### Page 2 of 186 Conservation and Health; and the National Council For Air and Stream Improvement, Inc. 1

1 Detailed information available at docket EPA-HQ-OPP-2008-0877 at www.regulations.gov



### 1.0 Introduction

The NRDC petition focused on the re gulation of 2, 4-dichlorophenoxyacet ic acid (i.e., 2, 4-D) raised a number of technical issues. Subsequent responses and additional comments by the 2, 4-D task force and other impacted organizations also raised additional t echnical issues. The purpose of this document is to provide a technical basis for the Ag ency response to these submissions . It describes the screening and review process which has been completed by the Agen cy for the various citati ons which were used by NRDC and in the subsequent related comments. Th ese documents referenced above cited a number of guideline and literature research st udies as well as other sources of information. This process involved several steps that included (1) in dentifying all pertinent informati on, (2) developing a framework for categorizing the information, referred to as “bins” , (3) completing a screen of each citation, and (4) completing further review of a study based on the results of the screenin g process if necessary.

The process used in this document for categorizing or “binning” the cited studies and the criteria used for screening purposes is described in Section 2: Overview Of Binning And Review

. Section 3: Overview of Hazard Data presents a brief overview of the fi ndings obtained by examining all data related to the hazard assessment and dose response ch aracterization. This topic is very broad and

includes issues related to muta genicity, endocrine disruption, ne urotoxicity, metabolism, and carcinogenesis. Section 4: Overview of Exposure/Epidemiological Data presents a brief overview of the findings obtained by examining all data related to exposure assessment. This topic is very broad and

includes issues related to diet ary exposure, epidemiological an alysis, population based biological monitoring, and the use of planned exposure st udies designed to elicit the impacts of specific

parameters. Section 5: Overview of Pet Incident Data presents a brief overview of the findings obtained by examining all data related to pet incidents asso ciated with 2, 4-D use. Section 6: Summary presents a brief overview of all findings obtained by ex amining the data. Several sources of data focused on ecological issues such as ecotoxicity. These have not been addressed herein but have been evaluated in a separate document unless they ar e pertinent to a human health relate d issue. [Note: Each study has

also been reviewed to determine if it needs a review for complian ce with ethical criteria for human research and are noted as such.]

Appendix A: Data Summary contains all citations with an associ ated code to describe the bins in which they have been placed for the purposes of this analysis and which indicates the type of review received (e.g., screen only). Appendix B: Hazard Data Summaries includes the reviews of the data prepared for those types of information. Similarly, Appendix C: Exposure Data Summaries includes the reviews of the data prepared for those types of information. Appendix D: Pet Incident Summaries includes the reviews of the studies focused on impacts for pets as a result of 2, 4-D exposure. A tiered approach was used to evaluate ea ch study cited by NRDC and in the subsequent comments. In this process each study was screened which entailed devel oping a summary that includ ed a descrip tion of the methods and results and a description of the credib ility related to how it wa s used by each specific commenter or in the NRDC petition itself. If further analysis was required, a more extensive review was completed. In some cases, information was cited by various submitters that were not applicable to the groupings described above. Generally this type of information required no fu rther summary outside of the screening exercise. The screening summarie s for these types of doc uments are included in

### Page 3 of 186 Appendix E: Meta Information . This is the only mention of these papers in this document.

Overview Of Binning Process

A variety of data were cited in the NRDC petiti on and the associated, subsequent documents. For efficiency purposes each citation was categorized based on a coding system that delineated the data based on the type of content and the level of review required. The codes that were used to delineate the data based on type of content includ e: hazard data, exposure data, pet in cident data, and ecological data. As indicated above, the category of hazard data is very broad and includes issues related to mutagenicity, endocrine disruption 2 

### , neurotoxicity, metabolism, and carcinogenesis. Each of these subcategories has a unique code associated with it fo r descriptive purposes. The codes for the various types of hazard data include:

• MUT: For studies that a ddress possible issues re lated to mutagenicity.

• EN: For studies that address possi ble endocrine disruption issues.

• MET: For studies that address i ssues related to metabolism.

• NEU: For studies that address possible neurotoxic effects.

• DA: For studies that address dermal absorption issues.

• CAN: For studies that ad dress carcinogenesis.

• IP/SUBQ: For studies that involved interper itonel or subcutaneous dosing regimens.

• NC: Relates to a hazard issue but does not pe rtain directly to a category described above.

Along with varied types of hazard da ta, a number of different types of exposure data were cited. These categories were also assigned separate codes, which include:

• EPI: For studies that address epidemiological data.

• PB: For studies that address population-base d biological monitoring or other sampling.

• PL: For studies that address planned exposur e studies such as required by guideline.

Studies that address pet incidents were fairly limited and are denoted by the code “PI”.

It should be noted there are severa l studies that address a variety of ecological issu es (e.g., ecotoxicity). In these cases, for track ing purposes, the studies are denoted by the code “ECO” . They have been reviewed in this document only if their results relate to another category associated with human exposure or risk. 3 

### In these cases the study will also be co ded with the other pe rtinent category code (e.g., “ECO/EN” for an eco study which may provide info rmation on endocrine disruption issues). [Note: In some cases when books are cited, a docume nt has already been addressed in some manner, or

the material does not fit one of the cr iteria described above the code is “ NR ” (No Action Required) because a screening document or fu rther reviews are not required.]

2 Note that separate documents have been developed as part of the 2,4-D screen as an endocrine disruptor. Please refer to them for more detailed information (TXR No. 0055461). 3 EFED created a separate review document focused on the eco-r elated studies that do not co ntain any human health related

information. Please refer to it for more detailed information.

### Page 4 of 186 It should be noted that the Agency must comply with updated requirements for protecting human subjects involved in research (i.e., 40CFR26) a nd review, under certain circumstances by the Human Studies Review Board (see http://www.epa.gov/osa/hsrb/ for more detailed information). It is not clear if the studies cited in the petition and related docu ments would meet current Agency criteria that would allow for their use in a regulatory decision. All studies with possible issues have been noted with a “Yes” if they need further ethics review should they be relied upon by the Agency.

Overview of Hazard Data

A summary of the available hazard data is included be low. In some cases specific papers were utilized in the NRDC petition itself and in one or more subs equent, related comments. In other cases citations were utilized only in single comments . For clarity, this section summar izes the actions associated with each citation for each code and the results are presen ted in alphabetical order based on the name of the primary author. Additionally, it is note d where each citation has been used.

Bin: Neurotoxicity

Bortolozzi, et al. (2003) Asymmetrical Development of the Monoamine Sy stems in 2, 4-Dichlorophenoxy acetic acid (2,4- D) Treated Rats. [Bin: Neurotoxicity] [Cited: NYS DOH] [Human Studies Considerations Apply: No] The cited study is one of several research studies fr om the same group of investigators looking into possible mechanism of 2, 4-D neur otoxicity. Study examined the e ffects of 2, 4-D on endogeneous monoamines and their metabolites [noradrena line, dopamine, 3, 4-dihyrdoxyphenylacetic acid, homovanillic acid, serotonin, and 5- hydroxyindolacetic acid] in the brai ns of neonatal rats exposed during gestation [dietary exposure of pregnant rats from gestation day (GD) 16 to 23] and/or postnatally

via the diet up to postnatal day (PND ) 90. According to the au thors, results support view that during the critical period in which monoami nes can influence cell developmen t, neonatal exposure to 2, 4-D produces alterations on these neurotransmitter systems, which they equated to previous findings expressed in spontaneous circling activity towards the right side by adult offspr ing of dams exposed to 2, 4-D, indicating a correlation be tween monoamines brain level altera tions and this behavior. The study was cited by NYS DOH as an additional study that should be considered in any new assessment along with those identified in the NRDC petition. The cited study does not pr ovide any dose-response information since only one dose level was tested. When there is only one toxic do se administered to test animals, the effects seen do not generally provide in formation useful for risk assessment as do studies conducted with multiple doses, including dose levels at which effects are not seen. Having information

at dose levels above and be low a level at which effect s are seen provides useful information that can be

considered in a risk assessment. Additionally, the dose level tested (70 mg/kg/day) in the cited study

exceeds the rat’s renal clearance m echanism; i.e., it overwhelms the body’s ability to excrete 2, 4-D, which allows 2, 4-D to build up in the body and exert its toxic effects. By design, studies investigating possible mechanisms of toxicity use dose levels that will cause the e ffect (toxicity) being investigated. The cited study does not provide information regardi ng potential effects of 2, 4-D on neurotransmitters

at dose levels below the saturation level for rena l clearance. An extended 1-generation reproductive toxicity study (MRID 47972101) on 2,4-D (submitted to Agency in response to the data requirements identified in the 2005 RED) includes an assessm ent of developmental neurotoxicity (including

### Page 5 of 186 neurobehavioral tests) at dose le vels that do not saturate metabo lic processes. In the extended 1- generation reproductive toxicity study, 2, 4-D was administered to young adult rats for ≈ four weeks prior to mating and continuing through mating, gest ation, and lactation. The young rat was exposed during gestation, lactation via the milk, and directly via the diet following weaning. Offspring were subjected to a developmental ne urotoxicity (DNT) assessment, wh ich incorporated a functional observational battery (FOB), which included cage-s ide, hand-held, and open-field observations of behavior, and measurements of body weight, rectal temperature, grip strength, and landing foot splay; motor activity using an automated system for measurin g motor activity; and an assessment of the startle response to auditory stimuli. On PND 60, a neuropathological exam was conducted on the brain (including the cerebrum, thalamus/ hypothalamus, cerebellum, and medu lla), spinal co rd, dorsal root ganglia, dorsal and ventral roots, peripheral nerves, and skeletal musc le. The examination of the brain included assessment of brain weight and gro ss measurements, microscopic measurements (morphometrics), and brain myelin. Three dose levels were tested. There were no treatment-related adverse effects on any of the numerous parameters a ssessed across life stages, which included multiple neurotoxicity-related endpoints simila r to those in the study cited by NRDC. The parental rats were assessed for clinical signs of toxicity and maternal behavior, am ong other parameters. Maternal behavior towards the offspring was comparable among the group s, and there were no c linical signs, such as circling to the right observed in the dams or offspring or signs of maternal ne glect. Although monoamine levels were not monitored in this study, neonatal exposure was simila r to that in the cited study, and the dose levels were adequate (appr oaching saturation of renal clearance). The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protect iveness of the existing Points of Departure as to neurotoxic

effects. Bortolozzi, et al . (1999) Behavioral Alterations Induced in Rats by a Pre- and Postnatal Exposure to 2, 4- Dichlorophenoxyacetic Acid. [Bin: Neurotoxicity] [Cited: NYS DOH] [Human Studies Considerations Apply: No] Study examined the effects of 2,4-D on physical/sexua l development [fur grow th, eye opening, testes descent, vaginal opening); surface righting (considered a reflection of subcortical maturation); negative geotaxis (tests labyrinthine and cerebellar integr ation; hindlimb support (t ests neuromuscular and locomotor development); open field tests (measures activ ity and habituation; # cro sses, rearings, vertical head movements, grooming episodes); 5-HT syndr ome behaviors [forepaw movements (treading, excessive placement or tapping), hunching (back arch ing), sprawling of limbs, backing, hindlimb abduction, mobility (ability for locomotion despite gait abnormalities, including paddling), abnormal head movements (vertical or lateral side-to-side repetitive movements), wet-dog shakes (including head shakes), lying on side, limb extension dystonic, and flat body posture; circling behavior; measurement of catalepsy] of neonatal rats exposed during gestation (d ietary exposure of pregnant rats from GD 16 to 23) and/or postnatally via the diet up to PND 90. According to th e investigators, 2, 4-D exposure at a dose producing no overt signs of toxicity may cause long- term alterations in functional state of rat CNS, detected through a neurobehavioral te st battery; however, they indica ted that exposure during pre- and postnatal development induced behavioral abnormalitie s that seem to disappear in adulthood after 2,4-D cessation. The study was cited by NYS DOH as an additional study that should be considered in any new 2, 4-D assessment, along with those identified in the NRDC petition. This study is one of several

### Page 6 of 186 studies by the same investigators performed to a ddress the mechanism of 2,4- D neurotoxicity, and it does not provide any dose-response information. The onl y dose used (70 mg/kg/ day) exceeds rat renal clearance levels and does not provide inform ation regarding potential effects of 2, 4-D on

neurotransmitters at dose levels below the satura tion level for renal clearance. In the extended 1- generation reproductive toxicity study on 2,4-D (MRID 47972101), young a dult rats were administered 2, 4-D for ≈ four weeks prior to mating and continuing through mating, ge station, and lactation. The young rat was exposed during gestation, lactation, a nd directly following weaning. Offspring were subjected to a developmental neur otoxicity (DNT) assessment, which included functiona l observational battery (FOB), motor activity and ac oustic startle response (ASR), similar parameters as assessed in the cited study. Additionally, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation and br ain morphometry, and a special stai n was used to evaluate brain myelination. The extended 1-gene ration reproductive toxicity study on 2, 4-D was performed at three dose levels up to the saturation level for renal cl earance. There was no evidence of developmental neurotoxicity in this study that i nvestigated the same parameters as assessed in the cited study. The no effect dose is greater than the poi nts of departure (PODs) utilized fo r the human health risk assessment supporting the 2, 4-D RED. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by th e Agency, and the results do not yield information that would cause the Agency to modify its conclu sions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects. Bortolozzi, et al. (2004) Effects of 2, 4-Dichlorophenoxyacetic Acid Expo sure on Dopamine D2–Like Receptors in Rat

Brain. [Bin: Neurotoxicity] [Cited: NYS DOH] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on neonatal rats exposed during gestation (dietary exposure of pregnant rats from GD 16 to 23) and/or postnatally via the diet up to PND 90. Ne onates were sacrificed at several ages for membrane r eceptor binding assays employing [ 3 

### H] nemonapride. According to the investigators, regional increases in D2–type receptor density may explain certain behaviors, such as catalepsy and right-turning preferen ce in 2, 4-D exposed rats observed in earlier studies by the same investigators. When 2, 4-D expos ure stopped, DA D2–type receptor density apparently recovered and stabilized to control level, sugges ting reversible vulnerability of D2–type receptors to 2, 4-D exposure. This study is one of several re search studies by the same inves tigators performed to address the mechanism of 2, 4-D neurotoxicit y, and it does not pr ovide any dose-response information. The only dose used (70 mg/kg/day) exceeds rat renal clearance levels and does not provide information regarding potential effects of 2, 4-D on neurotransmitters at dose levels below the saturation level for renal clearance. The study was cited by NYS DOH as an a dditional study that should be considered in any new assessment along with those identified in the NRDC petition. An extended 1-generation reproductive toxicity study on 2,4-D, in which offspr ing were exposed to 2, 4-D during gestation,

lactation via the milk, and directly via the diet through PND 22, 70, 139, in cludes an assessment of developmental neurotoxicity (incl uding neurobehavioral tests) at dose levels that do not saturate metabolic processes. In the extended 1-genera tion reproductive toxicity study on 2, 4-D (MRID 47972101), young adult rats were administered 2, 4-D for ≈ four weeks prior to mating and continuing through mating, gestation, and lact ation. The young rat was exposed during gestation, lactation, and directly following weaning. Offspring were subj ected to a developmenta l neurotoxicity (DNT) assessment, which included functional observational battery (FOB), motor activity and acoustic startle

### Page 7 of 186 response (ASR), similar parameters as assessed in the cited study. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropat hology evaluation and brain morphometry, and a special stain wa s used to evaluate brain myelin ation The extended 1-generation reproductive toxicity study on 2,4-D was performed at three dose levels up to the saturation level for

renal clearance. There was no evidence of devel opmental neurotoxicity. The cited study does not identify a more sensitive endpoint for human hea lth risk assessment than already identified by the Agency and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neur otoxic effects. Bortolozzi, et al . (2001) Intracerebral Administration of 2, 4-Dichlo rophenoxyacetic acid Induces Behavioral and

