2,4-D Human Health Risk Assessment DP No

. D389455

UN I TED STATES EN VIRON MENTAL PROTECTION AGENCY
WASHINGTON, D .C. 20460

MEMORANDUM

Date: August 8

, 2013 OFFICE OF CHEMICAL S AFETY AND POLLUTION P REV ENTION

SUBJECT: 2,4-D. Human Health Risk Assessment for a Propo sed Use of2,4

- D Cho li ne on H e r bicide- Tolerant Com and Soybean

PC Code: 03000 I
, 05 1 505 Decision No.: 447951

. 442244 Petition No.: NA Risk Assessment Type: Single Chemica l 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 -/ # · - Danette Drew, Chemist

## ~ Linda Taylor, Toxicologist

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

## L Risk Assessment Branch V 

## NIT**~** Health Effects Di vision (7509P)

THROUGH: ldeliz Negron Encarnacion, Chern).s J c=

. Michael Metzger, Branch Chief 

# fV( t
Risk Assessment Branch V 

### Nil Health Effects Division (7509P)

TO: Michael Walsh Registration Division (7505P)

Page 1 of962

, 4-D Human Health Ri s k A s sessment 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 Recommendat i ons 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 U ncertainty in the Exposure Database

................................................... 15 4.5 Toxicity E ndpoint 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 U sed in Human Risk Assessment

....... . .. . ...... . .......... . ...... . ... . ...... . . ... . .......... . .... .. ...... . ... . ... . ... . .... . .. . .......... .. ....... . ........... 18 

### 5.0 Dietary E xposure 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/Degr a date Residue Profile

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

.. .. ....... .. .... ..... ....................... 20 Summary of E nvironmental Degradation

.. .. ....... . ... . .... . ......................................... 20 Comparison of Metabolic Pathways

.......... .. ...................................................... .. . .. 21 Residues o f Concern Summary and Rationale

................................. .. .... .. ............. 2 1 Food Residue Profile

....... .... .... ... ....... . ............... .. ....................... .. ............................. . .. 22 Water Residue Profile

............... . .................................................................................. 23 Dietary Risk Assessment

.......................... . ..... .... .... .. ......... . ..... .. .......... . .. . ..................... 24 Description of Residue Data U sed in Dietary Assessment

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

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

.. ... .............. .. .... .. ............................... . ................. . .. 25

Pa ge 2 of 962,4

- 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 Ex ecutive 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

................................ ................................ .............. 962,4

- 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

- 1 2 ) 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) applicat ion 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, g rowth stage) application to soybean at a maxi mum 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 appl ication 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 applicat ion 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 a nd developmental toxicity were observed at high dose levels exceeding the threshold of saturation of renal clearance. T here are no residual uncertainties for
pre

- and/or postnatal toxicity . HED recommends that the 10X FQPA Safety Factor (for the protecti on 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 che mical, i.e., not classifiable as to human carcinogenicity.

Dietary Exposure and Risk Assessment: Acute and chronic aggregate (food + dietary d rinking2,4

- 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 wat er. 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 approp riate, 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 95 th percentile of the exposure distribu tion for the general population and all population subgroups. The res ulting acute risk estimate for children 1 to 2 years old (the subgroup with the greatest exposure) was 14 % of the aPAD at the 95 th percentile of the exposure.

Th e 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: T here 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 n o potential hazard via the dermal route f or 2,4

- D, therefore the handle r assessment included only the inhalation route of exposure and the post

application 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 fo r 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 ap propriate 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 d eposition 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 proposed2,4

- 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 o ccur 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 95 th 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.

The re 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 n o potential hazard via the dermal route f or 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 n o potential hazard via the dermal route f or 2,4

- D ; therefore, a quantitative occupational post

- application dermal risk assessment was not completed. Furthermore, a quantitative post

application inhalation risk assessment was not performed f or 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. T he 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

- heal th 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 recommend s 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 independent ly 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 satisf ied . 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 wi th 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 conj ugated, determined as the acid ”

.

N o new tolerances are required to support the requested use of 2,4

- D choline on the herbicide

re sistant 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/graz ing 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 commoditi es are listed in the table below.

Table 2.2.3

. Tolerance Summary for 2,4

- D2,4

- 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 0.0 1

2.2. 3 Revisions to Petitioned

- For Tolerances

The petitioner proposed that existing tolerance s 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 t he existing tolerances for residues of 2,4

- D on corn and soybean commodities are adequate to cover the proposed use of 2,4

- D chol ine on AAD

- 1 corn and AAD

- 12 soybean. However,

r evisions to current tolerances are req uired 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 MRL s 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 correspon ding 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 see d 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 allo w 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 Recomme ndations f rom Residential Assessment

None.

3.0 In troduction

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 0515052,4

- 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 expect ed to volatilize 2

.
2,4

- D is non

- persistent (t 1/2 =6.2 days) in terrestrial (aerobic) environments, moderately persistent (t 1/2 =45 days) in aerobic aquatic environments, and highly persistent (t 1/2 = 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 (K oc =61.7) in soil and aquatic environments.

T oxicokinetic studies conducted in pregnant rats show that 2,4

- D is transferred through maternal mi lk 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 maxi mum single application rate of 1.0 lb ae/A

. For post

- emergence appl ications 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 applica tion 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. Th e 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=812,4

- D Human Health Risk Assessment DP No. D389455

Page 11 of 96 eyewear; a chemical

- resistant apron is required when mixing or loading, cle aning 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 postemerg ence 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 t o 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 non

occupational settings, including during pesticide application as well as potential for post

application 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 post

application 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 Justice2,4

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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 risk s 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 patt erns are compiled by the USDA under the National Health and Nutrition Examination Survey, What We E at 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 d ietary 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 applicato rs 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 mo dels 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 toxicol ogy 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 tox icity (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

- g eneration 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), sub chronic 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 (rat2,4

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Page 13 of 96 and rabbit) studies available on the amine salts a nd 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 absorbe d orally, undergoes limited metabolism, and is eliminated quickly from the body primarily unchanged in the urine by active saturable renal transport. The observed dose

dependent, non

- linear pharmacokinetics of 2,4

- D is primarily due to the saturation of th is 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 dif ference 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. D ue 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

m agnitude 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, hyperplasi a 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 rab bits 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 proxim al 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 abo ve the2,4

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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 (l ethal) 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 we re 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 do se 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 d uring 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 chang e 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 F actor 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 deve lopmental 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 o f the Toxicology Database2,4

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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,

re productive 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 N eurotoxicity

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 st udy, r elative 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. N europathological effects were not obs erved in any study.

