayed a slightly lower 

body weight at the 3- and 6-month intervals [96% of control]. Body-weight gains were 

decreased significantly at all dose levels in the females at the 3-month interval [93%, 

94%, and 86% of control at the low-, mid-, and high-dose, respectively], and the highdose females continued to display a reduced body-weight gain at the 6-month [91% of 

control] and 12-month [91% of control] intervals. There were no consistent changes in 

food consumption in the female groups. 

Ophthalmology and hematology parameters [RBC, HGB, HCT, platelets] were 

comparable among the groups [both sexes]; no thyroid parameters were monitored.

There was a dose-related increase in kidney weights in both sexes. In males, increased 

kidney weights [absolute and relative] were observed at the mid- and high-dose levels at 

the terminal sacrifice only. In females, increased kidney weights were observed at the 

mid- and high-dose levels at both the interim and terminal sacrifices.
2,4-D Human Health Risk Assessment DP No. D389455

Page 80 of 96

Gross pathology findings were comparable among the groups [both sexes]. 

Microscopically, there was an increased incidence of lesions in the kidneys of both sexes 

at the mid- and high-dose levels. In males at the interim/terminal sacrifices, renal lesions 

were characterized as degeneration with regeneration of the descending limb of the 

proximal tubule in the mid- [20%/50%] and high-dose [100%/96%] males vs none in the 

low dose or control males; decreased vacuolization of the renal proximal tubule in mid-

[80%/78%] and high-dose [100%/96%] males vs 0% in the low-dose and control males. 

Additionally at the terminal sacrifice, there was an increased incidence of mineralization 

of the tubule(s) in the mid- [58%] and high- [72%] dose males compared to the low and 

control groups [38% and 32%, respectively]. In females, the renal lesions were 

characterized by hypercellularity in the descending part of the proximal tubule at both the 

interim and terminal sacrifices at the mid- [80%/64%] and high-dose [100%/50%] levels 

vs 0% in the low- and control females. There was no treatment-related increase in any 

tumor type in either sex.

The NOAEL is 5 mg/kg/day. The LOAEL of 62 mg/kg/day [males]/150 mg/kg/day 

[females] is based on an increased absolute and/or relative kidney weights and an 

increased incidence of renal microscopic lesions. There was no treatment-related 

increase in the incidence of any tumor type.

This carcinogenicity study is classified ACCEPTABLE/Guideline, and it satisfies the 

guideline requirement [OPPTS 870. 4200; §83-2] for a carcinogenicity study in the 

mouse.

NOTE: In the mouse study, there was an increase in hemangiosarcomas of the spleen in 

male mice at the low and mid doses, which was not sustained at the highest dose; 

however, the slides from all male mice at the low and mid doses were not evaluated. The 

HED Carcinogenicity Peer Review Committee [CPRC; TXR No. 0050017] requested 

that the slides of the low- and mid-dose spleens of the males be evaluated. These 

additional data were submitted [MRID 44284502]. The spleen of 35 male mice in the 

low-dose [5 mg/kg/day] group and 33 in the mid-dose [62.5 mg/kg/day] group were 

examined by light microscopy. No neoplasms were observed. When all of the data on the 

50 male mice/group were combined, there was no dose-related increase in 

hemangiosarcomas or in possible preneoplastic lesions, such as extramedullary 

hematopoiesis. It was concluded that the additional data do not alter the findings of the 

study. There was another mouse carcinogenicity study [MRID 40061801] conducted at 

dosages of 1, 15, and 45 mg/kg/day. No treatment-related increase in tumor incidence 

was found; however, this latter study was classified unacceptable because the high dose 

was considered inadequate to assess the carcinogenic potential of 2,4-D. 

A.5.6 Mutagenicity

Gene Mutation
2,4-D Human Health Risk Assessment DP No. D389455

Page 81 of 96

870.5100, Bacterial reverse mutation test

41409801 Acceptable/guideline

No evidence of bacterial mutation with or without S9.

870.5450, Unscheduled DNA synthesis assay

41409807 Acceptable/guideline

No evidence of induction of unscheduled DNA synthesis.

870.5395, In vivo mouse micronucleus test

41409804 Acceptable/guideline

No significant increase in frequency of micronucleated 

polychromatic erythrocytes in bone marrow at any time point.

Literature studies Office of Pesticide Programs, EPA, Reevaluation of the Genetic 

Toxicology Profile of 2,4-D (December 12, 2011).

A.5.7 Neurotoxicity

870.6200 Acute Neurotoxicity Screening Battery

In an acute neurotoxicity study (MRID 43115201), Fischer 344 rats (10/sex/dose) were 

orally administered 2,4-D (96.6% a.i.; Lot# 909) once via gavage at doses of 0 (corn oil), 

15, 75, or 250 mg/kg (actual dose: 0, 13, 67, or 227). Neurobehavioral evaluations, 

consisting of Functional Operational Battery (FOB) and motor Activity, were conducted 

pre-study (-Day 1), on Day 1 (≈5-6 hours post dose, peak-effect time), and on Days 8 and 

15 post dose. At terminal sacrifice (Day 15), rats were euthanized and neuropathological 

examination was performed on control and treated rats (5/sex/dose).