Neurochemical Alterations in the Rat Brain. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D in male rats follo wing intrastriatal injection. Injection of 2,4-D into the striatum produced a marked depression in lo comotor activity and elicited a moderate circling towards the right side post dose. These changes we re accompanied by a decrease in serotonin and an

increase in homovanillic acid levels. Administra tion into the nucleus accumbens induced similar behavioral and neurochemical patter ns, although rats did not present right turning. The Agency notes that the route of exposure is not ap propriate for human risk assessment; human exposure would not be by direct injection into the brain. Additionally, the re lationship of the levels of 2, 4-D attained in the brain following intracerebral injection to those atta ined following oral ingestion is not known. NRDC cited the study, among others, as evidence that postna tal exposures to 2, 4-D during the critical period of development of the infant brain rais e serious scientific concerns. The Task Force states that this study cited by NRDC was not a developmental neurotoxicity study but a very high dose a dult toxicity study in which 2, 4-D was directly administered into the rat’ s brain, which is a route not relevant to human exposures. The Task Force pointed out that the estim ated concentrations of 2, 4-D in each brain area were 2-4 mM, which was 40- to100-fold the concentra tion in brain after systemic treatment. As noted above, in the extended 1-generation reproductiv e toxicity study on 2,4-D (MRID 47972101), young rats were exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning and were subjected to a developmen tal neurotoxicity (DNT) assessment, which included functional observational battery (FOB), motor activity and acoustic startle response (ASR). Additionally, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation and brain morphometry, and a special stain was used to evaluate brain myelination. The extended 1- generation reproductive toxicity stud y on 2, 4-D was performed at three do se levels up to the saturation

level for renal clearance. There was no evidence of developmental neurotoxicity. These no effect doses are greater than the points of depa rture (PODs) utilized for human hea lth risk assessme nt supporting the RED. The cited study does not identify a more sensitiv e endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protect iveness of the existing Points of Departure as to neurotoxic

effects.
Brusco, et al . (1997) 2, 4-Dichlorophenoxyacetic Acid Through Lactation Induces Astrogliosis in Rat Brain. Molecular and Chemical Neuropathology. [Bin: Neurotoxicity]

### Page 8 of 186 [Cited: NYS DOH] [Human Studies Considerations Apply: No] The goal of the study was to detect the effect of 2,4-D on astrocytes in different CNS regions of 25-day old pups exposed to 2,4-D through mother’s milk [dams exposed i .p. during lactation days (LD) 9-25] using 2 rat astroglial markers. The investigators poi nt out that one of the most widely documented cellular reactions following nervous system damage ha s been hypertrophy of astrocytes, often referred to as a reactive gliosis or astrogliosi s characterized by rapid synthesis of glial fibrillary acidic protein (GFAP) intermediate filament (a sensitive and early biomarker of neurotoxicity). A glial reaction was

detected at the level of serotone rgic nuclei and extreme astrogliosis in the hippocampus and cerebellum in 2, 4-D pups. There was a significant increase in the number, size, number of processes, and density of immunostaining. The investigators c oncluded that exposure during the fi rst days of life modifies the astroglial cytoarchitecture in parallel with previo us neuronal changes observed by these investigators. The study does not provide any dose-response inform ation (discussed previously) and the dose to pups is unknown. The only dose tested (70 mg/kg/day) is one expected to exceed rat renal clearance levels. The study was cited by NYS DOH as an additional study that should be considered in any new assessment along with those identified in the NRDC pe tition. The cited study is one of several research studies from the same group of investigators looki ng into possible mechanism of 2, 4-D neurotoxicity. As noted above, in the extende d 1-generation reproductive toxi city study on 2,4-D (MRID 47972101), young rats were exposed duri ng gestation, lactation ( via the milk), and directly (via the diet) following weaning, and were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (FOB), motor activit y and acoustic startle res ponse (ASR). On PND 60, the young rats were perfused for central nervous sy stem (CNS) and periphera l nerve neuropathology evaluation and brain morphometry, and a special stain was used to evaluate brain myelination. Myelination was also evaluated in the PND 22 pups . At the highest dose tested, which exceeded in males and was approaching the level of renal saturation in females, there was no evidence of developmental neurotoxicity/ne uropathology. The cited study does not identify a more sensitive endpoint for human health risk assessment than alre ady identified by the Agency, and the results in the cited study do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects. Duffard, et al . (1995) Developmental Neurotoxicity of the H erbicide 2,4-Dichlorophenoxyacetic Acid. [Bin: Neurotoxicity; Endocrine] [Cited: NRDC petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] The citation is only an abstract, which has few details. Based on the title of the c itation, article was binned under neurotoxicity. Study examined the effect s of 2, 4-D on neonatal ra ts exposed at a single dose level during gestation ( i. p. exposure of pregnant rats fro m GD 16 to PND 21) and postnatally via the diet up to adult life. Vaginal smears were mon itored, and prolactin and pr ogesterone levels were measured. Authors stated that these preliminary results demonstrate a 2, 4- D adverse effect on the endocrine system. Study does not provide any dose-response information. The only dose used (70 mg/kg/day) is one expected to exceed rat renal cl earance levels following oral exposure. The study does not provide information regarding pot ential effects of 2, 4-D on estrous cycle or hormone levels at dose

levels below the saturation level fo r renal clearance. NRDC cited this study as demonstrating endocrine disrupting effects of 2,4-D. The Task Force cited it as one of a series of studies that NRDC contends provides evidence of 2, 4-D endocrine activity. The Task Force does not consider this study as providing compelling evidence of potential environmental or human endocrine health concerns, since it was

### Page 9 of 186 conducted at a high dose (well above saturation of renal clearance) a nd, therefore, is of limited value for risk assessment. The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The ci ted study was considered by the Endocrine Disruptor Review Team (TXR No. 0055461) . An extended 1-generati on reproductive toxicity study on 2,4-D, in which offspring were exposed to 2, 4-D during gestation, lactation, and directly via the diet through PND 22, 70, 139, includes an assessm ent of the endocrine system; evaluation of potential effects of 2, 4-D on parental male and female reproductive functi on; potential effects in offspring on survival, growth, development of ner vous, immune, and reproductive systems; endocrine and selected systemic toxicity parameters; include s estrous cycle evaluation (female adult rats and offspring), evaluation of sperm parameters, thyr oid hormone assessment; anogenital distance, nipple retention and puberty onset. There were no treatment-related adve rse effects on any of the numerous parameters evaluated to assess the endocrine system following 2, 4-D exposure at dose levels approaching saturation of re nal clearance. The changes in estrous cy cle noted in the ci ted study were not replicated in the exte nded 1-generation reproductiv e toxicity study. The cite d study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. Additionally, the extended one-genera tion reproduction study confirms th e protectiveness of the existing Points of Departure as to neurotoxic effects. Duffard, et al . (1996) Central Nervous System Myelin Deficit in Rats Exposed to 2,4-Dichlorophenoxy acetic Acid Throughout Lactation. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force; NCASI] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on 25-day old pups exposed to 2, 4-D through mother’s milk (dams exposed i.p. during LD 9-25, 9-15, or 15-25). Myelin defi cit was observed in pups nursed from PND 9- 25 or PND 15-25; in latter group, body and brain weight were not affect ed, so deficit in myelin cannot

be attributed to undernourishment. There was a clea r deficit in myelin staining and myelin chemical markers. The Agency notes that the study does not provide any dose-res ponse information. The only dose used (100 mg/kg/day) exceeds rat renal clea rance levels and does not provide information regarding potential effects of 2, 4-D on myelination at dose levels below the saturation level for renal clearance. The cited study was previously consider ed by the Agency in its rationale for additional toxicology data (DNT) in the 2005 RED. NRDC cited this study and others that they conclude identify adverse effects in offspring afte r lactational exposure. One of se veral studies cited by NRDC as providing evidence that postnatal exposures to 2, 4- D during critical period for development of the

infant brain raise serious scientif ic concerns. The Task Force noted that the study was conducted at an excessively high dose (level expected to exceed the th reshold level for saturatin g renal clearance of 2, 4- D) and included the use of an unrepresentative route and mode of administration (intraperitoneal) that is not relevant for human health pesticide risk assessment. Although the dams were exposed i.p ., the Agency notes that the pups were exposed via maternal milk. The dose to the pups is not known. The Task Force also considered this paper and other NRDC cited papers from the same group of investigators to be methodologically flawed [litter was not used as th e unit for statistical analyses (to avoid bias due to litter effects) ; the reporting of hist opathological evaluation limited, etc.]. NCASI referenced this study as one of the studies cited in the NRDC petition as supporti ng the claim that infants are at risk; however, NCASI argues that doses used in the study are extremel y high and likely exceed renal clearance levels and are in range known to produ ce frank maternal toxicity, which is inappropriate dose selection for developmental toxicity studies. NC ASI considers the fact that high doses are needed to elicit the reported effects as support of previous EPA conclusion that 2, 4-D use according to label

### Page 10 of 186 direction does not pose a risk to humans. The cited study is one of several research studies from the same group of investigators looking into po ssible mechanism of 2, 4-D neurotoxicity. In the extended 1-generation reproductive toxicity study on 2, 4-D (MRID 47972101), yo ung adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (F OB), motor activity and acousti c startle response (ASR). An evaluation of myelin in the P ND 22 and PND 60 offspr ing following exposure during gestation and lactation was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation a nd brain morphometry. The extended 1-generation reproductive toxicity study on 2, 4-D was performed at three dose levels up to the saturation level for renal clearance. At the highest dos e tested, there was no evidence of developmental neurotoxicity, and there was no adverse effect on myelination. The c ited study does not identify a more sensitive endpoint

for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modi fy its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-genera tion reproduction study confirms th e protectiveness of the existing Points of Departure as to neurotoxic effects. Evangelista de Duffard, et al . (1995) Altered Behavioral Responses in 2,4-Dichloro phenoxyacetic Acid Treated and Amphetamine Challenged Rats. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force; NCASI ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D following acute exposure ( i. p .) to rats. Various components of the (5-HT) serotonin syndrome (behavioral response induced by stimulation of 5-HT receptors) were

observed in rats after acute dose of 2, 4-D plus amphetamine, but not with 2, 4-D alone. The cited study

is one of several research studies from the same group of investigators looki ng into possible mechanism of 2, 4-D neurotoxicity. NRDC cited study as s howing that 2, 4-D result s in delays in brain development and abnormal behavior patterns, including apat hy, decreased social in teractions, repetitive movements, tremors, immobility, with females being more sensitive. The Task Force stated that the study does not assess brain development, and the findi ngs could be non-specific effects rather than any selective neurological effect. NCASI st ated that the NRDC petition fails to mention that dose levels used likely exceed renal clearance levels and the route of exposure is not appropriate. The dose levels used (50 and 100 mg/kg/day) are at or exceed rat re nal clearance levels. An extended 1-generation reproductive toxicity study on 2, 4-D, in which offspring were exposed via an appropriate route to 2, 4- D during gestation, lactation, and directly through PND 22, 70, 139, includes an assessment of developmental neurotoxicity (includi ng neurobehavioral tests) and neuropathology at dose levels that do not saturate metabolic proce sses. The young rats were exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Offspring were subjected to a devel opmental neurotoxicity (DNT) assessment, which included functional observ ational battery (FOB), mo tor activity and acoustic startle response (ASR). An evaluation of mye lin in the PND 22 and PND 60 offspring following exposure during gestation and lactation was perform ed. On PND 60, the young rats were perfused for central nervous system (CNS) a nd peripheral nerve neuropathology ev aluation and brain morphometry. The extended 1-generation reproductiv e toxicity study on 2, 4-D was perf ormed at three dose levels up

to the saturation level for renal clearance. At th e highest dose tested, th ere was no evidence of developmental neurotoxicity and no adverse effect on myelination. The cited study does not identify a

### Page 11 of 186 more sensitive endpoint for human h ealth risk assessment than alrea dy identified by the Agency. The extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects. Ferri, et al . (2007)

Selective Oxidative Stress in Brain Areas of Ne onate Rats Exposed to 2,4-Dichlorophenoxyacetic Acid Through Mother’s Milk. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Study examined the effects of 2,4-D on 25-day old pups exposed to 2,4-D through mother’s milk (dams exposed i.p. during LD 9-25 to dose level of 100 mg/kg/day). Reactive oxygen species (ROS) levels increased in the prefrontal cortex, striatum, and midbrain; GSH content decreased in striatum and midbrain of 2, 4-D pups compared with basal levels of 25-day-old pups. The investigators concluded that the results suggest that the de veloping brain areas have different su sceptibilities to th e adverse effect of 2, 4-D, especially those areas related to the dopaminergic system, and that oxidative stress is one 2, 4- D mechanism of toxicity. One of several studies cited by NRDC rega rding lactational exposure and the need to consider lactational expos ure in the aggregate RA. The Task Force referenced this study as another publication cited by NRDC that was conducted at a dose well above pharmacokinetic nonlinearity and included the use of an unrepr esentative route and mode of administration (intraperitoneal) that is not releva nt for human health pesticide risk assessment, which they conclude would further amplify nonlinear pharmacokinetic perf ormance. The Agency agrees that the route of exposure of the maternal rats is not relevant for human pesticid e risk assessment; however, the pups were exposed via the maternal milk, which is relevant. NRDC is correct that lactational exposure was not addressed in the 2, 4-D di etary exposure assessment. The Ag ency acknowledges that there are remaining uncertainties associated with attempting to characterize exposures from breast milk. In order to address this uncertainty, EPA is seeking monitoring data for 2,4-D in human breast milk (e.g., EPA’s National Children’s Study). The cited study is one of several research studies from the same group of investigators looking into possible m echanism of 2, 4-D neurotoxicity. In the extended 1-generation reproductive toxicity study on 2,4- D (MRID 47972101), young adult rats were administered 2, 4-D via

the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic pr ocesses. The young rat was exposed during gestation, lactation ( via the milk), and directly (via the diet) follo wing weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, whic h included functional obser vational battery (FOB), motor activity and acoustic startle response (ASR). An evaluation of myelin in the PND 22 and PND 60 offspring following exposure duri ng gestation and lactat ion was performed. On PND 60, the young rats were perfused for central nervous system (CNS) a nd peripheral nerve neuropa thology evaluation and brain morphometry. The extended 1- generation reproductive toxicity study on 2, 4-D was performed at three dose levels up to the saturation level for rena l clearance. At the highe st dose tested (82 mg/kg bw/day in males/59 mg/kg bw/day in females), th ere was no evidence of deve lopmental neurotoxicity and no adverse effect on myelination. The cited study does not identify a more sensitive endpoint for human health risk assessment than already iden tified by the Agency. The extended one-generation reproduction study confirms the protect iveness of the existing Points of Departure as to neurotoxic

effects. Garcia, et al . (2004) Study of Tyrosine Hydroxylase I mmunoreactive Neurons in Neonate Rats Lactationally Exposed to 2,4-Dichlorophenoxyacetic Acid. [Bin: Neurotoxicity]