4.4.3 Evidence o f Sensitivity/Su sceptibility in the Developing o r 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 pr e

- /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 ab ortions, 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 a re 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 r efinements 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 D eparture Selections2,4

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Page 16 of 96 4.5.1 D ose - 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 an y 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 (14 th 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 popula tion, 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) a nd 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 extende d 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 incr eased 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 Concentration s (HEC)/Human Equivalent Dose s (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 acc ount for the PD differences), a 10X uncertainty factor was applied to account for intra

- species variability, and a 10X UF LOAEL→NOAEL was applied to a ccount for the lack of a2,4

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Page 17 of 96 NOAEL. A lthough 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 treatment

related, 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 o ccur 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. T he use pattern indicates that dose levels required to exceed the renal clearance mechanism would not be attained following human inhalation exposure.

N o 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 develo pmental 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) a lthough 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 (↓ T 3 and T 4 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 tha t 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 lin ks human cancer to 2,4

- D
exposure.2,4

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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 f

or Risk Assessment Study and Toxicological Effects

Acute Dietary ( Females 13

- 49 years old ) Developmental

NOAEL = 25 mg/kg/day UF A = 10x

UF H =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 (14 th rudimentary ribs ) Acute Dietary (General Population, including Infants and Children) NOAEL = 67

mg/kg/day UF A = 10x

UF H =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= 2 1

mg/kg/day UF A =10x

UF H =10x FQPA SF = 1x cRfD =cPAD =

0.2 1 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 Intermediate

Term (1

- 30 days and 1

- 6 months) NOAEL = 21

mg/kg/day UF A = 10x

UF H =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 incidenc e 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 do se levels that exceed renal clearance mechanism (dermal equivalent doses of 750 and 900 mg/kg/day); d ose levels required to exceed the renal clearance mechanism would not be attained following dermal exposure to humans

.2,4

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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 f

or Risk Assessment Study and Toxicological Effects

Inhalation ( all durations) L OAEL = 0.05

mL/kg/day

HEC=0.013 mg/L/day A (residential)

HEC= 0.056 mg/L/day A (occupational)

HED=1.9 mg/kg/day B (residential)

HED= 2.68 mg/kg/day B (occupational) UF A = 3X

UF H = 10X UF LOAEL→NOAEL = 10X Residential

and Occupational LOC for MOE = 300 Subchron ic 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 fol lowing 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. UF A = extra polation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF L = use of a LOAEL to extrapolate a NOAEL. UF S = use of a short

- term study for long

- term risk assessment. UF D B = 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 appl icable.
1 T he 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. 01 3 mg/L ; Occupational HEC (portal of entry endpoint) = 0.05 mg/L * (6/8; 0.75) * (5/5; 1) * Tracheobronchial RDDR ( 1.490 ) = 0. 0 56 mg/L
B Residential HE D (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/day2,4

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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 r esidue in plants. The predominant residues in soybean forage and hay were 2,4

- D, 2,4

- DCP, acetyl

- glucose

- DCP conjugate and a g lucose conjugate of 2,4

- DCP. In soybean seed the primary identified residues were 2,4

- D, g lucose conjugate of 2,4

- DCP and 2,4

- DC P disaccharide . The study indicates that t he 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.

T wo 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 maj or re sidue identified in forage and fodder. These p lant 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 deter mined 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 re striction 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 (t 1/2 =6.2 days) in terrestrial (aerobic) environments, moderately persistent (t 1/2 =45 days) in aerobic aquatic environments, and highly persistent (t 1/2 = 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 (K oc =61.7) in soil and aquatic environments.2,4

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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 envir onmental fate studies for 2,4

- D, 1,2,4

- benezenetriol, 2,4

- di chlorophenol (2,4

- DCP)

, and c hlorohydroquinone (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

c holine.

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 i s 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 occ urrence and/or low toxicity.

In non

- transgenic crops, milk, tissue, and poultry, the primary resid u e 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 non

transgenic corn and soybean. Although the metabolite 2,4

- DCP f ound in transgenic corn and soybean was not a major metabolite in rat metabolism, s ince 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).

F or 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

amou nts 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

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Page 22 of 96 Table 5.1.4 Summary of Metabolites and Degradates to be included in th e Risk Ass essment 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 th e 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 th e 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 di ssociate 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; resi dues 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 non

detectable (<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 co rn grain2,4

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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 m ay concentrate in AGF and meal but do not concentrate in oil and hulls. The observed concentration factors w ere 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 1 3 months in corn grain, forage, fodder , starch, flour and oil ; Storage stabi lity d ata 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 corr ected 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 g reater than the tolerance. Since tolerance level residues for corn commodities were included in the dietary risk assessment, tha t 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 e xpected 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 livestoc k 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 System2,4

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Page 24 of 96 (PRZM/EXAMS) models for surface water and Screen ing 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/EFE D 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 136 a 15 c Chronic (non

- cancer) 23 b

a From the Tier II PRZM

- EXAMS

- Index Reservoir model. 1 in 10 year peak exposure. I nput 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, D403 774 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 d rinking 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 non

transgenic 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 so ybean 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 D40 0085 (D. Drew, 7/11 /13) for more information.2,4

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Page 25 of 96 A livestock feeding/grazing restriction for the soybean use is proposed; those are not consid ered feedstuffs associated with the proposed use. T herefore, 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 cor n 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 t he 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. F or 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 dri nking 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 I I 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, indi vidual 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 95 th percentile of the exposure distribution for the general population and all
p opulation subgroups. T he res ulting acute risk estimate for children 1 to 2 years old, the subgroup with the greatest exposure, was 14 % of the aPAD at the 95 th percentile of the exposure. Refer to the summary table in 5.4.6. The acute dietary assessment is unrefined; t o 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 Assessment2,4

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Page 26 of 96 For chronic dietary exposure assessment, an estimate of the residue level in each food or food

form (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 popu lation was children 1 to 2 years old uti lizing 15 % of the cPAD. Refer to the summary table in 5.4.6. The chronic dietary assessment is unrefined; t o 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 bee n 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 SOPs 3 along with

3 Available: http://www.epa.gov/pesticides/science/residential

- exposure

- sop.html2,4

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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 D 413002 , 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 a re 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 Mi xing/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 SOPs 4

.
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.