No treatment-related mortalities occurred during the study. No significant differences 

were noted in the mean body weights or mean body weight gains in either sex. Clinical 

signs and neurobehavioral evaluation revealed treatment-related changes. During the Day 

1 FOB evaluations, increased incidences of incoordination (6/10 males; 4/10 females) 

and slight gait abnormalities, described as forepaw flexing or knuckling, were observed 

in high-dose rats (8/10 males; 8/10 females). Slight gait abnormalities, observed in a 

single mid-dose female, were not judged to be treatment-related since no other signs of 

toxicity were evident. Minimal gait abnormalities, not judged to be treatment-related, 

were observed in one low-dose female and one each mid- and high-dose male. On the 

Day 2 and Day 3 clinical examinations, incoordination was noted in high-dose rats. The 

incidence of incoordination decreased to control levels by Day 4 in males and Day 5 in 

females. In high-dose rats, total motor activity was significantly lower at Day 1 only. No 

treatment-related gross or neuropathological findings were present.

Based on the results of this study, the LOAEL for systemic toxicity was not determined 

in males and females; the NOEL for systemic toxicity was 227 mg/kg in males and 

females. The LOEL for neurobehavioral effects was 227 mg/kg in males and females; the 

NOEL for neurobehavioral effects was 67 mg/kg in males and females.

This study is classified as Acceptable/Guideline, and it satisfies guideline requirements 

(§81-8) for an acute neurotoxicity screening battery in the rat.

870.6200 Subchronic Neurotoxicity Screening Battery

See under Chronic toxicity/carcinogenicity - rat
2,4-D Human Health Risk Assessment DP No. D389455

Page 82 of 96

870.6300 Developmental Neurotoxicity Study

See under 2-generation reproduction study (EOGRT)

A.5.8 Metabolism

870.7485 Metabolism - Rat

The metabolism of [phenyl-U-C14]-2,4-D was studied in male & female Fischer 344 rats 

(MRID 47417901). The phenyl ring-labeled compound was administered as a single oral 

dose of 1.04 - 1.05 or 97.1 - 97.4 mg/kg, or as a single oral dose of 1.06 mg/kg following 

a 14 day pretreatment with unlabeled 2,4-D at approximately 1 mg/kg/day. 

At least 85.5-93.7% of an oral dose was absorbed from the GI tract. Among the orally 

dose groups, approximately 85.5-93.7% of the dose was eliminated in urine and 3.6-

10.5% of the dose was eliminated in feces. In the IV-dose groups approx. 90.94-91.84% 

of the dose was eliminated in urine and 1.99-2.16% of the dose was eliminated in feces. 

At sacrifice, total radioactive residue in the carcass was less than 0.52-0.69% of the dose 

at the low oral dose and 1.17-2.57% at the high oral dose. No differences between the 

sexes were found as to extent of absorption or excretion at any dose level. At the high 

dose level, it appears that a non-linear region (of decreased clearance) was reached in the 

disposition of 2,4-D. 

Parent 2,4-D was the major metabolite found in the urine, amounting to 72.9-90.5% of 

the dose among the orally dosed animals of the main experiment. Small amounts of 

uncharacterized compounds A and B (0.6-1.3% and 0.0-0.7% of the dose, respectively), 

were found in the urine. Classified as acceptable.

870.7600 Dermal Absorption - Rat

There is an extensive set of high quality human research results. Ross (2005) notes that 

“the degree of uncertainty and variability associated with human dermal absorption for 

2,4-D is better defined than for virtually any other pesticide... .”Ref 91 Ross. EPA 

principally relied on an in vivo human study, which showed an average dermal absorption 

of 5.8% (Feldman). EPA also considered four other in vivo human studies. (Refs. 89, 96, 

97 and 98). These studies involved 8 separate trials using a total of 34 participants and 

had an average dermal absorption value of 5.7 percent. (Ref. 91 at 84, Table 2) To 

account for potential variability EPA selected a value of 10 percent.

A.5.9 Immunotoxicity

870.7800 Immunotoxicity

In an extended dietary one-generation reproductive toxicity study (MRID 47972101), 

2,4-dichloro phenoxyacetic acid (2,4-D; 97.85%-98.6% a.i.; lot # 2006 2433 8006-USA)

was administered to 27 Crl:CD(SD) young adult rats/sex/dose via the diet at dose levels 
2,4-D Human Health Risk Assessment DP No. D389455

Page 83 of 96

of 0, 100, 300, or 600 (females)/800 (males) ppm [equivalent to 0, ≈5, 15, or 30 

(females)/40 (males) mg/kg/day] for approximately four weeks prior to mating and 

continuing through mating (up to 2 weeks), gestation, and lactation. F1 offspring were 

evaluated for potential effects on the immune system. F1 offspring were maintained on 

the test diet until ≈PND 70 (Set 2a F1 offspring) or ≈PND 90-139 (Set 2b F1 offspring). 