### Page 12 of 186 [Cited: NRDC petition; 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on 25-day old pups exposed to 2,4-D through mother’s milk (dams exposed i.p. during LD 9-25 to dos e levels of 70 or 100 mg/kg/day). The investigators concluded that tyrosine hydroxylase immunoreactivity is significantly decreased, prob ably as a consequence of an inhibitory serotonin (5-HT) effect. This 2, 4-D inhi bitory effect on the dopaminergic neurons probably mediated by 5-HT may lead to biochemical and functional consequence like alterations in DA biosynthesis, storage, release, feedback control systems or receptor sensitivity. This study identifies effects on pup brain following lactational exposure . NRDC cited study as s upport for the need to consider lactational exposure to 2, 4-D in aggregate RA. NRDC is correct that lactational exposure was not addressed in the 2, 4-D di etary exposure assessment. The Ag ency acknowledges that there are remaining uncertainties associated with attempting to characterize exposures from breast milk. In order to address this uncertainty, EPA is seeking monitoring data for 2,4-D in human breast milk (e.g., EPA’s National Children’s Study). The Task Force referenced this study as another pub lication cited by NRDC

that was conducted at a dose well above pharmacokinetic nonlinearity and includ ed the use of an unrepresentative route and m ode of administration (intra peritoneal) that is not re levant for human health pesticide risk assessment, which they state would further amplify nonlinear pharmacokinetic performance. The cited study is one of several resear ch studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. In the extended 1-generation reproductive toxicity study on 2,4-D (MRID 47972101), young a dult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and c ontinuing through mating, gestation, a nd lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included func tional observational batter y (FOB), motor activity and acoustic startle response (ASR ). An evaluation of myelin in the PND 22 and PND 60 offspring following exposure during gestation and lactati on was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropat hology evaluation and brain morphometry. The extended 1-genera tion reproductive toxicity study on 2,4-D was performed at three dose levels up to the saturation level for renal cl earance. At the highest dose tested, there was no evidence of developmental neurotoxi city and no adverse e ffect on myelination. The cited study does not identify a more sensitive endpoint for human hea lth risk assessment than already identified by the Agency. The extended one-generation reproduction st udy confirms the protectiv eness of the existing Points of Departure as to neurotoxic effects. Garcia, et al. (2006) Dopamine- β -Hydroxylase immunohistochemical Study in the Locus Coeruleus of Neonate Rats Exposed to 2,4-Dichlorophenoxyacetic Acid Through Mother’s Milk. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Study examined the effects of 2,4-D on 22-day old pups exposed to 2,4-D through mother’s milk (dams exposed i.p. during LD 9-22 to dos e levels of 70 or 100 mg/kg/day). The investigators concluded that tyrosine hydroxylase immunoreactivity is significantly decreased, prob ably as a consequence of an inhibitory serotonin (5-HT) effect. This 2, 4-D inhi bitory effect on the dopaminergic neurons probably mediated by 5-HT may lead to biochemical and functional consequence like alterations in DA biosynthesis, storage, release, feedback control systems or receptor sensitivity. This study identifies effects on pup brain following lactat ional exposure. NRDC cited study as showing potential effects on the brain of neonatal rats (alterations in brain enzymes and neurotransmitters) and as support for the

### Page 13 of 186 need to consider lactatio nal exposure to 2, 4-D in aggregate RA. The Task Force referenced this study as another publication cited by NRDC that was conducted at a dose well above pharmacokinetic nonlinearity and included the use of an unrepr esentative route and mode of administration (intraperitoneal) that is not relevant for human hea lth pesticide risk assessmen t, which they state would further amplify nonlinear pharmacokinetic performance. The Agency agrees that the route of exposure

of the maternal rats is not relevant for human pesticide risk assessment; however, th e pups were exposed

via the maternal milk, which is relevant. As noted pr eviously, the cited study is one of several research studies from the same group of inve stigators looking into a possible m echanism of 2, 4-D neurotoxicity. All of these research studies incorp orate a dose level that will elicit the response being investigated (in this case, alterations in neurotra nsmitters). In the exte nded 1-generation reproduc tive toxicity study on 2,4-D (MRID 47972101), young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mati ng, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly (via the diet) following weaning. Offspring were subjected to a de velopmental neurotoxicity (DNT) assessment, which included functional observational battery (FOB), motor activity and acoustic startle response (ASR). An evaluation of myelin in th e PND 22 and PND 60 offs pring following exposure during gestation and lactation) wa s performed. On PND 60, the young ra ts were perfused for central nervous system (CNS) and peripheral nerve neuropa thology evaluation and brain morphometry. The extended 1-generation reproductive toxi city study on 2, 4-D was performed at three dose levels up to the

saturation level for renal clearance. At the highest dose tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelinati on. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. The extended onegeneration reproduction study confirms the protectiven ess of the existing Points of Departure as to

neurotoxic effects. Giasson, et al . (2000)

New Link Between Pesticides a nd Parkinson’s Disease. [Bin: Neurotoxicity] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] This citation does not mention 2, 4-D. Paper discusses another paper [Bet arbet et al., 2000; rats exposed chronically ( i.v .) to rotenone], which reports that chroni c administration of ro tenone can induce the major features of Parkinson’s diseas e (PD) in rats. The paper states th at this “remarkable observation not only provides a new animal model for the study of PD, but will also reinvigorate interest in the possible role of exposure to pesticides and other toxins as a cause of human neurodegenerative disease.” This paper was cited by Task Force in response to NRCD’s use of the Ton, et al., 2006 zebrafish paper to support their (NRDC) claim that 2, 4-D is associated with devel opmental neurotoxicity in mammals, including decreased motor activity and Parkinson’s-like tremors. Task Force states that the support for the Ton claim is a “news and view commentary” by Giasson and Lee, 2000, which discusses using the pyrethroid insecticide and pesticide rotenone as a model for Parkinson’ s disease. As discussed earlier, the extended 1-generation reproduc tive toxicity study on 2, 4-D addr esses neurotoxicity endpoints including assessments of motor ac tivity, functional operational battery (FOB), and myelination. At the highest dose tested, there was no evidence of devel opmental neurotoxicity and no adverse effect on motor activity or myelination. The no adverse effect levels observed in the extended 1-generation reproductive toxicity study are grea ter than the PODs utilized fo r human health risk assessment supporting the RED.

Konjuh, et al . (2008)

### Page 14 of 186 Neonatal hypomyelination by the herbicide 2,4-dichlorophenoxy acetic acid. Chemical and

ultrastructural studies in rats. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force; NCASI ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on myelinati on in 22-day old pups exposed to 2,4-D through mother’s milk (dams exposed to 70 mg/kg/day i.p. during LD 9-22). Study referenced by NRDC as one that shows that 2, 4-D impairs normal deposition of my elin in developing brain. NRDC states that the effects of 2,4-D make it likely that fetuses, infants, and children will be more susceptible to long-term adverse health effects from exposure to 2. 4-D. Study does not provide any dose-response information (do not know dose to pups via the milk). The only dose used (70 mg/kg/day administered i.p. to the

maternal animal exceeds rat renal clearance levels following oral exposure and does not provide information regarding potential effect s of 2, 4-D on myelination at dose levels below the saturation level for renal clearance. Task Force pointed out the excessive dose and route of exposure were not appropriate among other study deficien cies. NCASI stated that the petition fails to mention that dose levels likely exceed renal clearance levels and rout e of exposure is not appr opriate. The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. In the extended 1-genera tion reproductive toxicity study on 2, 4-D (MRID 47972101), young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those th at saturate metabolic processes. The young rat was expose d during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Offspring were subjected to a developmenta l neurotoxicity (DNT) assessment, which included functional observational battery (FOB ), motor activity and acoustic startle response (ASR), An evaluation of mye lin in the PND 22 and PND 60 o ffspring following exposure during gestation and lactation) was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation and brain morphometry. The extended 1- generation reproductive toxicity stud y on 2, 4-D was performed at three do se levels up to the saturation

level for renal clearance. At th e highest dose tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelination at any dose level. The cited study does not identify a more sensitive endpoint for human h ealth risk assessment than alrea dy identified by the Agency. The extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.
Rosso, et al. (2000) 2, 4-Dichlorophenoxyacetic Acid ( 2,4-D) in Developing Rats Alt ers Behavior, Myelination and Regions Brain Gangliosides Pattern. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force; NCASI ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on neonatal rats following sc injections (70 mg/kg during PND 12- 25 or 100 mg/kg during PND 7-25) ever y 48 hours. It was reported that early exposure of pups to high dose resulted in a deficit in myelin deposition and behavior al alterations together with GM ganglioside reduction, which could be reflect ing a 2,4-D CNS injury, although a weak undernutrition could be involved also. When 2,4-D dose was lower, body a nd brain weight effects were not observed, but changes in GM1 contents and in myelin deposition, a nd alterations in muscular force and motor activity

were still observed. The dose levels are above leve l for saturation of renal clearance mechanism (oral route) and the route of exposure (sc) of pups is not relevant to human exposure and risk assessment. However, the reported deficit on myelin depos ition was also observe d following pup exposure via milk

### Page 15 of 186 (Duffard, 1996). NRDC referenced the study as one that shows that 2,4-D appears to impair normal deposition of myelin in developing br ain, and that effects of 2,4-D make it likely that fetuses, infants, and children will be more susceptible to long-term a dverse health effects from exposure to 2, 4-D. Task Force commented that effect may be due to under nutrition. The Task Force noted that the study was conducted at an excessively high dose (level expect ed to exceed the threshold level for saturating renal clearance of 2, 4-D) and included the use of an unrepre sentative route and mode of administration that is

not relevant for human hea lth pesticide risk assessment. The Task Force also considered this paper and other NRDC cited papers from the same group of inve stigators to be methodologically flawed [litter was not used as the unit for statistical analyses (to a void bias due to litter effects); the reporting of histopathological evaluation limited, etc.]. NCASI stated that petition fails to mention that dose levels likely exceed renal clearance levels and route of exposure is not appr opriate. The cited study is one of

several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The extended 1-generation reproductive toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and directly through PND 22, 70, 139, includes an assessment of developmental neurotoxici ty and neuropathology (inc luding an evaluation of myelin in the PND 22 and PND 60 offspring following exposure during gestation and lactation) at dose

levels that do not saturate metabo lic processes. At the highest dos e tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelination at any dose level. The cited study does not identify a more sensitive endpoint for human h ealth risk assessment than already identified by the Agency. The extended one-generation reproduction st udy confirms the protectiv eness of the existing Points of Departure as to neurotoxic effects.
Rosso, et al . (2000) 2,4-Dichlorophenoxyacetic acid disrupts the cytoskel eton and disorganizes the Golgi apparatus of

cultured neurons. [Bin: Neurotoxicity] [Cited: NYS DOH ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D in primary culture s of cerebellar granule ce lls (24-hr exposure). In

this in vitro study (cultured neurons), there was a dose-de pendent inhibition of neurite extension; paralleled by a significant redu ction in cellular conten t of both dynamic and stable microtubules, a disorganization of Golgi apparatus, and inhibition in synthesis of complex gangliosides. The investigators concluded that the primary toxic effects of 2, 4-D on neurons are most probably due to inhibition of microtubule polymeriza tion, and these observations raise th e possibility that at least one mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly. This mechanistic study is not directly ap plicable to risk assessment. The relationship of the dose (mM) to

levels attained in the brain in vivo is not known. The study was cite d by NYS DOH as an additional study that should be considered in any new assessmen t along with those identifie d in the NRDC petition.

This citation is one of several research studies fr om the same group of i nvestigators looking into possible mechanism of 2, 4-D neurotoxicity. In respons e to the required additi onal data, the registrant conducted an extended 1-generation reproductive toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation, and directly through PND 22, 70, 139, includes an assessment of developmental neurotoxicity and neur opathology (including an ev aluation of myelin in the PND 22 and PND 60 offspring follo wing exposure during gestation and lactation) at dose levels that do not saturate metabolic processes. At the highest dose tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelination at any dose level. The cited study does not identify a more sensitive endpoint for human h ealth risk assessment than alrea dy identified by the Agency. The extended one-generation reproduction study confirms the protectiveness of the existing Points of

### Page 16 of 186 Departure as to neurotoxic effects.
Rosso, et al . (1997) Effects of 2, 4-Dichloro phenoxyacetic Acid on Central Nervous System of Developmental Rats. Associated Changes in Ganglioside Pattern. [Bin: Neurotoxicity] [Cited: NYS DOH ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on neonatal rats following sc injections of 2, 4-D (70 mg/kg or 100 mg/kg) every 48 hours from PND 7- PND 17 or PND 7- PND 25. Re sults suggest a delay in CNS development when pups exposed to 100 mg/kg/day (s c); at lower dose, brai n capable of triggering biochemical mechanisms producing a pl asticity response, which is expr essed as changes in ganglioside content and composition. The dose levels would be exp ected to exceed the threshold level for saturation of renal clearance mechanism; route of exposure (sc) of pups is not relevant to human pesticide exposure or risk assessment. The study was cited by NYS DOH as an additional study that should be considered in any new assessment along with those identified in the NRDC petition. Th e cited study is one of

several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. In the extended 1-generation reproductive toxici ty study on 2, 4-D (MRID 47972101), young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly (via the diet) following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (F OB), motor activity and acousti c startle response (ASR). An evaluation of myelin in the P ND 22 and PND 60 offspr ing following exposure during gestation and lactation was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation a nd brain morphometry. The extended 1-generation reproductive toxicity study on 2,4-D was performed at three dose levels up to the saturation level for renal clearance. At the highest dose tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelination at any dose level. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. The extended onegeneration reproduction study confirms the protectiven ess of the existing Points of Departure as to

neurotoxic effects. Sturtz, et al . (2006) Detection of 2, 4-dichlorophenoxy acetic acid in rat milk of dams expo sed during lactation and milk analysis of th eir major components. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on the nutritional status of pups following e xposure of the dams to 2, 4-D at dose levels of 15, 25, 50, or 70 mg/kg/day via the diet during PND 1 to PND 16. It was reported that maternal dietary expos ure during lactation results in dama ge to pups nutri tional status; 2,4- D produced a dose-related reduction of polyunsaturated fatty acids (PUFAs ); significant changes in milk fatty acid composition indi cating a disturbance in th e supply of PUFAs to the neonate, which can alter normal development of the brain. Th e study provides data following oral doses to the maternal rat, which are below, at, and above renal clearance capacity. NRDC cited study as confirmation that offspring are exposed to 2, 4-D during lactation. Additionally, it was pointed out that this 16-day exposure at dose level of 15 mg/kg/day resulted in significant decreases in pup body weight gains, and 2, 4-D accumulated in maternal milk and altered the nutritional content of the milk. NRDC considered

### Page 17 of 186 the study especially relevant in light of EPA selecting a NOAEL of 25 mg/kg/day for short-term (1-30 days) oral exposure and states that EPA should re- do the short-term oral risk assessment using 15 mg/kg/day as a LOAEL rather th an 25 mg/kg/day for the NOAEL. Th e Agency notes that the cited paper only provides pup body weight gain data (in the form of a graph), and data on pup body weights were not provided. The study does not make clear wh ether it was measuring decrements in body weight gain or absolute body weight. Body weight gain is a calculated value and can be misleading. For example, say the control and treated group both show a body weight of 50.9 grams on day X. The control group gains 1.1 grams and the treated group gains 0.8 gram. This is a 24% decrease in body weight gain for the treated group compared to the control. Howeve r, 52 grams vs 51.7 grams is a 1% difference in body weight. The cited study does not show a clear dose re sponse effect. Although there is a greater effect on body weight (gai n) comparing the lowest and highe st doses, the body weight effects are essentially the same at 15 and 25 mg/kg/day ( 11.4% and 11.6%) and that same phenomena is seen at 50 and 70 mg/kg/day (17.8% and 16.7%). Additionally, the decr ease in body weight gain was observed from the 6th day of lactation to the end of the e xperiment. 2, 4-D Task Force pointed out that these results were not duplicated in a recent rat dietary life-stage study (Marty, 2009), which demonstrated decreased pup body weight only when dams were treated with 2,4-D ≥ 800 ppm in diet (premating through PND 21 (52, 61, 71-117 mg/kg/day); no e ffects on body weight at 400 ppm (25, 28, 37-57 mg/kg/day); at 800 ppm, 2,4-D pharmac okinetic behavior in lactating dams was distin ctly non-linear (Saghir, 2009); i.e., renal clearance mechanism satura ted. Based on the toxicokine tic profile, the doses in the extended one-generation reproduction study were adjusted duri ng the lactational period to prevent excessive dosing both to the maternal rat and to the pups during early lactation and due to a “double exposure” when pups are both nursi ng and starting to consume diet (as in the case on PND 16). Adjustments to the diet were also performed in the Sturtz study, a lthough the procedures used were different and may, to some extent, e xplain the results in the Sturtz study compared to the extended onegeneration reproduction st udy. At the highest dose tested in th e extended 1-generation reproduction study, which exceeded renal clearance, decreased pup body weights were obser ved throughout lactation,

and the no observed adverse effect level fo r pup body weight was established at 300 ppm ( ≈ 30 mg/kg/day) for the PND 22 offspr ing. This NOAEL is greater than the POD utilized for the human health risk assessment supporting the 2, 4-D RED. The extended one-generation reproduction study confirms the protectiveness of th e existing Points of Departure as to body weight effects. Sturtz, et al . (2000) Detection of 2, 4-Dichlorophenoxy Acetic Acid (2,4- D) Residues in Neonates Breast-Fed by 2,4-D Exposed Dams. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force; NCASI ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on the neonatal rats during lactation (PND 1-9 or PND 1-15); dams were exposed i.p. (0, 50, 70, 100 mg/kg) every other day to maintain 2, 4-D neonates’ exposure. Residues of 2,4-D were found in stomach content, blood, brain, kidneys of PND 4 pups and older; residues were dose- and exposure-time dependent; at 100 mg/kg/day, decreased pup BW and tissue weight and diminished stomach content were note d. The investigators concluded that the results demonstrate that 2,4-D is transfer red to neonates and decreased body a nd organ weights could be due to

a diminished milk intake or/and to the direct 2,4-D toxic effect. When 2, 4-D was withdrawn from dams, 2,4-D residues remained in stomach content of th e pups for at least a w eek. NRDC cited study as evidence of exposure of pups to 2, 4-D through maternal milk, thereby resulting in potentially significant exposures to th e nurseling. NRDC states th at despite knowing this research and route of exposure, EPA failed to include any lactational exposure in its aggreg ate risk assessment. The Task

### Page 18 of 186 Force considers the study as limited in value for hum an risk assessment since the dose levels to the lactating rats were in excess of saturation of re nal clearance of 2, 4-D and toxicity observed in the resulting non-linear pharmacokinetic range does not inform human risk assessment, because nonlinear doses are exceedingly unlikely to occur in humans. Additionally, the Task Force notes that EPA was aware, as a result of animal feeding studies using ex aggerated doses, that 2, 4-D may be present in milk.