Resid ential 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 SOPs 5

.

4 Available: http://www.epa.gov/pesticides/science/residential

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- sop.html2,4

- D Human Health Risk Assessment DP No. D389455

Page 28 of 96 Combining Exposures/Risk Estimates: There is n o potenti al hazard via the dermal route f or 2,4

- D . Only inhalation risk estimate s 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 g reater than the level of concern of 300 for inhalation) for all scenarios.

Table 6 .1. Residential Handler Non

- cancer Exposure a nd Risk Estimates for 2,4

- D

.

Exposure Scenario Level of Concern Inhalation Unit Exposure (mg/lb ai) Maximum

Application Rate 1 Area Treated or

Amount Handled Daily 2 Inhalation

Dose (mg/kg/day) 3 MOE 4

(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/gallon A 5 gallons 0.000068 28,000

Lawns/Turf, Ready

- to

use/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/gallon A 5 gallons 0.00053 3,600

Lawns/Turf, WP/DF, Manually

- pressurized handwand or Backpack 1.1 0.06 lb ae/gallon A 5 gallons 0.0041 460

Lawns/Turf, Granule, Push

type 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/ft 2 ,B 1200 ft 2 0.00002 94,000

Lawns/Turf, Granule, Spoon 0.087 0.0000344 lb ai/ft 2 ,B 100 ft 2 0.0000037 510,000

Lawns/Turf, Granule, Cup/ Shaker Can 0.013 0.0000344 lb ai/ft 2 ,B 100 ft 2 0.00000056 3,400,000

Lawns/Turf, Granule, Hand Dispersal 0.38 0.0000344 lb ai/ft 2 ,B 100 ft 2 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 = Inhal ation 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 ft 2 = 0.0000344 lb ai/ft 2

6.2 Post

- Application Exposure

5 Available: http://www.epa.gov/pesticides/science/residential

- exposure

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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 tre ated turf ( children 1 < 2 years old only) Incidental i ngestion 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 SOPs 6 . These lifestages are not the only lifestages that could be potentially exposed for these post

- a pplication 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 Expo sure 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 SOPs 6

.

Chemical

- specific TTR Data There are three transferable turf residue studies available for 2,4

- D, MRIDs 45033101, 44655703, and 44655702. These studies have be en determined to be acceptable for risk assessment purposes. The highest predicted day 0 TTR value is 0.28 μg/cm 2 (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/cm 2 on 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 D 413002 and/or the 2012 Residential SOPs 6

. Combining Exposure and Risk Estimates There is n o potential hazard via the dermal route f or 2,4

- D . Only incidental oral risk estimate s

were quantitatively assessed

. The incidental oral scenarios (i.e., hand

- to

- mouth and object

- to

mouth) should be considered inter

- related and it is likely that they occur interspersed amongst

6 Available: http://www.epa.gov/pesticides/science/residential

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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 g ranular 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 e xposure 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 a nd Risk Estimates for 2,4

- D.

Lifestage Post

- application Exposure Scenario Application Rate 1 Dose

(mg/kg/day) 2 MOEs 3 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 - 5 430,000 Episodic Granular Ingestion 3% ai; 1.5 lb

ae/acre 0.14 490 A

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 subme rged 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 w ors t case and is used as a surrogate for all other possible post

application 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 inge stion 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 expe cted 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

- applicat ion risk assessment for the swimme rs scenario and can be found in D 413002

. I nhalation 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 e ach post

- application scenario are based on an analysis provided as an Appendix in the 2012 Residential SOPs 7 . These lifestages are not the only lifestages that could be potentially exposed for these post

- application scenarios; however, the assessment of t hese 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 i ngestion of water during recreational swimming (both adults and chi ldren 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 wi th 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 b y 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 t he algorithms used to estimate residential post

application exposure from uses o f 2,4

- D for aquatic application are presented in D 413002

. 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

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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 agg regate assessment for 2,4

- D

. Inhalation exposures are not included in the aggregat e 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 rout ine 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 residenti al 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

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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 Post

application Total Exposure (mg/kg/day) Residential Post

application 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 fiel d. 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 co

distillation), or after as dried deposited residues volati lize. The volatilization assessment uses an analysis after sprays have dried.

A pproaches EPA has used previously to assess inhalation exposures to fumigant pesticides 8 were used for the assessment and is also consistent with the recommendations of the De cember 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 Agency 10 . 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. F or 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 transp ort 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 app lication 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 size s, and various meteoro logical 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 dataset s which have been used in th e 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: P robabilistic 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

- Scientifi c 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 AgroScie nces 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

av eraging 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 sin c e the 6 hour exposure period used as the basis for the comparison repr esents the peak emissions period after application.

Volatilization Risk Estimates The field volatility study suggest s 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 PERFU M 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 off

target 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 drif t 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, sh ould 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 exposur es 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
tur f 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 labe ls 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 ex pected 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.