Set 2a (10sex/dose): assessment of potential developmental immunotoxicity (DIT): 

examination of humoral immune function using the sheep red blood cell (SRBC) 

antibody-forming cell (AFC) assay on PND 70-74.

Set 2b (10/sex/dose): assessment of potential developmental immunotoxicity (DIT): 

examination of innate cellular immunity using the natural killer cell (NK) assay on PND 

87-93.

F1 Offspring Set 2a (PND 67-73): Developmental Immunotoxicity (Primary Immune 

Response to Sheep Red Blood Cells): There were no deaths. Slight decreases in body 

weights and body-weight gains were observed in males at 800 ppm (↓6%-10% and 

↓15%) and females at 600 ppm (↓8%-9% and ↓10%). Terminal body weights were 

comparable among the male and female groups. Both absolute (↓10%) and relative (↓8%) 

thymus weight decreases were observed in the males at 800 ppm and in the females at 

600 ppm [absolute (↓13%) and relative (↓10%)]. Males at 300 ppm showed a 17% 

decrease in thymus weight but no dose response. Spleen weights were slightly lower in 

females at 600 ppm [absolute (↓13%) and relative (↓14%)]. There was no significant 

difference in response for AFC/spleen and AFC/106 splenocytes among the male groups. 

Females at 600 ppm displayed a non-significant decrease of 54% for AFC/spleen and 

27% for the AFC/106 splenocytes. 

F1 Offspring Set 2b (PND 67-73): Developmental Immunotoxicity (Natural Killer 

Cell Activity): There were no deaths, and body weights/gains showed a similar slight 

reduction in males at 800 ppm as observed in the other offspring groups. Female body 

weights/gains were comparable among the groups. Terminal body weights (PND 87-93) 

were comparable among the groups (both sexes). There were no significant treatmentrelated effects on absolute or relative spleen or testes weights in males, and no significant 

treatment-related effects on spleen weights in females (only organs weighed). There were 

no significant, treatment-related differences in the percent target cell cytotoxicity at any 

dose level compared to control (both sexes), and 2, 4-D did not alter the cytotoxic ability 

of splenic NK-cells in male or female rats at any dose level. 

The DIT offspring (PND 139) LOAEL is >800/600 ppm (71.8 mg/kg/day in males, 

55.3 mg/kg/day in females), based on the lack of evidence of DIT [SRBC antibodyforming cell assay (PND 66-70) and Natural Killer Cell assay (PND 87-93)]. The 

DIT offspring NOAEL is 800/600 ppm (71.8 mg/kg/day in males and 55.3 mg/kg/day 

in females), the highest dose tested. 

This study is classified Acceptable/guideline, and it satisfies the guideline requirement 

(870.7800) for an immunotoxicity study. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 84 of 96

A.5.10 Special/Other Studies

(1) In a pharmacokinetic titration study (MRID 47417902), adult, non-pregnant female 

Sprague-Dawley Crl:CD(SD) rats (4/dose) were administered 2,4-D (99% a.i.; Lot # 

2006 2433 8006-USA)) in the diet at 0, 100, 200, 400, 600 or 800 ppm for 29 days. 

These doses (time-weighted average doses) were equivalent to 0, 7, 15, 30, 45, and 58 

mg/kg/day. The test material intake during the last week of dosing averaged 0, 7, 14, 27, 

41, and 56 mg/kg/day. The parameters evaluated included daily cage-side observations, 

weekly clinical examinations, body weights, food consumption, and gross pathological 

examinations. To calculate systemic exposure, diurnal area under the plasma 

concentration time curve (AUC24h) was determined by collecting 3 blood samples (6:00 

am, 9:00 am, and 5:00 pm) the day after the 4-week exposure period. 

All rats survived to scheduled sacrifice, and there were no treatment-related signs of 

toxicity. Females at the 600 ppm and 800 ppm dose levels displayed reduced bodyweight gains (↓29% and ↓58%, respectively) after one week of exposure and overall 

(↓18% and ↓33%, respectively), compared to control. At termination, body weights were 

slightly reduced (↓4% and ↓8%, respectively) at the 600 ppm and 800 ppm dose levels. 

No changes were observed at necropsy. 

The difference in the AUC24h between the 100 ppm (21.2 μg h mL-1) and 200 ppm (67.6 

μg h mL-1) dose groups was 3-fold, which is greater than the expected 2-fold from the 

actual doses ingested. Compared to the lowest dose (100 ppm), there was an 11- (233 μg 

h mL-1), 31- (650 μg h mL-1), and 60-(1285 μg h mL-1) fold difference in AUC24h at 400 

ppm, 600 ppm, and 800 ppm, respectively. 

The study report states that in all cases, Cmax was found to be at the time of the first blood 

sampling (6:00 am; Figure 1, from page 28 of the report), corresponding to active feeding 

right before the lights are turned on (Saghir, et al., 2006). However, the individual data 

show that one 200 ppm female and two 400 ppm females displayed higher levels at 9:00 

am than at 6:00 am; one 100 ppm female showed nearly identical levels at 6:00 am and 

9:00 am and the highest level was observed at 5:00 pm; one 200 ppm female and one 800 

ppm female displayed the highest level at 5:00 pm (Table 4).