There was a discussion of this i ssue in the 2, 4-D RED. Compared to the high doses used in these studies, the EPA estimates of the maximum dietary exposure from food to human females ages 13-50 is 0.01018 mg/kg/day and the average exposure is 0.000642 mg/kg/day (USEPA memorandum, 2002). These values range from 4900 to 1 million times lowe r than the values in the cited rat study. NCASI stated that the NRDC petition fails to mention that the dose levels likely exceed renal clearance levels and the route of exposure is not a ppropriate. Study provides dose-response informati on at dose levels at

or above level for saturation of renal cleara nce mechanism. Although the dams were dosed i.p., the pups were exposed via the milk; the dose to the pups is not known. However, this study, in conjunction with another Sturtz study and data from the extende d 1-generation reproductive toxicity study, which determined the levels of 2, 4-D in the maternal rat’s milk, may provi de information for determining pup

dose. In the extended 1-genera tion reproductive toxicity study on 2, 4-D (MRID 47972101), young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (F OB), motor activity and acousti c startle response (ASR). An evaluation of myelin in the P ND 22 and PND 60 offspr ing following exposure during gestation and lactation was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation a nd brain morphometry. The extended 1-generation reproductive toxicity study on 2, 4-D was performed at three dose levels up to the saturation level for renal clearance. At the highest dose tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelination. The cited study does not identify a mo re sensitive endpoint for human health risk assessment than already identified by the Agency. The extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects. Sturtz, et al . (2008) Effect of 2, 4-Dichlorophenoxyacetic acid (2,4-D) on rat maternal behavior. [Bin: Neurotoxicity] [Cited: NRDC petition supplement; Beyond Pesticides; NYS DOH ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on matern al behavior. Dams were administered 2,4-D via diet (0, 15, 25, 50 mg/kg/day) from PND 1-7; on PND 5 and PND 7, dams (12/ dose group) were tested for maternal behavior [nursing behavior with retr ieval, crouching, licking (of whole body of pup), anogenital licking, time spent out of nest and eating; latency and length of retrieval, latency of crouching]. Serum prolactin levels; endoge nous monoamines (noradrenaline, dopamine, 3,4- dihyrdoxyphenylacetic acid, homovanil lic acid, serotonin, and 5-hydro xyindolacetic acid) and their

metabolites were determined. Maternal nesting behavior was not affected. Differences in mother-pup interactions were observed at all dose levels (laten cies of retrieval; latenc ies of crouching; increased time for retrieval of 8 pups; d ecreased % dams licking pups; none of 2,4-D dams licked anogenital area of pups; increased time spent out of nest); decreased indoleamine le vels (dose-related); increased catecholamine levels. The doses used (15, 25, 50 mg/kg/da y) are all at or below the expected level of

renal clearance saturation. Prolactin le vels have been related to the on set and maintenance of maternal behavior in rats. Beyond Pesticides cited the study as showing effects that need to be considered along

### Page 19 of 186 with other scientific l iterature, which they say abounds with evidence that 2,4- D is a dangerous chemical. NRDC cited the study as one that shows that even short-term exposure to 2,4-D at dose levels below toxic levels severely inhibits normal matern al hormones in brain and thereby adversely affects maternal behavior. It is to be noted that pup body weight gain was not affected by 2, 4-D treatment during the test period (PND 1-7), whic h is not consistent with a prev ious study by these investigators (Sturtz, 2006 above) where a decrease in pup body we ight gain was reported from PND 6 on under similar exposure conditions. Additionally, it is not known whether the diets provided to the lactating dams were adjusted to take into account the fact that lactating dams consume greater amounts of food compared to non-lactating female rats; the doses actua lly ingested may have been greater than intended. One of the studies NYS DOH requests EPA to consid er in any new 2,4-D risk assessment. The cited study is one of several re search studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. It is to be noted that a maternal be havioral assessment is one of the many parameters assessed in the ex tended 1-generation reproductive toxi city study on 2, 4-D. There was no evidence of maternal negl ect in the clinical observations performed on the maternal rats in the 2, 4-D extended 1-generation reproductive toxicity study at dose levels approaching metabolic saturation. Additionally, there was no impact on the young with resp ect to any lack of maternal care. The extended one-generation reproduction study confirms the protectiv eness of the existing Poin ts of Departure as to

neurotoxic effects.
Ton, et al. (2006) Zebrafish as a Model for Developmental Neurotoxicity Testing. [Bin: Neurotoxicity] [Cited: NRDC petition; 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Study is a screening assay and not directly applicab le to human health risk assessment. These data suggest that zebrafish may be an appropriate alte rnative model to study the mechanism(s) responsible for the developmental neurotoxicity of pesticides a nd aid in identification of compounds that should be further tested in mammalian systems. Neurotoxicity was alre ady identified as a potential e ndpoint for 2, 4-D and more appropriate studies have been perfor med to assess 2, 4-D’s developmental neurotoxicity potential. In the extended 1-generation repr oductive toxicity study on 2, 4-D (MRID 47972101), young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (F OB), motor activity and acousti c startle response (ASR). An evaluation of myelin in the P ND 22 and PND 60 offspr ing following exposure during gestation and lactation was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation a nd brain morphometry. The extended 1-generation reproductive toxicity study on 2, 4-D was performed at three dose levels up to the saturation level for renal clearance. At the highest dose tested, there was no evidence of developmental neurotoxicity and no adverse effect on myelination. The cited study does not identify a mo re sensitive endpoint for human health risk assessment than already identified by th e Agency. The extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

### Page 20 of 186 Bin: Endocrine

Blair, et al. (2000) The Estrogenic Receptor Relative Binding A ffinities of 188 Natural and Xenochemicals: Structural Diversity of Ligands. [Bin: Endocrine] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D in a validated (standardized) estrogen receptor (ER) competitivebinding assay, which was used to de termine the ER affinity. 2, 4-D was one of many chemicals that were screened at high concentratio ns to determine whether the chemi cal competed with [3H]–estradiol for the ER. 2, 4-D did not e xhibit any estrogenic activity at the concentrations tested. This is one of several studies referenced by the Ta sk Force that they identified as demonstrating that 2,4-D does not bind to or activate estrogen receptors, contrary to NR DC’s assertions that 2, 4-D is a “potent” endocrine disruptor. The Agency used an exposure based approach to select the first list of chemicals to receive

Test Orders for the Endocrine Disr uptor Screening Program (EDSP) Ti er 1 screening ba ttery; 2,4-D was

included in the list of chemicals. Additionally it should be noted that this study was cited by the Test Order recipient as Other Scientifi cally Relevant Information (OSRI) in response to the Agency’s EDSP Test Order and was evaluated by the Endocrine Disruptor Review Team (TXR No. 0055461). The EDRT concluded that while 2, 4-D was reported as nega tive in this investigation, no data were supplied

to substantiate this c onclusion, and this study did not meet Te st Order requirements (TXR No. 0055461, page 17). EPA waived the Tier 1 tests most releva nt to human endocrine effects for 2,4-D due to the availability of the extended one generation repr oduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4- Dichlorophenoxyacetic Acid (2,4-D) - Report of the Endocrine Disruptor Review Team - Test Order #: EDSP–031001-120” (December 20, 2010). ) EPA has received all required final study reports and data from the Tier 1 battery of tests for 2,4-D, which include an in vitro Estrogen Receptor Binding Assay (890.1250) The submitted EDSP Tier 1 tests will be considered with regard to potential

environmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the

submitted Tier 1 in vitro studies may inform EPA on mechanisti c issues in mammalian systems (e.g., whether 2,4-D can bind to the estr ogen or androgen receptor in mamm als), the studies will not affect EPA’s conclusions on the quantitativ e endocrine risks posed by 2,4-D fo r humans given the availability of the extended one-gener ation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’ s interaction with endocrine system endpoints. Duffard, et al. (1995) Developmental Neurotoxicity of the H erbicide 2,4-Dichlorophenoxyacetic Acid. [Bin: Endocrine] [Cited: NRDC petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] The citation only provided an abstract with few deta ils. Study examined the effects of 2, 4-D on neonatal rats exposed during gestation ( i. p. exposure of pregnant rats from GD 16 to PND 21) and postnatally via the diet up to adult life. Vaginal smears were mon itored, and prolactin and pr ogesterone levels were measured. Authors stated that these preliminary results demonstrate 2, 4- D adverse effect on the endocrine system based on alterations in the estr ous cycle. Study does not provide any dose-response information. The only dose used (70 mg/kg/day) was one expected to exceed rat renal clearance levels. The study does not provide information regarding po tential effects of 2, 4- D on estrous cycle or

### Page 21 of 186 hormone levels at dose levels below the saturation level for renal clearance. NRDC cited this study as demonstrating endocrine disrupting eff ects of 2,4-D. The Task Force cited it as one of a series of studies that NRDC contends provides evidence of 2, 4-D e ndocrine activity. The Task Force does not consider this study as providing compelling evidence of pot ential environmental or human endocrine health concerns, since it was conducted at a high dose (well a bove saturation of renal cl earance) and, therefore, is of limited value for risk assessment. The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The citation was considered by the Endocrine Disruptor Review Team (TXR No. 0055461). It is to be noted that the Agency issued a data call-in for a study that assesses thyroid, gonada l, reproductive and other endocrinesensitive endpoints and while awaiting the study, im posed an additional 10X uncertainty factor to account for the data gap. In response to the data call-in, the Task Force submitted an extended 1- generation reproductive toxicity st udy on 2,4-D. This study examined potential effects on parental male and female reproductive function, offspring survival and growth, including endocrine and systemic toxicity parameters such as estrous cyclicity (f emale adult rats and offspring); sperm parameters; anogenital distance; nipple retention; puberty onse t (vaginal opening and balano-preputial separation); adrenal weight, thyroid/parathyroi d gland weight, pituitary gland we ight, testes and ovarian weight, thyroid hormone effects; and hist opathology of a wide range of tissu es including the thyroid, adrenal, pituitary, testes, and ovary. The extended one-gener ation study for 2,4-D showed no treatment-related effects on potential estrogenic eff ects or androgen-sensitive endpoint s (no adverse effects on anogenital distance, nipple retention, age at vaginal opening, estrous cycle length or pa ttern, mating, fertility, time to mating, gestation length, pre-implantation loss, nu mber of corpora lutea, sperm parameters, ovarian follicle counts, and reproductive organ weights a nd histopathology; no evidence of hyposadias, ectopic tests, or treatment-related testicular prostate or seminal vesicle hist opathology). Anti-androgenic effects in terms of decreased male reproducti ve organ weights were observed in some animals but they were not statistically significant and were associated with decr eased body weight. No tr eatment-related effects on reproductive organ histopathology were obs erved. Slight effects were seen in the thyroid (increases or decreases thyroid weight and in T3, T4, and TS H hormones in some animals) but no dose response relationship was shown. These effects were more si gnificant at the highest dose tested but still were considered adaptive and not adverse (i .e., the thyroid responded to insult and corrected itself) due to the

fact that this dose exceeded the renal saturation leve l. Accordingly, the highe st dose was considered a No Observed Adverse Effect Level (NOAEL) for t hyroid effects. In the ex tended one generation reproductive toxicity study, there were no treatment -related adverse effects on any of the measured parameters evaluated to assess th e estrogen, androgen or thyroid en docrine systems in intact rats following 2, 4-D exposure at dose levels approachi ng saturation of renal clea rance. The changes in estrous cycle noted in the cited st udy were not replicated in the extended 1-generation reproductive toxicity study.
Haddow, et al. (1999) Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of

the child. [Bin: Endocrine] [Cited: NRDC petition ] [Human Studies Considerations Apply: No] This is a general paper (not 2, 4-D specific) in whic h the children of women w ho had thyroid deficiency during pregnancy were assessed for various abilities (15 test s of intelligence, at tention, etc.). NRDC cited this study in support of their conclusion that EPA failed to incorporate information on the endocrine disrupting effects of 2, 4-D. NRDC states that thyroid hormone pl ays a critical role in development of brain; slight thyr oid suppression adversely affects ne urological development in fetus,

### Page 22 of 186 with lasting effects on child learning and behavior. Contrary to the statement in NRDC petition, thyroid effects were identified in the 2, 4-D RED and a data call-in was issu ed for a 2-generation reproduction study that specifically required examin ation of the thyroid. HED agrees with NRDC that protection from adverse effects in the thyroid in women of childbear ing age is important to protect the developing fetus from adverse outcomes. It is to be noted that 2, 4-D was tested in rats, which are much more sensitive to thyroid perturbations than are humans because the se rum half-life of the thyr oid hormone, thyroxine, is much shorter in rats (less than 1 day) than in humans (5-9 days). The 10X interspecies uncertainty factor applied to 2, 4-D in the Agency risk asse ssment to account for the possibility that humans are more sensitive than the test animals is, therefore, more than adequate to protect humans. The 10X intraspecies factor accounts for variability in se nsitivity among species and gives protection for women with iodine deficiency. A 10X FQ PA database factor was applied to 2, 4-D, which provided added protection pending the outcome of the required studi es. The F1-extended one-generation reproductive toxicity study of 2, 4-D provides an assessment of the thyroid; i.e., an evaluation of thyroid hormones (T3, T4 and TSH), thyroid weight and histopathol ogy of the thyroid acro ss life stages, brain morphometry data, and an evaluation of the audito ry startle response. In the extended 1-generation toxicity study in rats, the expect ed hormone response consistent with perturbation in thyroid function

was seen in the GD 17 satellite dams. These findings correlated with the thyroid alterations histologically. There was a comparable increase ( ↑ 9%) in thyroid weights at the low and high dose levels but no change in thyroid we ight at the mid dose. In the F1 PND 4 pups, there were decreases in T3 levels at the low ( ↓ 8%) and mid ( ↓ 13%) dose groups, but not at the hi gh dose. Decreased T4 levels were seen at the mid ( ↓ 15) and high ( ↓ 12%) dose groups, but there was no dose response. TSH level was increased ( ↑ 19%) at the high dose. In the F1 PND 22 o ffspring, there was a decrease in T4 ( ↓ 20%) only at the high dose. In the F1 PND 62-64 offspring, T3 level was increased ( ↑ 11%) only at the high dose. T4 levels were increased ( ↑ 19%) both at the mid and high dose levels. There was a dose-dependent increase in TSH levels at the mid ( ↑ 11%) and high ( ↑ 24%) dose levels. Female offspring (PND 70) displayed decreased absolute thyroid weights at the mid ( ↓ 9%) and high ( ↓ 5%) dose levels. No treatment-related histopathol ogical lesions were seen in the thyroid glands of P1 rats or F1 offspring. The thyroid effects noted in the ex tended 1-generation reproductive toxic ity study were considered to be adaptive and not adverse. Additionally, no robust effects on auditory startle or statistically significant brain morphometric changes were seen in the DNT component of the extended one-generation reproductive toxicity study; findings otherwise expected as sequelae to any marked thyroid hormone deficiency. The no observed adverse effect level ( NOAEL) for thyroid toxicity was established at ≈ 40 mg/kg/day. This NOAEL is greater than the PODs utiliz ed for the human health risk assessment supporting the 2, 4-D RED. The Agency used an exposure based approach to select the first list of

chemicals to receive Test Orders for the Endocri ne Disruptor Screening Program (EDSP) Tier 1 screening battery; 2, 4-D was included in the list of chemicals. The EDSP is based on a two-tiered approach. Tier 1 consists of a batte ry of eleven assays to screen chem icals for their potential to interact with estrogen, androgen or thyroid hormone pathways. The fact that a substance may interact with a hormone system does not mean that when the substan ce is used, it will cause a dverse effects in humans or ecological systems

. The purpose of Tier 2 testing is to identify and establish a dose response relationship for any adverse effects that might result from interactions with hormone systems identified in Tier 1 screening

. 