T he 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 comp ared 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 ac ute 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 95 th 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 ar e 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 LOC 1 Max

Allowable Exposure 2 mg/kg/day Average

Food and Water Exposure mg/kg/day Residential

Exposure mg/kg/day 3 Tota l

Exposure mg/kg/day 4 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 un certainty 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 Risk2,4

- 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 ; t herefore

, 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
gen eral 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
p lus 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 wh ich 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 ki nds 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 equip ment 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 f or 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 da ta 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

- submit ted 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, kno wn as “unit exposures”, are outlined in the “Occupational Pesticide Handler Unit Exposure Surrogate Reference Table 12 ”, which, along with additional information on HED policy on use of surrogate data, including descriptions of the various sources, can be fo und at the Agency website 13

.
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 one

month 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.pd f
13 Available: http://www.epa.gov/pesticides/science/handler

- exposure

- data.html2,4

- 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 sleev ed shirt, long pants, shoes plus socks, no prote ctive 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
pr otective 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 D 413002

. Combining Exposures/Risk Estimates: There is n o potential hazard via the dermal route f or 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 baseli ne 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 a nd Risk Estimates for 2,4

- D

.

Exposure Scenario Crop or Target Inhalation Unit

Exposure ( μ g/lb ai) 1 Maximum

Application Rate 2 Area

Treated Daily 3 Inhalation

Mitigation Level : No respirator Dose (mg/kg/day) 4 MOE 5

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 ( μ g/lb ai) × Conversion Factor (0.001 mg/ μ g) × Application Rate (lb ai/acre or gal) × Area Treated or Amou nt 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 re

entry exposure). Such exposures may occur when workers enter previ ously treated areas to perform job functions, including activities related to crop production, such as scouting for pests2,4

- 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 c rop 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 n o potential hazard via the dermal route f or 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 irritat ion . 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 i rritation or primary skin irrit ation 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 procedu res, to identify the need for and, subsequently, the way to incorporate occupational post

application 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 quantitat ive 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 mo st 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

- 1 2 (A A D

- 1 2 ) Gene.

Goodlow, T. 1/14/09. D339654. 2,4

- D: Residue Analytical Method for Livestock Commodi t i es

.
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. D rinking 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 Dioxygenase

1 (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 Herbicide

Tolerant 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 A ppendix 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 y es

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 yes2,4

- D Human Health Risk Assessment DP No. D389455

Page 43 of 96 A.2 Toxicity Profile s

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 LD 50 = 699 mg/kg I II
870.1200 Acute dermal [rabbit] 00101596 LD 50 = >2000 mg/kg III
870.1300 Acute inhalation [rat] 00161660 LC 50 = > 1.79 mg/L I II
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

( F i s ch e r 34 4 r a ts ) 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, a l t e r a tion s i n he m a t olog y

a nd c l ini c a l c he m i s tr y parame t e r s , a n d c a t a r a c t f or m a t io n i n fem a les. At HDT, e ff

ec ts list e d a b ove occurred more freque n tly an d /or to a greater extent, as well as hist o pat h ol o gical lesions in e y es, liver, testes,

a drenals, ki d neys, t h ymus, b o n e m arrow, spleen, t h yr o ids, l u n g s. 870.3150 Sub c hroni c or a l

( c a psu l e ) tox i c i t y

- dogs MRID 4173730 1 (1990)

A c c e pt a bl e / G uid e line

0

, 0.3

, 1

. 0

, 3.0

, a n d 1 0

m g/kg/d a y] NOAEL = 1 mg / kg/day LOAEL = 3 mg/kg/day, based on decre a sed bod y w e i g ht/bod y

- w e i g h t g a i n a n d f oo d

c on s u m ptio n ( m a l e s ), alterations in clinical che m istry para m

eters [ increased BUN (bot h

s e x e s )

, c r e a tin i n e ( m a l e s )]

, an d d e c r e a s e d t e s t i s

w e i g ht in m ales. A t t h e h i g h e st do se t e st e d,

m o rt a lity ( 2 m ales, 1 f

e m ale sacri f iced

m ori b u n d), he m atol o gy alterations, and

m icrosco p ic lesions of the k i dn e y s and testes [h y posp e r m a t o g en e s i s ] w e r e obs e r v ed. 870.3150 S u b c h r o n i c o r a l

( d i e t ) t o xi c i t y ( do g) MRID 4278000 1 (1993)

A c c ep t ab l e / G uid e line

0

, 0.5

, 1.0

, 3.75

, a n d 7. 5

m g / kg / d a y N O A E L = 1 m g / kg / d a y LO A EL = 3.7 5 m

g / kg / d a y , b a s ed on de c r e a s e d bod y

- w e i g h t gai n (bot h s e x e s ) a n d f ood

cons u m pti o n ( m ales), a s w ell as a l t erati o ns in cli n ical ch em i s tr y pa r a m

e t e r s [inc r e a s e d B U N
, c r e a t inin e , a n d a l a nine a m inot r an sf e r a s e ] i n bot h s e x e s , a n d d e c r e a s e d t e s t e s w e i g ht an d

s l i g htl y h i g h e r i n c i d e n c e o f h y p osp e r m a t o g en e s i s /

ju v en i le t e s t i s a n d in a c t i v e / ju v en i l e pro s t a t e w e r e obs e r v ed.2,4

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Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.3200 21

- Day dermal

toxicity (rabbit) MRID 41735301

,

4173530 4 (1990)

A c c e pt a bl e / G uid e line

0, 10

, 100

, 100 0

m g/kg/d a y NOAEL = 1 0 00 mg/k g /da y LOAEL > 1 0 00 mg/k g /da y , no adverse effe c ts 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)

A c c e pt a bl e / G uid e line 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 (port al

- 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

,

0013040 8 (1983)

A c c e pt a bl e / G uid e line

0

, 8

, 25

, an d 7 5

m g/kg/d a y Maternal NOAEL = 25 mg / k g /day M a t e rn a l L O A EL = 7 5 m g/kg/da y , b a s e d o n d e c r e a s e d body w e igh t ga i ns

. Su r viv a l w a s no t

a f f e c t e d b y t r e a t m en t

.