2, 4-D was eliminated from the plasma at the same rate constant corresponding to an 

elimination half-life of 4 to 8 hours. 

COMMENT: Although it is acknowledged that the dose groups are small (4 rats/dose), 

there is considerable variability among the rats, which resulted in the standard deviations 

being, in several cases, greater than the means.

This pharmacokinetic titration study in female, non-pregnant rats is classified 

Acceptable/non-guideline. There is no guideline requirement for this type of study. The 

purpose of the study was to better characterize the pharmacokinetics of 2, 4-D in adult, 

non-pregnant Sprague-Dawley rats following dietary exposure for use in setting dose 

levels for the extended 1-generation reproduction study (MRID 47972101). In the initial 
2,4-D Human Health Risk Assessment DP No. D389455

Page 85 of 96

dietary dose range-finding study (MRID 47417901; separate abbreviated DER), the dose 

spread at the lower levels (100 ppm and 400 ppm) in the female rats did not allow a clear 

definition of the dose at which nonlinear kinetics likely began. 

(2) In a dose range-finding/pharmacokinetics study (MRID No. 47417901), male and 

female Sprague-Dawley Crl:CD(SD) rats (10/sex/dose) were administered 2,4-D (99% 

a.i.; Lot # 2006 2433 8006-USA) in the diet at dose levels of 0, 100, 400, 1000/800, 

2000/1200, or 1600 ppm during the premating period (≈4 weeks). Due to marked effects 

on food consumption and body weights at the highest dose levels, dietary adjustments 

were made to the 1000 ppm and 2000 ppm dose levels on test day 20 (TD 20). The 

dietary concentrations provided doses of 6, 23, 50, 86, and 92 mg/kg/day for P1 males 

and 7, 27, 60, 91, and 103 mg/kg/day for P1 females (average values from pre- and postmating and postweaning intakes). A few days prior to breeding, 4 P1 adult rats/sex/dose 

were subjected to timed blood collection (3 samples/rat/day). Rats were mated for a 

maximum of 2 weeks, and exposure of the P1 males continued for ≈7 weeks after the 

start of the mating phase, with timed blood collection (3 samples/rat/day from 4 

males/dose) prior to termination. P1 females were exposed through gestation and with 

timed blood collection (same as for P1 males) on lactation days 4 and 14 (LD 4 and LD 

14), and post-weaning on TD 95, the day prior to termination. Milk samples were 

collected on LD 4 and LD 14 (4 females/dose). A satellite group of P1 female rats 

(6/dose) was included for an assessment of 2, 4-D pharmacokinetics during gestation. 

Satellite females were subjected to the same exposure schedule as the main study P1 

females through mating (≈4 weeks premating, up to 2 weeks during mating phase) and 

also during gestation until termination on gestation day (GD) 17. Blood was collected 

from satellite females on GD 17 with timed blood collection (3 samples/rat) from 4 

pregnant females/dose. All rats were examined twice daily for clinical signs of toxicity 

and morbidity. Clinical observations, body weights, and food consumption were 

monitored weekly (both sexes), and reproductive performance was evaluated. Kidney 

weights were recorded for all rats, and histopathology of the kidney was evaluated in the 

control and high-dose rats (both sexes). 

Litters were culled on postnatal (PND) 4 to 10 pups (5/sex when possible). Litter 

parameters (litter size, pup body weight, and sex ratio) were recorded. Pups were weaned 

on PND 21 and group housed from PND 21-28 to minimize post-weaning stress. 

Thereafter, pups were housed individually. On PND 4, 14, 21, and 28, terminal blood 

samples were collected from 1 pup/sex/litter from 4 litters/dose. Timed blood samples (3 

samples/rat from 4/sex/dose) were collected from PND 35 rats. During the exposure 

period, body weight, food consumption, and cageside observations were recorded in the 

F1 offspring.

There were no treatment-related effects on conception, time to mating, gestation length, 

or pup sex ratio. At 2000/1200 ppm, the fertility (80%) index was reduced compared to 

the control (100%). At 1600 ppm, both the mating (80%) and fertility (70%) indices were 

reduced compared to the control (100%). According to the investigators, the small sample 

size, omission of uterine staining for implantation sites, and the lack of gross or 

microscopic examination preclude a determination of whether the findings were 
2,4-D Human Health Risk Assessment DP No. D389455

Page 86 of 96

treatment-related. P1 males showed comparable body weight/gain among the groups, 

whereas P1 females at 2000/1200 and 1600 ppm dose levels displayed decreased body 

weight/gains (overall ↓50% and ↓63%, respectively) and food consumption during the 

premating and mating periods. Gestation body weight/gain were variable, but mainly 

lower at the 1600 ppm (GD 17: ↓16%) and 2000/1200 ppm (GD 17: ↓13%) dose levels. 