### The Tier 2 mammalian study is current ly the 2-Generation Reproduction and Fertility Effects Study (Note: the OECD Guide line for the Extended F1 One Generation Reproduction Test Guideline is also under consideration). The existing 2, 4-D hazard data base includes an Extended F1 One Generation Reproduction Test Guideline. Th e Agency has determined that the extended onegeneration reproductive toxicity study has fulfilled the requirements for the in vivo mammalian Tier 1 assays, specifically the Hershberger, Uterotrophic, Male Pubertal and Female Pubertal assays. The

### Page 23 of 186 extended 1-generation reproductive t oxicity study measured/evaluated endpoints that were not evaluated in the 2-generation reproduction study performed unde r the old guideline. These include an evaluation of potential effects on parental male and female reproductive function, offspring survival, growth including endocrine and systemic toxicity parameters such as estrous cyclicity (female adult rats and offspring), evaluation of sperm parameters, anogenital distance, nipple retenti on, puberty onset (vaginal opening and balano-preputial separation), and t hyroid gland weight, hor mone and histopathology

assessment. Hurst, et al. (2003) The potential for oestrogenic effects of pe sticides in headwater streams in the UK. [Bin: Endocrine] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] The referenced study [yeast two-hybr id screen; Yeast Estrogen Screen (YES)] is a sc reening study in which 2, 4-D was negative. The Task Force referenced this study to dispute NRDC’s claim that 2, 4-D is an endocrine disruptor. The Task Force noted that the Agency has on several occasions rejected the position that data gathered under Endocrine Disruptor Screening Program is a prerequisite to a safety determination. Concern for endocrine disruption wa s identified in the RED. The Agency used an exposure based approach to select th e first list of chemicals to receiv e Test Orders for the EDSP Tier 1 screening battery; 2, 4-D was included in this list of chemicals. This study was cited as Other Scientifically Relevant Information (OSRI) in res ponse to the EDSP Test Orde r and was included in the Endocrine Disruptor Review T eam (EDRT) assessment of 2, 4-D (TXR No. 0055461). EDRT did not accept yeast-based assays as OSRI primarily because some test chemicals are unable to penetrate the cell wall of the yeast cells and this may result in a false negative. Tier 1 screening assays are limited to evaluating whether a substance is ca pable of interacting wi th the endocrine system and are not sufficient to determine whether a chemical may have an effect in humans that is similar to an effect produced by naturally occurring hor mones. The fact that a substance may in teract with a hormone system does not mean that when the substance is used, it will cause adverse effects in humans or ecological systems. EPA has received all requir ed final study reports and da ta from the Tier 1 batter y of tests for 2,4-D. The

submitted EDSP Tier 1 tests will be considered with regard to potential environmental effects and the

need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian sy stems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the quantitative endocrine risks posed by 2,4-D for humans given th e availability of the extended one-generation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints. Jung, et al . (2004) Anti-estrogenic activity of fifty chem icals evaluated by in vitro assays. [Bin: Endocrine] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] The referenced study was conducted us ing a yeast two-hybrid assay syst em. 2, 4-D was not reported to inhibit estrogen activity. The Agency used an e xposure-based approach to select the first list of

chemicals to receive Test Orders for the EDSP Tier 1 screening battery; 2,4-D wa s included in this list of chemicals. This study was cite d as Other Scientifically Relevant Information (OSRI) in response to

the EDSP Test Order and was included in the Endocrine Disrupt or Review Team assessment of 2, 4-D (TXR No. 0055461). As stated above, EDRT did not accept y east based assays as OSRI primarily because some test chemicals are unable to penetrate the cell wall in yeast-based assays and this failure

### Page 24 of 186 may result in a false negative. EPA has received all re quired final study reports a nd data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 tests will be considered with regard to potential

environmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the

submitted Tier 1 in vitro studies may inform EPA on mechanisti c issues in mammalian systems (e.g., whether 2,4-D can bind to the estr ogen or androgen receptor in mamm als), the studies will not affect EPA’s conclusions on the quantitativ e endocrine risks posed by 2,4-D fo r humans given the availability of the extended one-gener ation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’ s interaction with endocrine system endpoints. Jungbauer, et al. (2002) Yeast reporter system for rapid det ermination of estrogenic activity. [Bin: Endocrine] [Cited: 2,4-D Task Force ] [Human Studies Considerations Apply: No] When the Agency used an exposure-based approach to select the first list of chemicals to receive Test

Orders for the EDSP Tier 1 screeni ng battery; 2, 4-D was in cluded in this list of chemicals. Jungbauer and Beck tested 2,4-D in a yeast ba sed system. The concentrations tested were not identified nor were data provided (TXR No. 0055461, page 17). This study was not cited as OSRI by the submitter due to the limitations of yeast-based assays . Some test chemicals are unable to penetrate the cell wall in yeastbased assays and this failure may result in a false negative. As noted above, EPA has received all required final study reports and data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 tests will be considered with regard to potential e nvironmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., wh ether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies w ill not affect EPA’s conclusions on the quantitative endocrine risks posed by 2,4-D for humans given the availability of the extended one-generation reproduction study (an

in vivo study in rats) that co mprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints

. Kim, et al. (2002) Mechanism of phenoxy compounds as an endocrine disruptor. (in Korean) [Bin: Endocrine] [Cited: NRDC petition; 2,4-D Task Force ] [Human Studies Considerations Apply: No]

This article was written in Korean and was not av ailable to the Agency in English. The Agency identified concern for potential endo crine effects in the RED (as noted a bove). In response to this and other concerns raised, the registra nt conducted the extended one-generat ion reproductive toxicity study. This study measures several key en dpoints. These endpoints include an evaluation of potential effects on parental male and female reproductive function, o ffspring survival, growth including endocrine and systemic toxicity parameters such as estrous cyclic ity (female adult rats and offspring), evaluation of sperm parameters, anogenital distance, nipple rete ntion, puberty onset (vag inal opening and balanopreputial separation), and thyroi d gland weight, hormone and hi stopathology assessment. In the extended 1-generation reproductive toxicity study on 2,4-D (MRID 47972101) submitted in response to the data call-in, young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was expose d during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Parameters assessed in clude sperm parameters, sex gland weights and histopathology; AGD, nipple retentio n; puberty timing. There were no tr eatment-related, adverse effects on any of these parameters.

### Page 25 of 186 Kim, et al. (2005) Effects of 2,4-D and DCP on the DHT-induced androgenic action in human prostate cancer cells. [Bin: Endocrine] [Cited: NRDC petition; 2,4-D Task Force ] [Human Studies Considerations Apply: No] Kim et al., (2005) studied th e ability of 2,4-D and its metabolite to increase th e proliferation of 22Rv1 human prostate cancer cells. Nega tive results were reported. Trancr iptional activation studies were conducted with various cell lines. All were reported negative, except 2,4-D was reported to increase the response when coadministered with testosterone. NRDC cited the paper as evidence of endocrine disrupting effects of 2, 4-D, sta ting that the combination of 2, 4- D and testosterone resulted in synergistic increases in the prolif eration of prostate cancer cells. The Task Force commented that this in

vitro mechanistic study cited by NRDC is based on the earlier report that 2, 4-D increased prostate weight in rats following oral (gavage) exposure at a high dose above the threshold for saturation of renal clearance, which they state renders the usefulness and interpretation of this in vitro study of questionable value to potential human risk. As discussed above, the Agency used an exposure-based approach to

select the first list of chemicals to receive Test Or ders for the EDSP Tier 1 screening battery; 2,4-D was included in this list of chemicals. This study wa s included in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461); it should be not ed that due to the limitations of these studies, the submitter did not claim them as OSRI. Effects of dietary 2, 4-D treatment on prostate weight and pathology were examined in extended 1-generation re productive toxicity study on 2, 4-D, and there were no treatment-related effects. The NOAEL (45 mg/kg/da y) is greater than the PODs utilized for the

human health risk assessment s upporting the 2, 4-D RED. The object ive of the EDSP Tier 1 battery

assays is to screen for the potential to interact with the estrogen, androge n and thyroid hormone pathways. EPA has received all required final study repor ts and data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 tests will be cons idered with regard to potential environmental

effects and the need for Tier 2 in vivo studies for effects in w ildlife. Although the submitted Tier 1 in

vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the quantitative endocrine risks posed by 2,4-D for hum ans given the availabil ity of the extended onegeneration reproduction study (an in vivo study in rats) that comprehe nsively examined the risks to human health from 2,4-D’s interaction with endocrine sy stem endpoints. The purpose of Tier 2 testing is to identify and establish a dose response relationshi p for any adverse effects that might results from interactions identified through Ti er 1 screening. The Tier 2 mammalian study is currently the 2- Generation Reproduction and Fertilit y Effects Study or the Extended F1 One Generation Reproduction Test Guideline. The existing 2, 4-D hazard da tabase includes an Extended F1 One Generation Reproduction Test Guideline. Kojima, et al . (2004) Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. [Bin: Endocrine] [Cited: 2,4-D Task Force ] [Human Studies Considerations Apply: No]

In an in vitro assay (hER α transactivation assay; report er gene assays for human ER α (hER α ) and

hER β and AR (hAR) expression), 2,4-D did not show es trogenic effects or antie strogenic properties in

the hER α transactivation assay; did not show androgenic transcriptional activity; and did not show an inhibitory effect on the androgenic activity induced by DHT. Concern for endocrine disruption potential was identified in the RED. The Agency used an e xposure-based approach to select the first list of

### Page 26 of 186 chemicals to receive Test Orders for the EDSP Tier 1 screening battery; 2,4-D wa s included in this list

of chemicals. This is one of seve ral studies cited as Other Scientifi cally Relevant Information (OSRI) in response to the EDSP Tier 1 Test Orders and by th e Task Force to refute the NRDC evidence of the endocrine disrupting effects of 2,4-D. This citation was evaluated as OSRI in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461, page 25). The citation does not provide any data to substantiate the reported negative results, and transcriptional assays do not measure receptor binding. The F1-extended one generation reproductiv e toxicity study of 2,4-D evaluated several key endpoints. These endpoints include an evaluation of pote ntial effects on parental male and female reproductive function, offspring survival, growth includ ing endocrine and systemic toxicity parameters such as estrous cyclicity (female adult rats and o ffspring), evaluation of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal ope ning and balano-preputial separation), and thyroid gland weight, hormone and histopathol ogy assessment. In addition the te st guideline includes options to evaluate development of the immune and nervous systems. The findings for 2,4-D in the extended onegeneration study determined there were no adverse e ffects observed on any of the numerous parameters evaluated, and the NOAEL was established at 45 mg /kg/day in males and 40 mg/kg/day in females. Both NOAELs are greater than the PODs utilized for the human health risk a ssessment supporting the 2,

4-D RED. Lin, et al . (2000)

In vitro studies of cellular and molecular deve lopmental toxicity of adjuvants, herbicides, fungicides, Minnesota. [Bin: Endocrine] [Cited: 2,4-D Task Force ] [Human Studies Considerations Apply: No] The Agency used an exposure-based approach to select the first list of chemical s to receive Test Orders for the EDSP Tier 1 screening battery; 2,4-D wa s included in this list of chemicals. Lin et al ., (2000)

was cited as Other Scientific ally Relevant Information in response to the Test Order. This was also one of several studies cited by the Ta sk Force to refute the NRDC ev idence of the endocrine disrupting effects of 2,4-D. Technical grade 2,4-D was positive in the assay, but the reagent grade was negative. It was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461, page 18); that the study may be useful supplementary inform ation but it did not satisfy the requirements of the EDSP Test Order. A cell proliferation assay does not measure receptor binding and may be affected by factors other than binding with the receptor. EPA ha s received all required fi nal study reports and data from the Tier 1 battery of tests for 2,4-D. The su bmitted EDSP Tier 1 tests will be considered with regard to potential environmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the quantitative e ndocrine risks posed by 2,4- D for humans given the availability of the extended on e-generation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints Liu, et al. (1996) The Direct Effect of Hepatic Peroxisome Prolif erators on Rat Leydig Cell Function in vitro. [Bin: Endocrine, Cancer] [Cited: NRDC petition; 2,4-D Task Force ] [Human Studies Considerations Apply: No] The referenced study is an in vitro study designed to address the m ode of action for Leydig cell adenomas and peroxisome proliferation. The results sugge st that peroxisome prolif erators, as a class of

### Page 27 of 186 compounds, directly modify the ster oidogenic function of Leydig cells in vitro . Investigators stated that further investigations were necessary to address the mechanism for the in vitro effects on Leydig cells and to clarify the apparent relationship between peroxi some proliferator-induced changes in Leydig cell function and the development of Leydig cell tumors. Th is study is one of severa l studies cited by NRDC as the basis for their concern for potential endocr ine disrupting effects of 2, 4-D. NRDC believes the findings establish the dangerous endocrine disrupting effects of 2,4-D that requires that 2,4-D be banned in light of the findings that 2,4-D causes slight decrease in testoste rone release and significant increase in estrogen release from testicular cells. The Task Force stated that no significant effects in hCGstimulated release of testosterone were seen in rat Leydig cell culture s. Effects on reproductive

hormones were identified in the RE D and were addressed in extended 1-generation reproductive toxicity study (MRID 47942101). The F1-extended one gene ration reproductive toxicity study of 2,4-D evaluated several key endocrine end points. These endpoints include an evaluation of potential effects on parental male and female reproductive function, offspring survival, growth including endocrine parameters such as estrous cyclicity (female adult rats and offspring), evalua tion of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and bala no-preputial separation), and thyroid gland weight, hormone and histopathology assessment. Th e F1-extended one generation reproductive toxicity study on 2,4-D de monstrated a lack of interaction with the endocrine system, as evidenced by the lack of effects on male and fe male reproductive function, anogenital distance (AGD), male and female sexual maturation, reproductive orga n weights, or histopat hology of reproductive and endocrine organs, or on sperm parameters and es trous cyclicity/periodicity (TXR No. 0055341). The NOAEL was established at 45 mg/kg/day in males and 40 mg/kg/day in females, which are greater than

the PODs utilized for the human health ri sk assessment supporting the 2, 4-D RED. Malysheva, et al. (1997) Morphological and histochemical changes in the thyroid gland after a single exposure to 2,4-DA herbicide (in Russian) [Bin: Endocrine] [Cited: Beyond Pesticides ] [Human Studies Considerations Apply: No] The study is in Russian and no translation is availabl e. There are no details re garding test methods or dose levels used, and no data are available to substantiate the re ported effects. As cited by Beyond Pesticides, decreases in thyroid gland transport and produ ction functions and impairment of hormone iodination in thyroid were observed af ter acute exposure to 2, 4-D. Thyroi d effects were identified in the 2, 4-D RED and a data call-in was issued for a 2-ge neration reproduction study that specifically required examination of the thyroid. The F1-extended one-g eneration reproductive toxicity study of 2, 4-D provides an assessment of the t hyroid following repeat exposure; i.e., an evaluation of thyroid hormones (T3, T4 and TSH), thyroid weight and histopathology of the thyroid acr oss life stages, and an evaluation of auditory startle and brain morphometry data (re ported to be sensitive to thyroid disruption). The thyroid toxicity NOAEL was established at ≈ 40 mg/kg/day, the highest dose tested. This NOAEL is greater than the POD utilized for the human hea lth risk assessment suppor ting the 2, 4-D RED. The cited study does not identify a more sensitive endpoint for human risk assessment than already identified by the Agency. McKinlay, et al . (2008) Endocrine Disrupting Pesticides: Im plications for Risk Assessment. [Bin: Endocrine] [Cited: Beyond Pesticides ] [Human Studies Considerations Apply: No] This paper is a review article that cites a paper by Kim, et al., 2005 (discussed above) in which, as cited