D e ve l op m en t a l N O A EL = 2 5 m g/kg/d a y Develo p mental LOAEL = 75 mg/ k g/ d ay, based on skeletal abnormalities/ malformations

. 870.3700b Prenatal

developmental in (New Zealand white rabbit)

GD 6

- 18 MR I D 4174760 1 [1990]

A c c e pt a bl e / G uid e line

0

, 10

, 30

, an d 9 0

m g/kg/d a y Maternal NOAEL = 30 mg / k g /day M a t e rn a l L O A EL = 9 0 m g/kg/d a y , b a s e d o n

c lin i c a l s igns [ a t a xi a , d e c r e a s e d m oto r a c tiv i ty , lo s s o f right i n g re f

l e x , c old extremities], aborti o n (2 )

, decre a sed bo d y

- weig h t gains. Surv i va l w a s no t a f

f e c t e d b y t r e a t m

en t

.

D e ve l op m en t a l N O A EL = 3 0 m g/kg/d a y Develo p mental LOAEL = 90 mg/ k g/ d ay, based

on aborti o ns.2,4

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Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.3800 Reproduction and

fertility effects (Fischer 344 rat) MRID 00150557 and

00163996 (19 85 ) acceptable/guideline*

*new 870.3800 required (see MRID 47972101 below) Parental NOAEL = 5 mg/kg/day. Parental LOAEL = 20 mg/kg/day

, based on decr e ased fe m ale body w eight/bo d y weight gain ( F 1) and male renal tubu l e alt e ration ( F 0 and F1). At HDT, excess toxicity and too few pups for second litter (dose group terminated).

Reproductive N OAEL = 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/d ay 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

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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 stud ies 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 lesi ons 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 tes ted. 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
prox imal 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 an d 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 (sa tellite GD 17 dams), sperm parameters, ovarian follicle counts, and reproductive organ histopathology).2,4

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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 de c r e a s e d bod y

- w e ig h t g ain ( f

e m a l e s ) an d f

o o d

c on s u m p t io n ( f

e m a l e s )

, a l ter a t io n s i n he m a t o lo g y [ d e c r e a s e d R B C ( f e m a l e s )

, H G B (f e males), platel e ts (both sexes)] and cli n ical chemist r y parame t ers [ increas e d crea t ini n e (both sex e s), alani n e and aspartate aminotransfe r ase (males )

, alkal i ne pho s phatase ( both sex e s), decr e ased T4 (both

sexes )

, glucose (f

e males), c holesterol ( both sexes), and triglyce r ides (fe m ales)], i n cr e ased thyroid weights ( b o t

h s e x e s a t s t ud y t e r m i na t io n )

, d e c r e a s e d t e s t e s an d o v a r ia n

w e ig h t s , an d mi c r o sc op i c l e si o n s i n t h e lu n gs (f e males). A t the high

- dose level, t h ere we r e microscopic lesio n s in the ey e s, liver, adipose tiss u e, and l ung s

.

There was no t r eat m ent

- r elat e d i n cr e ase 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 decr e ased bo d y

- w e ight gain (both sexes) and food consumption (f

e males), as well as alte r ations in clinical chemistry para m et e rs [incr e ased B U N
, cre a t inin e , a n d a l an i n e

a m in o t r an s f

e r a se , d e c r e a s e d gl u c os e ] i n bo t h

s e x e s , d e c r e a s e d b r ai n w e ig h t i n f e m a l e s , an d h i s t op a t h ol o gi c a l le si o n s i n t h e li ve r an d k i dn e y s . 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 (19 97 ) 0, 5, 75, or 150 mg/kg/day Acceptable/Guideline See under 870.4100a above

870.4300 Carcinogenicity

(B 6 C 3 F 1 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 tre a tmen t

r e la t e d in c r e a s e in ki d ney weights in both

sexes and mic r oscopic r e nal lesio n s in males. T h ere was no trea t ment

- rela t ed increase in the i n cidence of any tumor type. Doses not adequate to assess carcinogenic potential.2,4

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Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.4300 Carcinogenicity

(B 6 C 3 F 1 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 a n i n c r e a s e d ab s o l u t e a n d /o r r e la t iv e kidn e y w e ig h t s an d a n in c re a se d in c id e n c e

o f re na l m i c r o sc op i c lesions. T here w as no trea t ment

- rela t ed increase in the i n cidence of any tumor type. 870.5100 Salmonella

typhimurium TA98, TA100, TA1535, Ta1537, Ta1538 MRID 41409801(1990)

Acceptable/guideline 100

- 100 0 μg /

p l a t e w / S9 ; 66

. 7

- 667 0 μg / p l a t e w / ou t S9 N o e v i den c e of ba c t e r i a l mu t a t i o n w / a n d

w / ou t S9

870.5395 in vivo mouse

micronucleus assay ICR mouse (bone marrow) MRID 41409804

,

41870101 (1990) Acceptable/guideline 40

- 40 0 μg / kg Neg a ti ve.

870.5450 Uns c hedu l e d DN A

s yn t he s i s a s s a y MRID 4 1 409807 (1990)

Acceptable/guideline N o evidenc e o f i n d uct i o n o f u n schedule d D N A synthes i s 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 N O A E L = 6 7 m g /k g. LOAEL = 227 mg/kg/day, bas e d o n a n inc r e a se d in c id e n c e o f in c o or din a t i o n a n d

s l ig h t g a i t abn o r m a li t i e s ( d e s c r ib e d a s f o r e pa w f le xi n g o r knu c klin g ) an d d e c r e a s e d
t o t a l m o t o r a c t i vi t y. 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 NO A E L = 71/68 m g /k g /d a y. LOAEL = 141/139 mg/kg/day

, ba se d o n i n c r e a s e d re la t iv e f

o r e li m b g r i p s t re ng t h and increas e d i n cidence of bilateral r e tinal d egene r ation.