Body weights of the dams during lactation were comparable among the groups, although 

body-weight gains during the first week of lactation were reduced at the 2000/1200 ppm 

(↓26%) and 1600 ppm (↓22%) levels. Increased kidney weights were observed at 1600 

ppm (P1 males) and at ≥ 1000/800 ppm (P1 females), and multifocal, degenerative 

lesions in the proximal convoluted tubules of the renal medulla were found on 

microscopic examination in P1 males at ≥400 ppm and in females at ≥800 ppm. 

Pup survival was comparable among the groups through LD 7. At 1600 ppm, decreased 

survival was observed on LD 14, and this group was terminated prior to LD 21. By LD 

21, decreased survival was observed at 2000/1200 ppm (↓30%). Decreased pup body 

weights were observed by LD 4 at 1600 ppm (both sexes ↓15%-17%), by LD 7-LD 21 at 

2000/1200 ppm (males ↓21%-41%/females ↓24%-44%), and by LD 14 at 1000/800 ppm 

(males ↓14%-23%/females ↓13%-20%). At 1000/800 ppm, post-weaning (PND 28-35) 

body weights (males ↓33%-35%/females ↓30%-32%) and body-weight gains (males 

↓37%/females ↓36%) were decreased compared to the control. 

This study provides plasma levels of 2, 4-D for both the maternal rats and pups of both 

sexes, as well as 2, 4-D levels in maternal milk. An estimated dose to the pups was not 

provided. These data may be useful in future assessments regarding lactational exposure.

 

Dose levels for the extended one-generation reproduction study were selected based on 

the results of this study and a pharmacokinetic titration study (MRID 47417902; separate 

DER). The kidney findings confirm the gender-based difference in the renal clearance of 

2,4-D in adult rats. Based on this gender difference, different high-dose levels were 

selected for males and females for the definitive extended one-generation reproductive 

toxicity test. The male high dose was ≈40 mg/kg/day (dietary concentration 800 ppm), a 

dose that was anticipated to be slightly higher than the inflection point for nonlinear TK 

in male pups from PND 35 to adulthood. The female high dose was ≈ 30 mg/kg/day (600 

ppm), which was anticipated to be clearly higher than the inflection point in pups and 

female adults. The mid- and low-dose levels were the same for male and female rats. The 

low dose of 5 mg/kg/day (100 ppm) was predicted to identify a clear NOAEL and was 

consistent with the NOEL dose identified in the previously conducted 2,4-D dietary 2-

generation reproduction study (MRID 00150557; Tasker, 1985). If exposure-related 

effects were seen in the high-dose group, the mid-dose of 15 mg/kg/day (300 ppm) was 

expected to provide dose-response data relevant to human risk evaluation (unless these 

effects were limited to doses clearly above linear TK). It was anticipated the 300 ppm 

mid-dose would be slightly above the inflection point for non-linear TK in female adults 

and pups and within the range of linear TK for adult males and post-weaning male pups. 

The dose levels selected for use in the extended one-generation reproduction study on 2, 

4-D were 100, 300, or 600 (females)/800 ppm (males). During protocol review, a dose 
2,4-D Human Health Risk Assessment DP No. D389455

Page 87 of 96

level of 50 mg/kg/day was discussed as an appropriate high dose, which was considered 

adequate for assessing effects slightly above renal clearance saturation.

This dose range-finding study is classified Acceptable/non-guideline. There is no 

guideline requirement for this type of study. The purpose of the study was to provide 

information for use in setting dose levels for the extended 1-generation reproduction 

study (MRID 47972101).
2,4-D Human Health Risk Assessment DP No. D389455

Page 88 of 96

Appendix B. Metabolism Summary Table

TABLE B-1. Summary of Characterization and Identification of Radioactive Residues in AAD-1 Field 

Corn (event 278) Matrices Following Application of [14C]2,4-D DMA at 3.13 lb ae/A (level 

of exaggeration: 1X).

Metabolite Fraction Corn, Forage Corn, Grain1 Corn, Fodder

TRR = 3.020 ppm TRR=0.032 ppm TRR = 4.179 ppm

%TRR ppm %TRR ppm %TRR ppm

2,4-D 77.4 2.34 -- -- 56.5 2.36

Disaccharide conjugate of 2,4-DCP 13.0 0.39 -- -- 8.3 0.347

Glucose conjugate of 2,4-DCP 7.0 0.21 -- -- 11.9 0.497

2,4-DCP 2.3 0.069 -- -- 1.7 0.072

Unidentified extractable 0.3 0.009 -- -- 21.6 0.901

Pectin 3.4 0.104 -- -- 5.9 0.246

Lignin 1.7 0.053 -- -- 2.2 0.091

Hemicellulose/plant proteins (ADF extract) 1.2 0.037 -- -- 1.3 0.052

Cellulose (ADF nonextractable) 0.4 0.011 0.5 0.021

Starch 20.6 0.007

Starch extract -- -- 6.1 0.002 -- --

Total identified 99.7 3.01 -- -- 78.4 3.28

Total characterized 7.0 0.214 26.7 0.009 31.5 1.31

Total extractable 111.8 3.38 26.7 0.009 95.2 3.97

Unextractable (PES) 2-- -- 73.7 0.024 -- --

Accountability3112 100 96

1 Results of starch isolation for second subsample of corn grain.

2

 Residues remaining after exhaustive extractions.