### Page 28 of 186 by Beyond Pesticides, 2,4-D was reported to promote th e proliferation of andr ogen-sensitive cells by acting synergistically with its ma in metabolite, DCP. 2, 4-D did not promote the proliferation of androgen-sensitive cells alone. The F1-extended one generation reproductive toxicity study of 2,4-D evaluated several key endpoint s. These endpoints include d an evaluation of potenti al effects on parental male and female reproductive function, offspring su rvival, growth including endocrine and systemic toxicity parameters such as estrous cyclicity (fem ale adult rats and offspring), evaluation of sperm parameters, anogenital distance, ni pple retention, puberty onset (vag inal opening and balano-preputial separation), and thyroid gland we ight, hormone and histopathology as sessment. There were no adverse effects observed on any of the numer ous parameters evaluated in the extended 1-generation reproductive study (TXR No. 0055341), and the NOAEL was establishe d at 45 mg/kg/day in males and 40 mg/kg/day in females. Both NOAELs are greater than the PODs utilized for the human health risk assessment supporting the 2, 4-D RED. Nishihara, et al . (2000) Estrogenic Activities of 517 Chemic als by Yeast Two-Hybrid Assay. [Bin: Endocrine] [Cited:2, 4-D Task Force comments ] [Human Studies Considerations Apply: No] The referenced study is a yeast two- hybrid assay system, in which 2, 4- D was reported to be negative. When the Agency used an exposure-based approach to select the first list of chemicals to receive Test

Orders for the EDSP Tier 1 screen ing battery; 2,4-D was included in th e list of chemicals. This study was referenced in a document submitted to the Agency by the 2,4-D Consortium in response to the EDSP Tier 1 Test Order, however it was not cited as Other Scientifica lly Relevant Information (OSRI).

This citation was included in the Endocrine Disr uptor Review Team assessm ent for 2, 4-D (TXR No. 0055461). In yeast-based assays some test chemicals are unable to penetrate the ce ll wall and this failure may result in a false negative. As noted above, EPA ha s received all required final study reports and data from the Tier 1 battery of tests for 2,4-D. The su bmitted EDSP Tier 1 tests will be considered with regard to potential environmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the quantitative e ndocrine risks posed by 2,4- D for humans given the availability of the extended on e-generation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints. Oakes, et al. (2002) A Study of the Potential for a Herbicide Formul ation Containing 2,4-D an d Picloram to Cause Male-Mediated Developmental Toxicity in Rats. [Bin: Endocrine] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: Yes; the human data referenced in the citation were not used in the Agency’s assessment ] Study examines the effects of 2, 4-D on male rats following 9 weeks of exposure with a mixture of 2,4- D and picloram. The treated males were mated with untreated female rats, and the females were sacrificed on GD 20, and fetuses were weighed and ex amined for either structural malformations or skeletal development. The results did not show any evidence that repeated exposure of male rats was likely to cause male-mediated birth defects in offspring or other adverse reproductive outcomes. Information provided in the report includes 2,4-D se rum levels attained at 150 mg/kg/day dose level [daily dose Cmax values of 249-284 μg 2,4-D/mL se rum]; serum half-life of ~6 hours; commercial

### Page 29 of 186 applicators and forestry workers (no protection) estimated exposure s ranging from 0.9 to 160 μg/kg/day, which represents exposure to 0.1 -11 mg for 70 kg man (referenced Draper and Street, 1982; KolmodinHedman and Erne, 1980; Lavy, et al , 1987; Nash, et al, 1982) ; in terms of estimated serum levels, men given an oral dose of 5 mg 2,4-D/kg (=350 mg for 70- kg man) had a Cmax of 40 μg/mL (Kohli, et al., 1974); an 11-mg intake would give an estimated plas ma level of 1 μg/mL; negative study at doses up to 150 mg/kg/day 2,4-D, which exceeds renal excretion mechanism. The study was cited by the Task Force as providing evidence that exposur e to a herbicide formulation cont aining 2,4-D and picloram is not likely to cause male-mediated birth defects or ot her adverse reproductive out comes, and no effects characteristic of endocrine-mediated response were observed. The findings in the extended 1-generation reproductive toxicity study of 2, 4-D are consistent with th is conclusion. There were no adverse effects on any of the numerous parameters monitored in th e extended 1-generation reproductive toxicity study of 2, 4-D, which included a detail ed assessment of endocrine endpoint s. The NOAEL was established at 45 mg/kg/day, which is greater than the POD utilized for the human health risk assessment supporting the 2, 4-D RED. Soto, et al (1995) The E-SCREEN assay as a tool to identify es trogens: An update on estrogenic environmental pollutants. [Bin: Endocrine] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No]

This is one of several studies ci ted by the Task Force to demonstrat e that 2, 4-D does not bind to or activate estrogen receptors. The Task Force referenced this study to refute NRDC’s assertions that 2, 4- D has extensive hormone disrupting activity that may o ccur at low doses. The E-screen is a quantitative assay that compares the cell number achieved by sim ilar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 17 β -estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. The results for 2, 4-D in this assay were reported to be negative, however no data were provided to support this conclusion. A cell proliferation assay could supplement information provided by a binding st udy, but not substitute for it because it does not directly measure binding. In addition, factor s other than binding may s timulate proliferation.

As noted above, EPA has received all required final study reports and da ta from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 tests will be considered with regard to potential

environmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the

submitted Tier 1 in vitro studies may inform EPA on mechanisti c issues in mammalian systems (e.g., whether 2,4-D can bind to the estr ogen or androgen receptor in mamm als), the studies will not affect EPA’s conclusions on the quantitativ e endocrine risks posed by 2,4-D fo r humans given the availability of the extended one-gener ation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s inte raction with endocrine sy stem endpoints.

Bin: Dermal Absorption

Brand et al (2007) Transdermal absorption of the herb icide 2,4-dichlorophenoxyacetic acid is enhanced by both ethanol consumption and sunscreen application [Bin: Dermal Absorption] [Cited: NRDC Petition] [Human Studies Considerations Apply: No]

### Page 30 of 186 Both alcohol consumption and topical sunscreen applic ation act as transdermal penetration enhancers for model xenobiotics. The effect of combining these two treatments on transdermal absorption of the herbicide 2, 4-dichlorophenoxyacetic acid (2, 4-D) wa s examined. Skin from rats ingesting low (1.5 g/kg), medium (4.3 g/kg), or high (6 g/kg) etha nol doses or saline cont rol was treated with a commercially available sunscree n containing titanium dioxide and octyl methoxycinnimate and transdermal absorption of 2,4-D was monitored by ex cising the skin and placing it into a diffusion chamber. Ethanol increased penetration by a factor of 1.9, 2.0 and 2.5 for animals treated with 1.5, 4.3 and 6 g/kg respectively, demonstrati ng an ethanol-induced dose response. Sunscreen applic ation to skin from ethanol gavaged rats caused 2, 4-D absorp tion above that induced by ethanol alone by an additional factor of 1.3, 2.1 and 2.9 fo r 1.5, 4.3 and 6 g/kg respectively. This in vitro study indicates that

the use of sunscreens and ethanol can enhance the derm al absorption of 2, 4-D. The use of the study as described in the NRDC petition is reflective of the results of the study. Several human dermal absorption ( in vivo ) studies were considered in the Agency assessm ent as part of the weight of evidence for determining the dermal absorption factor. A mean-p ercent absorbed value of 5.7% of the applied dose

was derived from four different human dermal abso rption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the variab ility noted in the results. The studies considered by EPA involved exposure conditions that varied base d on application site (forearms, hands), topical dose rates (1.7 to 1100 μg/cm 2 

### ), form (acid or salt), applicati on media (water, ethanol, acetone), and exposure time. The overall average dermal absorpti on value for all of these data combined (N=34), regardless of design, was 5.7 percent. Examination of these variables, particular ly the use of different application vehicles and different anatomical sites, is likely to have captured much of the variability

measured in the sunscreen and alcohol in vitro studies. EPA used its standa rd uncertainty factors (UF A 

### interspecies and UF H 

### intraspecies) to establish a level of concern for this assessment even though human dermal absorption data were used and a protective value was selected for the risk assessment. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorp tion. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Finally, it should

also be noted that the UF H 

### factor is partially intended to account for uncertainties associated with the relative sensitivity to a pesticide within a populati on, which includes factors such as differences in dermal absorption that could possibl y be attributed to th e use of sunscreen, clot hing, or occlusion. Therefore, the Agency is confident that the dermal absorption value used does not under estimate dermal

risk.

Brand et al (2002)

Sunscreens Can Increase Dermal Penetra tion of 2,4-Dichlorophenoxyacetic Acid [Bin: Dermal Absorption] [Cited: NRDC Petition] [Human Studies Considerations Apply: No] The purpose of this work was to determine the effect of sunscreen use on the tr ansdermal absorption of a model herbicide, 2, 4-dichlorophenoxyacetic acid. The 2, 4-D was placed on top of the epidermis in an

in vitro diffusion chamber after pretreatment with one of 9 commercially available sunscreens for 24 hours (dorsal skin from hairless mice CRL:SK1). Th e total penetrating thr ough the skin in 24 hours ranged from 39.1 ± 1.7% for the no s unscreen control to 81.0 ±2.8% for Neutrogena Oil Fr ee Sunscreen. Six sunscreens led to a significant en hancement of total 2, 4-D penetrati on as compared to the control. Investigators concluded careful selection of sunscreen during pe sticide application could reduce potential exposure. The use of the study in the NRDC petition is reflective of the results. There are methodological issues associated with this study incl uding the use of hairless mice and whether or not

### Page 31 of 186 UV radiation was applied to the treated skin during th e test as UV radiation can possibly impact dermal absorption. In the 2005 risk asse ssment, it should be noted that EPA used a protective value for quantifying dermal absorption from a human voluntee r study. Further, the Agency still applied its standard uncertainty factors to establish a level of concern, even though thes e factors typically are

intended to establish levels of conc ern for risks based solely on animal toxicity data. EPA derived an initial dermal absorptio n factor of 5.7%, based upon the mean-p ercent absorbed value from four different human dermal absorption st udies. However, EPA ultimately us ed a value of 10%, in order to be protective, based on the upper range of th e variability observed in the results. Brand et al (2008) The Effect of Chronic Ethanol Ingestion on Skin Penetration of 2,4-D (2008) – title cited (could not find paper with this tit le; see below Brand, 2004,,). Chronic Et hanol Ingestion Alters Xenobiotic Absorption Through the Skin: Potential Role of Oxidative Stress. [Bin: Dermal Absorption] [Cited: NRDC Petition] [Human Studies Considerations Apply: No] Study examined the effects of ethanol ingestion on skin penetration of 2, 4-D. Rats pair-fed a diet containing 36% ethanol for 12 weeks; then switched to a non-alcohol diet and monitored for 4 weeks; non-invasive measurements for changes in dermal blood flow using laser Doppler velocimetry (LDV), damage to skin barrier via transepidermal water loss (TEWL), a nd changes in skin moisture content were performed. On 0, 1 day, or 1, 2, 3, 4 weeks after alcohol removal, rats sacrificed and their skin was analyzed for alcohol and aldehyde dehydrogenase, and lipid peroxidation. Tran sdermal penetration of DEET and 2, 4-D was determined also. Transderma l absorption, LDV, TEWL, skin alcohol and aldehyde dehydrogenase and lipid pe roxidation were signif icantly increased afte r continuous ethanol exposure; factors remained elevated for up to 4 weeks after alcohol consumption stopped, reflecting fundamental changes in the skin. It was concluded that alcohol consumption trigge rs increases in levels of enzymes associated with ethanol metabolism, induces physical changes in the skin leading to increased uptake of xenobiotics (e .g., 2, 4-D). One of several studies cited by NRDC (supplement) to support concern for enhanced dermal penetrati on of 2, 4-D following alcohol consumption. Many factors can alter dermal function (bar rier) and impact the magnitude of the dermal absorption factor (e.g. anatomical site of application, presence or absence of penetration enhancers in the pesticide formation, whether liquid formulations are aqueous or organic, species differences, magnitude of dose, and duration of exposure). Several human dermal absorption studies were considered in the Agency assessment as part of the weight of evidence for determining the dermal absorption factor. A mean-percent absorbed value of 5.7% of the applied dose was derived from four different human de rmal absorption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the variability noted

in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity among members of the human population (UF H 

### interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A 

### ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspeci es factor can either be reduced to 1 or a 3X. In this case, in spite of using a dermal absorption value from a human study, the full 10X UF H 

### was retained. It should also be

noted that the UF H 

### factor is partially intended to account fo r uncertainties associat ed with the relative sensitivity to a pesticide within a population, which includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscr een, clothing, or occlusion. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorp tion. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Therefore, the

### Page 32 of 186 Agency is confident that the dermal absorption valu e used does not under estimate dermal risk and is adequately protective of any potential enhancement to dermal absorption from sunscreen/alcohol use. Brand et al (2004) Effects of Chronic Alcohol Consumption on Dermal Penetration of Pesticides in Rats. [Bin: Dermal Absorption] [Cited: NRDC Petition] [Human Studies Considerations Apply: No] Alcohol consumption acts as a transdermal penetrati on enhancers for model xenobi otics. The effect of alcohol consumption on transdermal absorption of 2, 4- D was examined. Skin from rats ingesting liquid diets containing 36% ethanol for 6-8 weeks was placed in an in vitro flow-through diffusion cell system, and the epidermal side was exposed to 2, 4-D. Transd ermal penetration was enha nced by a factor of 2.2. This is a generic issue; alcohol consumption has been shown to adversely affect the skin itself, which can alter dermal function (barrier) a nd lead to increased dermal absorp tion. This paper is one of several research papers from the same investigators cite d by NRDC to show that the 2007 Brand study confirms prior research showing alcohol consumption e nhances 2, 4-D penetration. The existing dermal absorption value of 10% is based on the high end of the range of results in an extensive set of high quality human research results. The Agency considers this the best available data because these data eliminate the interpretative considerations necessar y to extrapolate from a different species and an in

vitro test methodology. Current OPP policy precludes using in vitro dermal absorption data as a standalone for quantitative use in risk assessment. Corroborating in vivo data is also required. This is

because OPP does not consider in vitro methods to have been sufficiently validated for stand-alone regulatory use i 

### . Related to this issue of validation is the issue that the studies cited by NRDC were conducted by a single laboratory, and so have not been independently replicated. Additionally, there are important limitations to in vitro models for dermal absorption, one of which is lack of an intact vasculature. This likely limits the use of such a t echnique for assessing the eff ects of ethanol on dermal absorption if vasodilation facilitate s increased dermal uptake after sy stemic absorption of ethanol.