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

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Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.7485 Metabolism and

pharmacokinetics (Fischer 344 rat) MRID 41737302 (19 90 ) 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 foll owing 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 fi rst 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 AUC 24h 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 gender

based difference in renal clearance of 2,4

- D in adult rats.2,4

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Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.7600 Dermal penetration

(human, male) F e ld ma n

. R

. J

. a n d

Mai ba c h

, H

. I

. ( 1 974 )

. P e r c ut a n e o u s P e n e t ra ti o n

o f S ome Pesticides and H e rbicides in Man. Toxi c ology 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 applic a tion was 5 .8 % ± 2.4 %; 10% DAF used in previous risk assessments

870.7800 Immunotoxicity

SRBC antibody

forming 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

- 492,4

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Page 51 of 96 Study Selected: Developmental T oxicity S tudy

- rat MRID No. : 00130407/00130408 Executive Summary : See Appendix A, Guideline § 870.3 7 00 a Dose and Endpoint for Risk Assessment: NOAEL = 25 mg/kg/day based on fetal skeletal malformations (14 th 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 t he 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 clear ance 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 ( UF A ) , 10X intraspecies variability ( UF H ), FQPA SF 1X].

A.3.2 Acute Reference Dose (aRfD)

- General Population

Study Selected: Acute Neuro toxicity S tudy

- rat
MRID No. : 43115201 Executive Summary : See Appendix A, Guideline § 870. 62 00 Dose and Endpoint for Risk Assessment: NOAEL = 67 mg/kg/day

, based on a n inc r e a se d in c id e n c e o f in c o or din a t i o n a n d s l ig h t g a i t abn o r m a li t i e s ( f o r e pa w f le xi n g o r knu c klin g ) an d d e c r e a s e d t o t a l m o t o r a c t i vi t y at the LOAEL = 227 mg/kg/day. Comments about Study/Endpoint/Uncertainty Factors: T he 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 ( UF A ) , 10X intraspecies variability ( UF H ), FQPA SF 1X].

A.3.3 Chronic Reference Dose (cRfD ) `

Study Selected: Extended 1

- G eneration R eproduction Toxicity S tudy

- rat MRID No. : 47972101 Executive Summary : Se e 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

m g/ k g/ d ay ; 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 assessm ent. 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 si nce 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, also2,4

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Page 52 of 96 have similar dose

- spacing i ssues 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 ( UF A ) , 10X intraspecies variability ( UF H ), FQPA SF 1X].

A.3.4 Incidental Oral Exposure (Short

- and Intermediate

- Term)

Study Selected: Extended 1

- G eneration R eproduction Toxicity S tudy

- rat MRID No. : 47972101 Executive Summary : Se e Appendix A, Guideline [§ 870. 3800; OECD 443/416] Dose and Endpoint for Ris k 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 assessment s . This study was selected instead of the rat developmental toxicity and rat subchronic oral toxicity studies used previously since there i s 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

- a nd 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 ( UF A ) , 10X intraspecies variability ( UF H ), FQPA SF 1X].

A.3.5 Dermal Exposure (Short

- and Intermediate

- Term )

A quantitative dermal assessment is not required f or 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 toxi city 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 s tudy; 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 f

indings 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 clearan ce mechanism would not be attained following human dermal exposure.

Additionally, a lthough there was no thyroid hormone assessment and the thyroid was not weighed in the dermal study, the rat extended 1

- generation reproduction toxicity (oral) study2,4

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Page 53 of 96 perfo rmed 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 ( ↓ T 3 and T 4 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. : 47 3987 01 Executive Summary : Se e Appendix A, Guideline [§ 870. 3465] Dose and Endpoint for Risk Assessment : No NOAEL for portal of entry effects was determined, a nd 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 total ly resolved following a 4

- week recovery period. Excessive salivation, labored breathing, and chromodacryorrhea were observed at the high

- dose level following the 12 th exposure and continu ed 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 c ells 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 (residen tial: 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 exte nded 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
sat uration (males 45/females 40 mg/kg/day). The changes in thyroid hormones (↓ T 3 and T 4 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 ( UF A = 3X, UF H = 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 metho dology was used, which takes into consideration the pharmacokinetic differences between2,4

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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 Calculation s
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 (σ g ), which is then used to determine the RDDR. The RDDR is a multiplicative factor used t o 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 ti ssue.

RDDR r = (RDD r

/Normalizing Factor) A / (RDD r /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 e asily calculated by using a software program designed specifically for computing the RDDR from the MMAD and σ g 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 Minut e SPECIES weight (g) Ventilation (VE, ml)

----------------------------------------------------------------------------- Rat 236 171.5 Human 8 0000 13800.0

---------------------------------------------------------------------- -------

Tracheobronchial SA ( c m^2) deposited fraction

-------------------------------------------------------------------------------- Rat 22.5 0.06 3 Human 3200. 0 0.0 752,4

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Page 55 of 96 ------------------------- ------------------------------------------------------- RATIO 0.00 7 0. 843

RDDR = (VE/SA) animal /(VE/SA) human * deposited fraction animal /deposited fraction human
= (171.7/ 22.5 ) animal /(13800/3200 ) human * 0.06 3 /0.0 75

= 1.490

HEC Calculations for Short

- and Intermediate

- term Occupational Exposure:

Assume worke r exposure for 8 hrs/day and 5 days/week and residential exposure for 24 hrs/day and 7 days/week:

HEC = NOAEL study * (daily duration of exposure animal /daily duration of exposure human ) * (days/week of
exposure animal /days/week of exposure human ) * RDDR

Occuaptional HEC = 0.0 5 mg/L * (6/8) * (5/5) * 1

. 490 = 0.05 6 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 Assessment s.

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 4 7398701 ) 28

- day TB 0. 0 5 Not attained 6 8 5 5 1

. 490 0.05 6 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

- Ter m 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 regi on

Route

- to

- Route Extrapolation

More information and details in: Memo, “Route

- to

- Route Extrapolations” J. Whalen and H. Pettigrew, 10/10/98.2,4

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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 u sed 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 re spiratory 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; durat ion 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: (0.0 5 6 mg/L) x 1 x 6 x 8 x 1 = 2 .6 8 mg/kg/day Residenti al 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 propose d 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 du e 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 numb er 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 arra y table reflects the time adjustment in the calculations.