3

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis) * 100. TRR were calculated by 

summing extractable and nonextractable radioactivity. For grain, based on normalized results as reported by the petitioner.

* Samples of forage were collected 42 days after the last application, and samples of grain, cob, and fodder were collected 66 

days after the last application. 
2,4-D Human Health Risk Assessment DP No. D389455

Page 89 of 96

TABLE B-2. Summary of Characterization and Identification of Radioactive Residues in AAD-1 Field 

Corn (event 474) Matrices Following Application of [14C]2,4-D DMA at 2.62 lb ae/A.

Metabolite Fraction Corn, Forage Corn, Grain Corn, Cobs Corn, Fodder

TRR = 3.531 ppm TRR=0.040 ppm TRR = 0.026 ppm TRR = 5.563 ppm

%TRR ppm %TRR ppm %TRR ppm %TRR ppm

2,4-D 67.5 2.38 7.1 0.003 -- -- 51.3 2.86

Disaccharide conjugate of 2,4-DCP 12.2 0.430 -- -- -- -- 9.5 0.529

Glucose conjugate of 2,4-DCP 4.8 0.170 -- -- -- -- 14.6 0.815

2,4-DCP 1.4 0.048 -- -- -- -- 1.1 0.063

Pectin 3.2 0.114 3.0 0.002 7.6 0.002 5.1 0.282

Lignin 2.3 0.080 14.0 0.006 5.0 0.001 2.3 0.131

Hemicellulose/plant proteins (ADF extract.) 0.8 0.029 35.2 0.014 -- -- 0.8 0.044

Cellulose (ADF nonextractable) 0.3 0.012 1.4 <0.001 -- -- 0.3 0.019

Methanol/water and/or ethanol 0.1 N NaOH -- -- 8.3 0.003 36.7 0.010 -- --

Starch (SS2)1-- -- 26.3 0.011 -- -- -- --

Total identified 85.9 3.03 7.1 0.003 -- -- 76.5 4.26

Total characterized 6.6 0.235 53.6 0.023 49.3 0.013 8.5 0.476

Total extractable 104.7 3.70 89.6 0.037 49.3 0.013 101.3 5.64

Unextractable (PES)2-- -- -- -- NR NR -- --

Accountability3105 95 104 102

1 Results reflect a separate subsample of nonextractable residues of grain and are not included in total characterized residues. 

2

 Residues remaining after exhaustive extractions. NR = Not reported. 

3

 Accountability = (Total extractable + Total unextractable or Cellulose)/(TRR from combustion analysis) * 100. For forage, 

based on extraction results prior to characterization of pectin and lignin.

* Samples of forage were collected 30 days after the last application, and samples of grain, cob, and fodder were collected 57 

days after the last application. 

TABLE B-3. Summary of Characterization and Identification of Radioactive Residues in AAD-12

Soybean (event 416) Forage and Hay Following Application of [14C]2,4-D DMA at 2.14 lb 

ae/A (2.401 kg ae/ha) for forage and 3.19 lb ae/A (3.578 kg ae/ha) for hay.1

Metabolite Fraction 7-DAT2 Forage 7-DAT3 Hay 22-DAT3 Hay

TRR = 50.227 ppm TRR = 35.565 ppm TRR = 37.008 ppm

%TRR ppm %TRR ppm %TRR ppm

2,4-D 85.8 43.12 64.3 22.885 59.4 21.98

2,4-DCP 7.1 3.558 3.6 1.295 1.4 0.518

Metabolite A 10.4 5.223 12.6 4.474 13.4 4.969

Metabolite B (X11963417) 0.8 0.381 5.4 1.919 7.0 2.573

Metabolite C 0.1 0.055 1.1 0.395 2.7 1.000

Other 5.1 2.557 2.5 0.901 22.3 8.256

Total identified 104.2 52.33 87.1 30.97 83.9 31.04

Total characterized 5.1 2.557 2.5 0.901 22.3 8.256

Total extractable2110.3 55.39 93.8 33.36 108.7 40.24

Unextractable (PES)32.8 1.393 4.2 1.478 8.2 3.027

Accountability4113 97.9 117

1

 Metabolite fractions were obtained from the summed ASE extracts (40-110 °C) as calculated by the petitioner.

2

 Calculated by the study reviewer by summing ppm in ASE extracts (40-110 °C).

3

 Residues remaining after exhaustive extractions.
2,4-D Human Health Risk Assessment DP No. D389455

Page 90 of 96

4

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis; see TABLE C.2.1) * 100. TRR 

were calculated by summing extractable and nonextractable radioactivity.

TABLE B-4. Summary of Characterization and Identification of Radioactive Residues in AAD-12

Soybean (event 416) Seed Following Application of [14C]2,4-D DMA at 3.133 lb ae/A (3.512 

kg ae/ha).