The Agency is already aware of the kinds of variables that impact the magnitude of the dermal absorption factor (e.g. anatomical si te of application, presen ce or absence of penetr ation enhancers in the pesticide formation, whether liquid formulations are aqueous or organic, speci es differences, magnitude of dose, and duration of exposure). The human volunteer dermal absorpti on studies that we re considered

in the 2005 risk assessment addressed several of these variables (skin site, dose le vel, organic or aqueous dose vehicles) and a conservative, upper-bound estimat e of 10% was selected that accounts for these variables. As such, the Agency considers the 10% DAF adequately protective for a wide range of the variables known to impact the magnitude of the dermal absorption factor. It is to be noted that there is a 10X uncertainty factor applied for potential vari ation in sensitivity am ong members of the human population (UF H 

### interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A 

### ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspecies

factor can either be reduced to 1 or a 3X. In this case, in spite of using a dermal absorption value from a human study, the full 10X UF H 

### was retained. The UF A 

### is typically used to acc ount for the uncertainty associated with the extrapolation of animal tes ting results to humans for risk assessment, including dermal absorption. As such, the risk assessment re sults are inherent ly more protective in this case because this uncertainty has been eliminated. Therefore, the Agency is confident that the dermal absorption value used does not under estimate dermal risk and is adequately protective of any potential enhancement to dermal absorption from alcohol use. Feldman et al (1974) Percutaneous Penetration of Some Pesticides and Herbicides in Man [Bin: Dermal Absorption]

### Page 33 of 186 [Cited: NRDC petition; 2,4-D Task Force] [Human Studies Considerations Apply: Yes] Twelve different radiolabeled ( 14 

### C) pesticides and herbicides, in cluding 2,4-D were applied to the forearms of human subjects an d their urinary excretion of ( 14 

### C) was quantified. Data were also obtained via I.V. dosing to correct skin penetration data fo r incomplete urinary recovery. Approximately 5.8 ± 2.4 % of the 2, 4-D administered dose was excreted. Investigators reported th e values for 2, 4-D and other chemicals and acknowledged that many factors can impact dermal absorption (e.g., skin loading rates). NRDC stated that the Agency did not adequately assess the eff ect of 2, 4-D soaki ng into clothing or skin exposure covered by clothi ng or gloves by using this study of six adults to estimate dermal absorption that didn’t use any form of occlusion. It is to be noted that long-sleeved shirts, long pants, and/or gloves are protective clot hing, the purpose of which is to minimize exposure. One would not expect exposure from soaking thro ugh clothing/gloves with proper use of 2, 4-D or any other pesticide. Several human dermal absorption st udies, which included th is study, were considered in the Agency assessment as part of the weight of evidence for determining the dermal absorption factor. A meanpercent absorbed value of 5.7% of the applied dos e was derived from four different human dermal absorption studies. Ultimately, a value of 10% was us ed by the Agency based on these data and given the variability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity am ong members of the human population (UF H 

### interspecies) and a 10X uncertainty factor for extrapolat ion from animal to human (UF A 

### ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspeci es factor can either be reduced to 1 or a 3X. In this case, in spite of us ing a dermal absorption value from a human study, the full 10X UF H 

### was retained. It should also be noted that the UF H 

### factor is partially intended to account for uncertainties associated with the relative sensitivity to a pesticid e within a population, which includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscreen, clothing, or

occlusion. The UF A 

### is typically used to account for the uncertainty associ ated with the extrapolation of animal testing results to humans for risk assessmen t, including dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Therefore, the Agency is confident th at the dermal absorption value used does not under estimate dermal risk and is adequately protective. Harris, et al (1992) Percutaneous penetration of 2, 4-dichlorophenoxya cetic acid and 2, 4-d dimethylamine salt in human

volunteers. [Bin: Dermal Absorption] [Cited: 2, 4-D Task Force] [Human Studies Considerations Apply: Yes] Study examined the effects of 2, 4-D and 2, 4-D di methylamine salt (DMA) following dermal exposure in five male volunteers (each dosed on separate o ccasions with both chemical s). Urine samples were collected for 144 hours following dermal application of 10 mg to dorsum of the hand and analyzed for 2,4-D. An average of 4.5% was recovered in ur ine following 2,4 –D exposure, and 1.8% following DMA; greater amounts of DMA (7.7%) were washed off at 6 hours compared to 2,4-D (5.4%); average

half-lives for excretion were ≈ 40 hours (2,4 –D) and 60 hours (DMA). 2,4-D was applied as a solution in acetone, which can be expected to increase penetra tion into corneum where it is resistant to removal by washing; thus overestimating dermal penetrati on under field conditions. These data were evaluated previously by HED (TXR No. 010044, 2/23/1993; seve ral publications includi ng bystander exposure, professional applicators); results were considered in the Agency assessment as part of the weight of evidence for determining the dermal absorption factor. This is one of several studies cited by the Task Force that pointed out that EPA’s decision regarding glove use is cons istent with robust peer-reviewed

### Page 34 of 186 literature, which repeatedly demons trates that use of protective rubbe r gloves alone dramatically reduces total absorbed dose of 2,4-D. Moody, et al (1990) Dermal absorption of the phenoxy herbicides 2,4-D, 2,4-D isooctyl, and 2,4,5-T in rabbits, rats, rhesus monkeys, and humans: a cross-species comparison. [Bin: Dermal Absorption] [Cited: 2, 4-D Task Force] [Human Studies Considerations Apply: Yes] This 1990 study was conducted to determine whether anim al models could be used reliably to predict pesticide dermal absorption in humans and the effect of the pesticide vehicl e and anatomic site on pesticide absorption. Dermal ab sorption was compared among male New Zealand rabbits and male Sprague-Dawley rats (shaved mid-dorsal region), ma le rhesus monkeys (shaved middorsal forearm or forehead regions), and human volunteers (for ehead). A constant dose rate (4 μg/cm 2 

### ) was used and nonocclusive gauze patches were used to protect dosed areas for 24 hours (except forehead). Radiolabel dissolved in water, acetone or Es teron LV96. Urine samples (animals) collected at 4 and 8 hrs on first day, 24-hr intervals for 14 days; huma n urine collected at 4, 8, and 12 hrs and daily until post-treatment day 7; dose site washed at 24 hr s post dose (all); % dermal absorptio n and excretion half-lives were calculated. The investigators indi cated that the use of volunteers wa s approved through the institutional review board of the University of California at San Francisco. The inve stigators concluded that the data

demonstrate that none of the animal models consis tently approximated dermal absorption in humans. Discussion section of paper states: it is noteworthy that the i.m. correction factor used for the animal data permits interspecies comparison of dermal abso rption data and circumvents a potential problem that differential interspecies excretion of the pesticide ra diolabel between urinary a nd fecal routes precludes direct extrapolation. They note that their human data were not corrected for non-urinary excr etion since

iv percent recovery was 100% for 2, 4-D acid in humans (Feldmann, 1974). Assuming a non-urinary

excretion of the amine salt and isooctyl ester similar to that obtained in monke ys would tend to increase the human dermal absorption at most by 26-28%, a value consistent with the 20-25% estimated by Grover, et al. (1986) for non-urinar y excretion and body storage of 2,4- D acid ingested by humans. Task

Force listed this reference but no comment s specific to this paper were found. Moody, et al (1992) Dermal Absorption of the Phenoxy herbicide 2,4-D Dimethylamine in Humans: Effect of DEET and

Anatomic Site. [Bin: Dermal Absorption] [Cited: NRDC Petition; 2, 4-D Task Force] [Human Studies Considerations Apply: Yes] Percutaneous absorption of 14 

### C-ring labeled 2, 4-D-dimethylamin e was examined following topical application to palm and forearm of human volunteers. The effect of two vehicles (water and acetone) and DEET on dermal absorption was also investigate d. Urinary data demonstrated that there was no significant difference between palmar and forearm skin permeability to 2, 4-D-amine; DEET had no significant effect on total cumulative palmar permeability, and there was no significant difference between % recoveries obtained with acetone or aqueous vehicles. Results show that 10 ± 11.5% of the

14 

### C -2, 4D-amine was absorbed derm ally from the human palm and 7 ± 6.2% was absorbed from the human forearm. The reported forearm data for the monkey was 6 ± 3.4%, which is comparable to that found in this study. The investigators concluded that, from regulatory vi ewpoint, these data should raise concern since 2, 4-D-amine has been shown to permeate the palm, and hands are the anatomical sites most often contaminated by pesticide applicators. Although this exposure may be somewhat reduced by wearing gloves, NRDC noted that the study shows significant derm al absorption even following

### Page 35 of 186 rigorous soap and water washing at 24 hr post treatment. NRDC cited th is research as support for their concern that the presence of DEET on skin significan tly enhances absorption of 2,4-D. Task Force noted that EPA previously responded to this allegation by pointing out that the conclusion that the data demonstrate a difference between exposure with (14±4.5%) and without (10±11.5%) DEET is not supported given the magnitude of the standard deviat ion. Task Force also pointed out that in the 2005 RED, the PPE changes implemented mitigated con cerns for potential enha ncement by DEET, etc. Several human dermal absorption studies were consid ered in the Agency assessment as part of the weight of evidence for determining the dermal ab sorption factor. A value of 10% was used by the Agency based on these data and given the variab ility noted in the results. Additionally, a 10X uncertainty factor applied for pot ential variation in se nsitivity among members of the human population (UF H 

### interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A 

### ; intraspecies) were applied. It is to be noted that although a dermal absorption value from human data was used, the full 10X UF H 

### was retained to account for uncerta inties associated with the relative sensitivity to a pesticide within a population. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal tes ting results to humans for risk assessment, including dermal absorption. As such, the risk assessment re sults are inherent ly more protective in this case because this uncertainty has been eliminated. Therefore, the Agency is confident that the dermal absorption value used does not underestimate de rmal risk and is adequately protective. Moody, et al (1992) Effect of the mosquito repellen t DEET and long-wave ultraviolet radiation on penetration of the herbicide 2, 4-D and the insecticid e DDT in natural rubber gloves. [Bin: Dermal Absorption] [Cited: NRDC Petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] The study examined total 14 

### C-ring labeled 2,4-D permeability across a 1.5 cm 2 

### cut-out of natural rubber latex gloves incubated in labor atory apparatus at 37°C for 48 continuous hours (mean thickness

0.51 ± 0.008 mm; n=4). DEET itself rapidly permeated the glove, but no significan t effect of DEET was observed on glove permeability to 2,4-D (2.4 ±1.8% with DEET vs 3.2±3.46% without DEET); photoenhanced (long-wave ultraviole t (UVA) radiation) permeation of 2,4-D was observed in a natural rubber glove (6.2% ± 0.73% +UVA vs 0.3% ± 0.14% -UVA). NRDC cited study as showing that these gloves commonly worn by farmers will not afford adequate dermal protection claiming DEET alone enhanced 2,4-D permeability. NRDC states the gloves were highly perm eable to 2,4-D penetration when there was simultaneous exposure to DEET and sunli ght. The Task Force noted that the study does not provide definitive conclusions for enhanced risk fo r following reasons: 1) authors concluded DEET did not significantly impact 2,4-D penetr ation through rubber; 2) n=2; pe rmeability of 2,4-D was increased to 6.2±0.73% when rubber was simultaneously expos ed to UV lamp emitting UVA (not “sunlight”; nonUVA control 0.3±0.14% 2,4-D permeation); 3) neither DEET alone nor UVA alone rendered the rubber “highly permeable to 2,4-D despite the relatively severe experimental conditions; 4) only permeation of total [ 14 

### C] was measured and thus aut hors could not exclude that the postulated enhanced permeation with UVA co-treatment was represented by 2,4-D photolys is product(s); and 5) authors stated the field

relevance of observations remained to be established. As described elsewhere, Agency is confident that the dermal absorption value used does not under estim ate dermal risk and is adequately protective. Pelletier, et al (1989) Disposition of 2,4-Dichlorophenoxyacetic Acid Dime thylamine Salt by Fischer 344 Rats Dosed Orally and Dermally. [Bin: Dermal Absorption] [Cited: 2, 4-D Task Force]

### Page 36 of 186 [Human Studies Considerations Apply: No] This 1989 study was designed to compare the pharmac okinetics of 2, 4-D administered orally and dermally to better understand the relationship between oral toxicity studies and typical dermal field exposure. It was stated that 2, 4-D is rarely commercially available in th e acid form due to its poor water solubility and that commercial formulations are usua lly composed of alkali or amine salts or esters. Dimethylamine salt of 2,4-D was administered to rats orally and dermally. Th e dermal dose (10 mg/kg) was within upper range that might be expected from typical agricultural use a nd the oral dose (1 and 0.4 mg/kg), based on preliminary work, which indicated a 4-10% absorpti on factor for 2,4-D amine through skin, as compared to almost complete absorption from oral route. At 7 hrs, skin wash removed 63% of applied dermal dose. Based on urinar y excretion values, the systemic av ailability was about 10% of the dermal dose, while that of the oral dose was virtua lly 100 % within 72 hours afte r exposure. In terms of 2, 4-D concentration, about 1 mg of 2, 4-D was excreted following either a dermal dose of 10 mg/kg or an oral dose of 1 mg/kg. Peak concentrations in blood and kidneys were achieved within 30 minutes (both routes). Oral dose almost co mpletely excreted within 24 hours, whereas about 13% of the dermal dose was systemically available at 72 hours. This paper was referenced by the Task Force, but no specific discussion of it was found. Pont, et al (2004) Active ingredients in sunscreens act as topica l penetration enhancers for the herbicide, 4- dichlorophenoxyacetic acid. [Bin: Dermal Absorption] [Cited: NRDC Petition] [Human Studies Considerations Apply: No] The purpose of this work was to determine if the ac tive ingredients in sunscreen also act as dermal penetration enhancers. Full-thicknes s dorsal skin of female hairless mi ce was placed into a flow through diffusion chamber with epidermal side facing donor chamber; 2, 4-D amine in ethanol; 9 sunscreen ingredients. Investigators conclude d none of the chemicals tested signi ficantly inhibite d 2, 4-D diffusion across skin. Based on the data, a sunscreen containi ng only octocrylene would be optimal to prevent sunscreen-induced penetration enhancement; however, octocrylene only absorbs in the UVB range and, therefore, cannot give broad-spec trum protection alone. The penetration enhancement seen across

hairless mouse skin also occurred with human skin (abdominal skin from healthy human volunteers obtained after plastic surgery). EPA agrees that the in vitro studies indicate that the use of sunscreens could potentially enhance the dermal absorption of 2,4-D under the tested cond itions. As a matter of policy, OPP does not agree that in vitro dermal absorption data provide ev idence sufficient to be used quantitatively in risk assessment. As a matter of science, the Agency considers the present dermal absorption factor adequately protective, since it is ba sed on human studies that utilized different vehicles and anatomical sites of exposure, the same vari ables that would be impact ed by sunscreen, DEET,

and/or ethanol use. The human volunteer dermal ab sorption studies that were considered in the 2005

risk assessment addressed several of these variab les (skin site, dose level, organic or aqueous dose vehicles) and a conservative, upper-bound estimate of 10% was selected that accounts for these variables. As such, the Agency considers the 10% DAF adequately protective for a wide range of the variables known to impact the magnit ude of the dermal absorption factor. The use of the study in the NRDC petition is reflective of the results. EPA used its standard uncertainty factors (UF A 

### interspecies

and UF H 

### intraspecies) to establish a level of concer n for this assessment even though human dermal absorption data were used and a protective valu e was selected for the risk assessment. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorp tion. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Finally, it should

### Page 37 of 186 also be noted that the UF H 

### factor is partially intended to account for uncertainties associated with the relative sensitivity to a pesticide within a populati on, which includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscreen. Therefore, the Agency is

confident that the dermal ab sorption value used does not under estimate dermal risk. Riviere, et al (2003) Percutaneous absorption of topical N, N-diethyl- m-toluamide (DEET): effect s of exposure variables

and coadministered toxicants. [Bin: Dermal Absorption] [Cited: NRDC Petition] [Human Studies Considerations Apply: No] The purpose of the study was to de termine the effect of co-exposur e factors that could modulate transdermal flux of topically appl ied DEET. Studies were conducted us ing isolated perfused porcine skin flaps (IPPSFs), in vitro porcine skin, and silastic diffusion cells . Results suggest that co-exposure to a number of chemicals that potentially could be encountered in a military environment may modulate the percutaneous absorption of topically applied DEET beyond that seen for normal vehicles at typically applied concentrations. Also obser ved was that covering DEET dose area s with an occlus ive dressing, or even fabric, enhances absorption. NRDC states th is study shows occlusion can significantly enhance skin absorption. The Agency is aware that occlusive coverings increase skin hydration status by preventing water loss, and that hydrate d skin is generally more permeable to pesticides than un-hydrated skin. Therefore, occlusive coverings would be exp ected to increase exposure via the dermal route, resulting in increased risk. Howe ver, in EPA’s evaluati on of this study, it found th at the Agency’s risk assessment and margins of safety established through the application of uncerta inty factors, already account for any uncertainty raised by NRDC re lative to the findings in this study.