A. 5 Executive Summaries

A. 5 .1 Subchronic Toxicity

870.3100 90

- Day Oral Toxicity

- Rat2,4

- 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 fec es [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 1 00 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 [bo th 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 co ntrol] 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 poste rior 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 i n proximal tubular cells [kidney, both sexes]. Many of the lesions correlated well with the alterations2,4

- 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 decrea sed body weight/body

- weight gain, alterations in some hematology [decreased platelets (both sexes)] and clinical chemistry [decreased T 3 (females) and T 4 (both sexes)] parameters, and cataract formation in females at the LOAEL of 100 mg/kg/day.

This subch ronic 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 toxi city 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 f emale [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 observ ed 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 dos e 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 o bserved 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); d ecreased 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 grou p. 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 al terations, 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 m g/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 bea gle 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 ra nge

- 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 s exes 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 body

weight 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 r espective 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 i n 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 dec rease in testes weight [absolute, relative to body, and relative to brain], although statistical significance was not2,4

- 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. Micro scopically, 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 m g/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 subc hronic 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 [ne at 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 attaine d 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 f emales 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; 8 70.3200] 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 b y 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

e ffects 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 (da y 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 high

do se 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 patholog y 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/da y, 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 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) is 0.05 mg/L, the lowest dose tested.

This subchronic inhalation toxicity study in the rat is A cceptable/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 7 5 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 a nd 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 body

weight 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 p regnancy 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 lo sses, 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 w eight 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 7 th cervical ribs [4 fetuses in 3 litters vs none in the control], presence of 14 th rudimentary ribs [4 fetuses in 3 litters vs none in the control]; malaligned sterneb rae [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 vs 2 fetuses in 1 litter of the control]; and unossified sternebrae # 5 or #6 [73 fetuses in 22 litters (3.32 fetus es/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, 14 th rudimentary ribs, and reduced ossificat ion 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 [2 nd wavy ribs, lumbar ribs] and mis sing 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/k g/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 N o. 003887] was inadequate, and the study was re

- evaluated [Document No. 011934]. The2,4

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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 no

observed

- adverse

- effect level [NOAEL] and lowest

- observed

- adverse

- effect level [LOAEL]. Additionally, the presence of 7 th cervical ribs and 14 th rudimentary ribs are considered skeletal malformations; previously listed as skeletal variations.

870.3700b Prenatal D evelopmental 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% methylc ellulose], 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 righti ng 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 peri od [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 da ys 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 wer e 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 l osses, 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 displ ayed domed head [hydrocephaly]. This finding is not considered treatment

related. There were no apparent differences in the incidence of external, visceral, or skeletal variations, anomalies, retardations, or malformations among the groups.

The maternal t oxicity NOAEL is 30 mg/kg/day, based on abortions, decreased body

weight 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 d evelopmental 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 lev els 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
expose d 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, parturi tion 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 numb ers). 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 gener al 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 wi de 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 fro m 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 p ost

- 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 ge neration 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. Af ter 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 parameter s were comparable among the groups (both sexes). P1 males displayed increased kidney weights2,4

- 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, t reatment

- 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 ind ices 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 sacr ifice, 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 s light 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 n ot 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 wer e 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) th at 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, treatment

related difference in absolute or relative anogenital distance in either sex and no differences in nipple/areolae retention between c ontrol 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
ext ent 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 Of

fspring (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 we re 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 neuropathologi cal 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. Male s 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. Plate let 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 t he 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) wer e 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
p pm 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 ppm2,4

- 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 tubu le 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 a nd 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%). Duri ng 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 com pared 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. Th ere 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 syste m 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 Respo nse 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 dos e 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/10 6 splenocytes among the male groups. Females at 600 ppm displayed a non

- significant decrease of 54% for AFC/spleen and 27% for the AFC/10 6 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 s exes). There were no significant treatment

related 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

- rela ted differences in the percent target cell cytotoxicity at any2,4

- 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): R eproductive Toxicity: There were no treatment

related 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 compare d 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 a bsolute (↓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 6 00 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), b ased 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 m g/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 antibody

forming 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, s atellite 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 reproducti ve 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 f

or 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

- g eneration reproductive toxicity study guideline (OECD 443, 416; November, 2010).

COMMENT: The major change is a change in t he 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 lit ters 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 F 1 generation (302,4

- 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 fee d 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 gr oups 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 m ales 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 high

dose F0 dams displayed a significant reduction in body weight gains during both gestation periods, with the greater deficit being observed during the s econd 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

- d ose 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,
t he 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 kid ney 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 level s, 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 litt ers ↓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 com parable 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 litt er 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 signifi cantly 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 bod y 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 b ody 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 th e 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 histopatho logical 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 ti ssues (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 t he 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 dos e 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/g roup 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 cli nical 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 wa s very slight in the high

- dose males2,4

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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 female s 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

- mo nth 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 dec reased 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 in creased 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 value s 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

- do se 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 high

dose, 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, althou gh 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 high

dose 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 st atistical 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 high

dose 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 w eights 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 [dose

related].

Gross pathology findings included decreased fat in high

- do se 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 opacity2,4

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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 sexe s 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 degenerati on 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. Tumo r 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 (b oth 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 l ungs. 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 mal e 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 re peat 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 mid

dose 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 of2,4

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Page 76 of 96 the males be evaluated. These additional dat a 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

w eight/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. D ecreased 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 l evel 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 we re 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 low

dose females also displayed a decre ase [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 lev els, 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%, 6 3%, 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 ob served 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 mi d

- and high

- dose levels. Males at the high dose also displayed decreased brain weight, but statistical significance was not attained. In general,2,4

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Page 77 of 96 the organ

- weight effects observed can be attributed to the lower body weight, although the decreased testes we ights 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.