1

Metabolite Fraction 41-DAT3 Seed

TRR = 0.372 ppm

%TRR ppm

2,4-D 1.4 0.005

2,4-DCP 0.1 <0.001

Metabolite B (X11963417) 3.0 0.011

Metabolite C 4.6 0.018

Other 56.9 0.211

Hexane (triglycerides/fatty acids) 10.0 0.037

Precipitates from base hydrolysis 13.4 0.050

Total identified 9.1 0.035

Total characterized 80.3 0.298

Total extractable290.9 0.338

Unextractable (PES)32.6 0.010

Accountability493.5

1 Metabolite fractions were calculated by the study reviewer by summing results from ASE extraction (40-70 °C) for combined 

methanol 40 °C, combined methanol/water 70 °C, and base hydrolysis of nonextractable residues.

2

 Calculated by the study reviewer by summing ppm in ASE extracts (40-70 °C) and base hydrolysis of nonextractable residues.

3

 Residues remaining after exhaustive extractions.

4

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis; see TABLE C.2.1) * 100. TRR 

were calculated by summing extractable and nonextractable radioactivity.
2,4-D Human Health Risk Assessment DP No. D389455

Page 91 of 96

Appendix C. Physical/Chemical Properties

Table C1. Physico-Chemical Properties

Parameter Value Reference

Molecular Weight 221 g/mol DP#403774 and 404562, F. Khan, 

10/02/12

pH Not available

DP#309452, T. Jimerson, 10/13/04

Water solubility (20°C) 569 mg/L at 20 °C

Solvent solubility (20°C to 25°C)

g/100 g at 25 °C

acetone 85.0

benzene 1.07

diethyl ether 220

ethanol 130.0

isopropanol 31.6

toluene 0.067

xylene 0.58

Vapor pressure (25°C) 1.4 x 10-7 mm Hg at 25 

°C

Octanol/water partition coefficient, log KOW

(25°C)

In 0.001 M solution:

2.14 at pH 5

0.177 at pH 7

0.102 at pH 9

Aerobic soil metabolism half-life (days)- median 3.2

DP#403774 and 404562, F. Khan, 

10/02/12 Anaerobic aquatic metabolism half-life (days) 321

Soil partitioning coefficient (Koc; ml/goc) 76
2,4-D Human Health Risk Assessment DP No. D389455

Page 92 of 96

Appendix D. Review of Human Research

Human Studies Review

This risk assessment relies in part on data from studies in which adult human subjects were 

intentionally exposed to a pesticide or other chemical. These data, which include studies from 

PHED 1.1; the AHETF database; the Outdoor Residential Exposure Task Force (ORETF) 

database; and the Residential SOPs, are (1) subject to ethics review pursuant to 40 CFR 26, (2) 

have received that review, and (3) are compliant with applicable ethics requirements. For certain 

studies, the ethics review may have included review by the Human Studies Review Board. 

Descriptions of data sources, as well as guidance on their use, can be found at the Agency 

website14. 

 

14 http://www.epa.gov/pesticides/science/handler-exposure-data.html and 

http://www.epa.gov/pesticides/science/post-app-exposure-data.html
2,4-D Human Health Risk Assessment DP No. D389455

Page 93 of 96

Appendix E. Occupational Exposure/Risk Summary Tables

Table E-1. Occupational Handler Non-Cancer Exposure and Risk Estimates for 2,4-D.

Exposure Scenario Crop or 

Target

Inhalation Unit 

Exposure (μg/lb 

ai)1

Maximum

Application 

Rate2

Area 

Treated 

Daily3

Inhalation

Mitigation Level: 

No respirator

Dose 

(mg/kg/day)4 MOE5

Mixer/Loader

Mixer/Loader, 

Liquid, Groundboom

High Acreage 

Crop 0.219 1 lb ae/acre 200 acres 0.000548 4,900

Applicator

Applicator, Spray, 

Groundboom

High Acreage 

Crop 0.34 1 lb ae/acre 200 acres 0.00085 3,200

1 Based on the “Occupational Pesticide Handler Unit Exposure Surrogate Reference Table” (March 2013); Level of mitigation: Baseline.

2 Based on proposed labels.

3 Exposure Science Advisory Council Policy #9.1.

4 Inhalation Dose = Dermal Unit Exposure (μg/lb ai) × Conversion Factor (0.001 mg/μg) × Application Rate (lb ai/acre or gal) × Area Treated or 

Amount Handled Daily (A or gal/day) ÷ BW (kg).

5 Inhalation MOE = Inhalation NOAEL (mg/kg/day) ÷ Inhalation Dose (mg/kg/day).
2,4-D Human Health Risk Assessment DP No. D389455

Page 94 of 96

Appendix F. Residue Summary Table

TABLE F-1. Summary of Residue Data from Soybean Field Trials with 2,4-D.

Commodity Total 

Applic. 

Rate

(lb ae/A)

PHI 

(days)

Residue Levels1

(ppm)

n Sample

Min.

Sample

Max.

LAFT2 HAFT2 Median Mean Std. Dev.

2,4-D

Soybean, 

early forage

1.97-2.10 6-8 24 <0.01 4.014 <0.01 3.930 0.272 0.696 1.146

Soybean, 

forage

2.97 - 3.13 6-8 24 <0.01 18.424 <0.01 18.282 0.202 1.420 3.900

Soybean, 

early hay

1.97-2.10 6-8 24 <0.01 21.885 <0.01 20.281 0.754 2.552 4.758

Soybean, hay 2.97 - 3.13 6-8 24 <0.01 60.413 <0.01 58.783 0.613 3.769 11.903

Soybean, 

seed

2.97 - 3.13 51-103 24 <0.01 <0.01 <0.01 0.01 0.01 0.01 N/A

2,4-DCP

Soybean, 

early forage

1.97-2.10 6-8 24 <0.01 25.195 <0.01 22.451 10.772 9.521 7.264

Soybean, 

forage

2.97 - 3.13 6-8 24 <0.01 36.966 <0.01 32.080 14.729 13.329 9.783

Soybean, 

early hay

1.97-2.10 6-8 24 <0.01 190.589 <0.01 190.347 32.338 40.018 48.419

Soybean, hay 2.97 - 3.13 6-8 24 <0.01 132.961 <0.01 131.509 36.701 40.509 35.918

Soybean, 

seed

2.97 - 3.13 51-103 24 <0.01 0.054 <0.01 0.047 0.014 0.019 0.012

1 Except for sample min/max, values reflect per trial averages; n = no. of field trials. For calculation of median, mean, and 

standard deviation, the LOQ (0.01 ppm) was used for any results reported as ND (0.003 ppm) or between the LOD and LOQ in 

Table 12. N/A = Not applicable.

2

 LAFT = lowest average field trial; HAFT = highest average field trial.

TABLE F-2. Summary of Residue Data from Field Corn Crop Field Trials with 2,4-D.

Commodity Total 

Applic. 

Rate

(lb ae./A)

PHI 

(days)

Residue Levels1

(ppm)

n Sample

Min.

Sample

Max.

LAFT2 HAFT2 Median Mean Std. Dev.

2,4-D

Corn, field, 

early forage

2.95 - 3.11 27-42 24 <0.01 2.988 <0.01 2.612 0.410 0.642 0.687

Corn, field, 

forage

39-78 25 <0.01 4.373 <0.01 3.125 0.254 0.698 0.954

Corn, field, 

grain

73-139 24 <0.01 <0.01 <0.01 <0.01 0.01 0.01 --

Corn, field, 

stover

73-139 25 <0.01 8.920 <0.01 8.077 0.738 1.349 2.064

2,4-DCP

Corn, field, 

early forage

2.95 - 3.11 27-42 24 <0.01 3.997 <0.01 3.056 0.791 1.031 0.977
2,4-D Human Health Risk Assessment DP No. D389455

Page 95 of 96

TABLE F-2. Summary of Residue Data from Field Corn Crop Field Trials with 2,4-D.

Commodity Total 

Applic. 

Rate

(lb ae./A)

PHI 

(days)

Residue Levels1

(ppm)

n Sample

Min.

Sample

Max.

LAFT2 HAFT2 Median Mean Std. Dev.

Corn, field, 

forage

39-78 25 <0.01 5.007 <0.01 3.900 0.483 0.877 1.122

Corn, field, 

grain

73-139 24 <0.01 <0.01 <0.01 <0.01 0.01 0.01 --

Corn, field, 

stover

73-139 25 <0.01 5.842 <0.01 5.377 0.373 0.911 1.217

1 Except for sample min/max, values reflect per trial averages; n = no. of field trials. For calculation of median, mean, and 

standard deviation, the LOQ (0.01 ppm) was used for any results reported as ND (0.003 ppm) or between the LOD and LOQ.

2

 LAFT = lowest average field trial; HAFT = highest average field trial.
2,4-D Human Health Risk Assessment DP No. D389455

Page 96 of 96

Appendix G. International Residue Limits 

2,4-D (PC Code: 030001; Date of Request: 07/16/13)

Summary of US and International Tolerances and Maximum Residue Limits 

Residue Definition:

US Canada Mexico2 Codex3

40 CFR 180.142:

Plant/Livestock: 2,4-D (2,4-

dichlorophenoxyacetic acid), both 

free and conjugated, determined as 

the acid

(2,4-

dichlorophenox

y)acetic acid

2,4-D

Commodity1 Tolerance (ppm) /Maximum Residue Limit (mg/kg)

US Canada Mexico2 Codex3

Corn, field, forage 6.0 10 maize forage

Corn, field, grain 0.05 0.05 field corn 0.05 maize

Corn, field, stover 50 40 maize fodder (dry)

Soybean, seed 0.02 0.01 (*) soya bean (dry)

Completed: M. Negussie; 07/16/13

1

Includes only commodities of interest for this action. Tolerance values should be the HED recommendations and 

not those proposed by the applicant.

2 Mexico adopts US tolerances and/or Codex MRLs for its export purposes.

3 * = absent at the limit of quantitation; Po = postharvest treatment, such as treatment of stored grains. PoP = 

processed postharvest treated commodity, such as processing of treated stored wheat. (fat) = to be measured on the 

fat portion of the sample. MRLs indicated as proposed have not been finalized by the CCPR and the CAC.