T he 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, decrease d 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/da y [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 contr ol] 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%/high

dose 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 with2,4

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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 plat elet 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 bu t 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 sex es 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 h igh

- 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 foc i 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 dos e], 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. T he 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 (m ales), 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

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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), liv er (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 stre ngth. 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/ g uideline

, and it satisfi es 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 B 6 C 3 F 1 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 [i nterim 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 displaye d 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, respec tively], and the high

dose 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 relat ive] 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

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Page 80 of 96 Gross pathology findings were comparable a mong 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 reg eneration 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. T here 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 rena l 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 stu dy 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 Muta tion2,4

- D Human Health Risk Assessment DP No. D389455

Page 81 of 96 870.5100, Bacterial reverse mutation test 4 1409801 Acceptable/guideline No evidence of bacterial mutation with or without S9

.

870.5 450

, Unscheduled DNA synthesis assay 4 140980 7 Acceptable/guideline No evidence of induc tion of unscheduled DNA synthesis

.
870.5395, In vivo mouse micronucleus test 4 1409804 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 evaluatio ns, 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 neurop athological 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 si gns 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 observ ed 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 classif

ied 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

- rat2,4

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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 approxima tely 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 a pprox. 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 do se. 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 t he 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 urin e. 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 bette r 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 levels2,4

- 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 imm unotoxicity (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 mal es 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/10 6 splenocytes among the male groups. Females at 600 ppm displayed a non

- significant decrease of 54% for AFC/spleen and 27% for the AFC/10 6 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 treatment

related effects on absolute or relative spleen or testes weights in males, and no significant
treat ment

- 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 cytot oxic 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 antibody

forming 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 re quirement ( 870.7800) for an immunotoxicity study.2,4

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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 plas ma concentration time curve (AUC 24h ) 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. F emales at the 600 ppm and 800 ppm dose levels displayed reduced body

weight 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 AUC 24h 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 exp ected 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 AUC 24h at 400 ppm, 600 ppm, and 800 ppm, respectively.

The study report states that in all cases , C max 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 sm all (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 initial2,4

- 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 def inition 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 post

mating a nd 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 t he 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), a nd 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 bloo d 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 t he 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 fert ility (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 whethe r the findings were2,4

- 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 d uring 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, althou gh 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 lesi ons 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%/f emales ↓ 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 contro l.

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 lactatio nal 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). T he kidney findings confirm the gender

- based difference i n 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 (dieta ry 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 tha n 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 pre viously 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 ad ult 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 dose2,4

- D Human Health Risk Assessment DP No. D389455

Page 87 of 96 level of 50 mg/kg/day was discussed as an app ropriate 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 A ppendix 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 [ 14 C]2,4

- D DMA at 3.13 lb ae/A (level of exaggeration: 1X)

. Metabolite Fraction Corn, Forage Corn, Grain 1 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 2.3 4

-- -- 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 3.2 8
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

-- -- Accountability 3 112 100 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 applicat ion.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 [ 14 C]2,4

- D DMA at 2.6 2 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 2.8 6 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 5.6 4 Unextractable (PES) 2

-- -- -- -- NR NR

-- -- Accountability 3 105 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

- 1 2

Soybean (event 416 ) Forage and Hay Following Application of [ 14 C]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 F orage 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 extractable 2 110.3 55.39 93.8 33.36 108.7 40.24 Unextractable (PES) 3 2.8 1.393 4.2 1.478 8.2 3.027 Accountability 4 113 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 Resi dues in AAD

- 1 2

Soybean (event 416 ) Seed Following Application of [ 14 C]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 extractable 2 90.9 0.338 Unextractable (PES) 3 2.6 0.010 Accountability 4 93.5

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 combust ion 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 K OW (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) 762,4

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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 Residen tial 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 website 14

.

14 http://www.epa.g ov/pesticides/science/handler

- exposure

- data.html and http://www.epa.gov/pesticides/science/post

- app

- exposure

- data.html2,4

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Page 93 of 96 Appendix E . Occupational Exposure/Risk Summary Tables

Table E

- 1. Occupational Handler Non

- Cancer Exposure a nd Risk Estimates for 2,4

- D

.

Exposure Scenario Crop or Target Inhalation Unit

Exposure ( μ g/lb ai) 1 Maximum

Application Rate 2 Area

Treated Daily 3 Inhalation

Mitigation Level : No respirator Dose (mg/kg/day) 4 MOE 5

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 ( μ g/lb ai) × Conversion Factor (0.001 mg/ μ g) × Application Rate (lb ai/acre or gal) × Area Treated or Amou nt Handled Daily (A or gal/day) ÷ BW (kg). 5 Inhalation MOE = Inhalation NOAEL (mg/kg/day) ÷ Inhalation Dose (mg/kg/day).2,4

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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 Levels 1 (ppm)

n Sample Min. Sample

Max. LAFT 2 HAFT 2 Median Mean Std. Dev.

2,4

- D
So ybean

, early forage 1.97

- 2.10 6

- 8 24 <0.01 4.014 < 0.01 3.930 0. 272 0.6 96 1.146

Soybean

, forage 2.9 7

- 3.1 3 6

- 8 2 4 <0.01 18.424 < 0.01 18.282 0.2 02 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 2.9 7

- 3.1 3 6

- 8 24 <0.01 60.413 <0.01 58.783 0.613 3.769 11.903 Soybean, seed 2.9 7

- 3.1 3 51

- 103 2 4 <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 2.9 7

- 3.1 3 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 2.9 7

- 3.1 3 6

- 8 2 4 <0.01 132.961 < 0.01 131.509 36.701 40.509 35.918 Soybean, seed 2.9 7

- 3.1 3 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 betw een 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 Levels 1 (ppm)

n Sample Min. Sample

Max. LAFT 2 HAFT 2 Median Mean Std. Dev.

2,4

- D
Co rn, 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 0.9772,4

- 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 Levels 1 (ppm)

n Sample Min. Sample

Max. LAFT 2 HAFT 2 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 = l owest 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 Mexico 2 Codex 3
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

Commodity 1 Tolerance (ppm) /Maximum Residue Limit (mg/kg) US Canada Mexico 2 Codex 3 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 quan titation; 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 n ot been finalized by the CCPR and the CAC.