In the 2005 risk assessment, it shoul d be noted that EPA used a prot ective value for qua ntifying dermal absorption from a human volunteer study. Further, th e Agency still applied its standard uncertainty

factors to establish a level of con cern, even though these factors typically are intended to establish levels of concern for risks based solely on animal toxici ty data. EPA derived an initial dermal absorption factor of 5.7%, based upon the mean-percent absorb ed value from four different human dermal absorption studies. However, EPA ul timately used a value of 10%, in order to be protective, based on the upper range of the variab ility observed in the results. Also, as previously indicated, EPA used its standard uncertainty factors (UF A 

### interspecies and UF H 

### intraspecies) to establish a level of concern for this assessment even though human dermal absorption data were used and a protective value was

selected for the risk assessment. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assess ment, including dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Finally, it s hould also be no ted that the UF H 

### factor is partially intended to account

for uncertainties associated with the relative sensitiv ity to a pesticide within a population which includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscreen, clothing, or occlusion. Ross, et al (2005) Dermal absorption of 2, 4-D: a review of species differences. [Bin: Dermal Absorption] [Cited: 2, 4-D Task Force] [Human Studies Considerations Apply: Yes] This is a review paper th at provides a comparison of dermal abso rption studies in animals (rat, mouse, rabbit, and Rhesus monkey) and humans. Several conc lusion were presented, including an estimate of

### Page 38 of 186 potential human (consumer or pr ofessional worker) daily absorbed doses of 2, 4-D following skin exposures to liquid formulations across a range of applied dose densities (1-1000 μg of 2, 4-D per cm 2 

### of skin), and for contact durations of 24 hours or less. A mean percent absorbed value of 5.7% of the applied dose (derived from four different human derm al absorption studies) was considered appropriate. The use of the results of this study in the Task Force Comments on the NRDC petition appears reasonable. Several human dermal absorption st udies, which are included in this review, were considered in the Agency assessment as part of th e weight of evidence for determining the dermal absorption factor. A mean-percent absorbed value of 5.7% of the applied dose was derived from four different human dermal absorption studies. Ultimatel y, a value of 10% was used by the Agency based on these data and given the vari ability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensiti vity among members of the human population (UF H 

### interspecies) and a 10X uncertainty factor fo r extrapolation from animal to human (UF A 

### ; intraspecies). Generally when an endpoint is de rived from a human study, the 10X in traspecies factor can either be reduced to 1 or a 3X. In this case, in spite of using a dermal absorption value from a human study, the

full 10X UF H 

### was retained. It should al so be noted that the UF H 

### factor is partially intended to account

for uncertainties associated with the relative sensitivity to a pe sticide within a population, which includes factors such as differences in dermal absorp tion that could possibly be attributed to the use of sunscreen, clothing, or occlusion. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Th erefore, the Agency is confident that the dermal absorption value

used does not under estimate dermal ri sk and is adequately protective. Wester, et al (1998) Percutaneous Absorption of Salicylic Acid, Th eophylline, 2,4-D Dimethyl amine, Diethyl Hexyl Phthalic Acid, and p-Aminobenzoic Acid in the Iso lated Perfused Procine Skin Flap Compared to

Man In Vivo. [Bin: Dermal Absorption] [Cited:2, 4-D Task Force] [Human Studies Considerations Apply: Yes] Study compares percutaneous absorption in the isolated perfused porcine skin flap (IPPSF) system with

that in man in vivo . Six human subjects (males and postmenopausal women) were topically dosed 2, 4-D (dimethylamine salt) in etha nol on the ventral forearm (site not oc cluded). Urine collected for day of dosing and subsequent 6 days; at 24 hr, site washed with soap and wa ter. At 7 days after application, skin-dosing site was cellophane ta pe-stripped 10 times. Percutaneous absorption was determined from

14 

### C–urinary excretion. Five IPPSF were surgically prep ared, harvested and pe rfused in a non-recirculating chamber and then dosed with [ 14 

### C] 2, 4-D –dimethylamine one hour after equilibrium; a 10-

cm 2 

### area on the IPPSF was dosed in a manner identical to the in vivo human study. Total penetration

after 8 hours was estimated as th e radiolabel detected in perfusate plus treated skin. Human in vivo percutaneous absorption was 1.1±0.3 % dose absorbed compared to penetrated dose in IPPSF (3.8±2.4 % dose absorbed). It was concluded that the IPPSF system appears to be a good model for predicting percutaneous absorption relative to man. This pape r was referenced by the Task Force, but no specific

comments were found.

### Page 39 of 186 Bin: Metabolism

Saghir, et al (2006) Strategies to Assess Systemic Exposure of Che micals in Subchronic/Chro nic Diet and Drinking

Water Studies. [Bin: Metabolism] [Cited:2, 4-D Task Force] [Human Studies Considerations Apply: No] Study objective was to devel op a practical method to determine the st eady-state daily systemic dose of a chemical to animals in repeat dose toxicity studies by determining the area under the blood/plasma concentration time curve (AUC-24 h) for compounds of different blood/plasma ha lf-life. The approach

involved collection of a number of blood samples within a 24 hours pe riod and then examining various combinations of 1-3 time points predictive of the actual AUC values. This information was considered to provide a scientific basis (e .g., sampling times in reference to light/dark cycle) for choosing the minimum number and best combination of time poi nts in order to predict the daily systemic bioavailability of a chemical (AUC- 24 h) as well as daily highest (C

max 

### ) and lowest (C min 

### ) body burden and the time of their occurrence. The study also inve stigated the use of term inal blood concentrations from a rat subchronic study collected after 18 h of fasting/removal of diet to roughly estimate elimination half-life of chemicals, as well as the use of ti me-course urinary eliminat ion as an indicator of the circulating blood levels/AUC-24 h at steady-state exposure levels. Rats were dosed with 2, 4-D at dietary levels of 5 mg/kg/day and 100 mg/kg/day. 2,4-D concentratio ns in plasma and AUC values increased in a distinctly nonlinea r fashion between 5 and 100 mg/kg/ day (4-week exposure); nonlinear increase was apparent from the 89-fold increase in Cmax, and a 69-fold higher plasmaAUC-24 hr, far exceeding the expected 20-fold difference between dos es; higher than expected plasma AUC-24 hr was also supported by the saturation of urinary excretion at high dos e; only 28-fold higher 2,4-D was recovered during 18 hr of collection instead of 69-fold as expected from differenc e in circulating levels. The study demonstrates that a dose of 2,4-D of 100 mg /kg/day can be considered an excessive dose. This information (saturation of the renal excretion mechanism) was available previously and consider in the RED. The Task Force cited this paper with re spect to Sturtz (2000) ci tation by NRDC regarding lactational exposure at 50, 70, 100 mg/ kg/day. Task Force states that t oxicity observed in the resulting non-linear pharmacokinetics range does not inform hu man risk assessment beca use nonlinear doses are unlikely to occur in humans. Similar data by these authors were submitted (MRID 47414901 and 47414902) in conjunction with th e extended 1-generation reprodu ction study on 2, 4-D and are incorporated into the overa ll assessment for the study. Saghir, et al (2009) A Dietary Dose Range-Finding and Toxicokinetic (TK) Study of 2,4-Dich lorophenoxyacetic Acid (2,4-D) in Adult Crl:CD(SD) Rats an d Their Offspring. 1. Toxicokinetics. [Bin: Metabolism] [Cited:2, 4-D Task Force] [Human Studies Considerations Apply: No] The citation is an abstract. Study examined the eff ects of 2, 4-D dietary expos ure to rats from prebreeding through weaning and F1 offspring exposur e through PND 35. 2, 4-D AUC 24 h was calculated from 3 plasma samples collected at 6 am, 9 am, and 5 pm on designated sample day. 2,4-D in milk determined on lactation days 4 and 14 and in plasma of nursing pups by collection of single 9 am sample on PND 4, 14, 21, and 28. Due to saturation of renal cl earance, nonlinear TK was observed in parental males at 1600 ppm (test day 28) and 1200 ppm (test day 71). Onset of nonlinear TK in parental females was observed at <400 ppm (test day 29 and 95; AUC 100 ppm = 25; AUC 400 ppm = 201 μg h mL-1);

### Page 40 of 186 during gestation and lactation, fe male 2,4-D levels were higher du e to higher diet consumption; nonlinear TK occurred at <400 ppm (GD 17 and LD 14) and > 400 ppm and <800 ppm (LD 4); a shift in nonlinear TK on LD 4 may have been due to excretion into milk (levels ≈ 50% of female plasma); plasma 2,4-D in pups 30-70% lower than dam on LD 4 (M/F TK nonlinear <800 pp m) but similar to or higher than dam on LD 14 (M/M TK nonlinear < 400 ppm). Maximal 2,4-D in pups was reached on PND 21 when pups were consuming 2,4-D via the diet in addition to nur sing, but dropped to levels equal to dams on test day 29 and 95 levels on PN D 35. Study results showed a kinetically-derived maximum dose of 400 ppm ( ≈ 25 mg/kg/day) for females and 1200 ppm ( ≈ 65 mg/kg/day) for males. Increased dam and pup AUCs during lactation supported dietary adjustment duri ng lactation. It was concluded by the investigators that the TK data support a definitive study high dose for females and pups lower than that derived by traditional toxic ity testing. These dose selection data (MRID 47414901) were discussed with the Agency prior to the ex tended 1-generation reproduc tion study initiation. The Task Force cited this study with respect to Stur tz (2000) citation by NRDC regarding lactational exposure at 50, 70, 100 mg/kg/day. Task Force states that toxicity obser ved in the resulting non-linear pharmacokinetics range does not inform human risk assessment because nonlinear doses are unlikely to occur in humans. The results are incorporated into the overall assessment of the extended 1-generation reproduction study. van Ravenzwaay, et al (2003) Comparative Metabolism of 2,4-Dichlor ophenoxyacetic Acid in Rat and Dog. [Bin: Metabolism] [Cited:2, 4-D Task Force] [Human Studies Considerations Apply: No] Study examined the effects of 2, 4-D on rats and dogs of both sexes dosed orally with either 5 mg/kg or 50 mg/kg 14 

### C-2, 4-D. The rates and routes of excretion a nd plasma toxicokinetics and biotransformation were assessed. Elimination of 2, 4-D from rat plasma was significantly faster than in the dog (T 1/2 

### were 1.3-3.4 hrs for rat and 99-134 hr for dog) following 5 mg/kg or 50 mg/kg, respectively; AUC for rat was 21-57 μ eq.hg-1 and 4889-5298 μ eq.hg-1 for dog at 5 mg/kg; 122-2358 μ eq.hg-1 for rat and 34100- 44296 μ eq.hg-1 for dog at 50 mg/kg; major route of excre tion for rat was in urine; essentially complete after 24 hrs (low)/48 hrs (high) dos e; elimination incomplete in dog over sampling period (50% of dose recovered); urine principal route at low dose, but equal amounts excreted in urine and feces at high dose over 120 hrs; body burden of 2, 4-D in dog significantly hi gher than in rat for an equivalent dose, which is consistent with the increased sensitivity of dog to 2,4-D toxicity. It was concluded that in the case of certain organic acids (2, 4-D), the rat is a better mode l for assessing the relevance to man of the toxicity of these compounds than the dog. These data we re considered previously by the Agency. The toxicological endpoints for use in human risk assessmen t for 2, 4-D were selected from the rat database, based on the determination that the rat is a better pred ictor than the dog of the pot ential toxicity of 2,4-D to man. The data at 50 mg/kg in the rat suggested the clearance mechanism was saturated. The Task

Force used this reference in its discussion of lactational exposure (S turtz 2006 study) where ↓ pup BW observed at 15 mg/kg/day. Task Force points out that th is effect level was not replicated in 2009 Marty dietary life-stage study, where BW decreases were not observed at 400 ppm (25, 28, 37-57 mg/kg/day). Effects were observed at 800 ppm, which occurred under conditions in whic h 2,4-D pharmacokinetic behavior in lactating dams was di stinctly non-linear, thus rendering th ese responses of limited relevance to human risk assessment; also, cited with respect to Sturtz (2000) citation by NRDC regarding lactational exposure at 50, 70, 100 mg/kg/day; toxicity observe d in the resulting non-linear pharmacokinetics range does not inform human risk assessment because nonlinear doses are unlikely to occur in humans. The extended 1-generation reproduc tion study on 2, 4-D will provide data on potential effects following lactational exposure. No further action required.

### Page 41 of 186 Bin: Mutagenicity

Arias 2003, 2007 Sister chromatid exchange induction by the herb icide 2, 4-dichlorophenoxyacetic acid in chick embryos Cytogenetic Effects of Short- and Long-term Exposu re of Chick Embryos to the Phenoxyherbicide 2, 4-D. [Bin: Mutagenicity] [Cited: NRDC Petition; NYS DOH ] [Human Studies Considerations Apply: No] In the first study, concentrations of 0, 0.5, 1, 2, a nd 4 mg/embryo of either the pure grade of 2,4-D (99%) or the commercial formula tion, Agroxone containing 37% 2,4-D were injected into the air cell of White Leghorn chicken eggs (9-12/dose). Treatment la sted for 3 days; eggs were automatically rotated at 37.8oC and BrdU (150 μg/embryo) was injected into the air cell. On Day 4, embryos were removed following a 2-hour exposure to colchicine (0.05%). Chromosome preparations were made from harvested embryos and slides were stained using th e fluorescent-plus-Giemsa technique; the report did

not indicate whether slides were coded. A total of 30- 40 second-division metaphases were scored for SCE in the first five pairs of macrochromosomes. The frequency of first (M1), second (M2) and third (M3) division metaphases from 200 cells were scored for mitotic delay and MIs were determined from the count of 1000 cells. No tabular presentation of the data was available. Results, shown in graphs, indicated a significant (p <0.05) increase in SCEs for the high concentration of the commercial formulation (~1.43 SCEs/ cell vs. ~1.22 SCEs/cell for the solvent control, acetone) and a nonsignificant increase (~1.41 SCEs/ cell vs. ~1.22 SCEs/cell fo r acetone) for pure 2,4-D. Although the dose response was concentration related for both test materials, the increase at the high dose was only 1.2-fold higher, compared to the solvent control and 1.2-fold compared to the av erage spontaneous background of 1.15 SCEs/cell. Additionally, data were presented as the mean and standard error of the mean, which suggests wide variation in the data. The investigator also st ated that cell cycle de lay was observed at the high concentration (2 and 4 mg/embr yo) and that the MI wa s significantly (p<0.01) reduced by both test materials at the high dose (4 an d 8% reductions for the commercial and pure 2, 4-D, respectively). Based on these considerations, the values for SCE induction, cell cycling and MIs provide questionable evidence of a positive response. The study looks only at DNA damage (SCE), not mutagenic effects.

For the second study, Arias (2007) used the same purity of the test mate rials at similar concentrations and the same protocol with the exception of in cluding a 7- and 10-day extended exposure time and increasing the sample size to 35 embryos/concentrati on. Results were similar to the previous study showing “significant (p<0.05)” increas es in SCEs/cell at 4.0 mg/embr yo (1.1-fold increase) at 4, 7 and 10 days for the commercial product and at 4.0 mg/emb ryo (1.1 fold-increase) at 7 and 10 days for pure 2,4-D. Similarly, slight but signifi cant (p<0.01) effects were reported on cell cycle delay and the MI ( ≈ 23.3 for the commercial product and ≈ 23.1 for the pure substance at th e high dose vs. 24 for acetone), which was not associated with prolonged exposure. Although these effects were statistically significant, the biological relevance is unclear.

### Page 42 of 186 Charles et al (1999a) Ames Assays and Unscheduled DNA Synthesis Ass ays on 2,4-Dichlorophenoxyacetic Acid and its

Derivatives. [Bin: Mutagenicity] [Cited: 2, 4-D Task Force; NCASI ] [Human Studies Considerations Apply: No] Charles et al (1999a) reported the results of the i ndependently conducted Ames test plate incorporation and the preincubation modification to the plate incorporation assay. Additionally, independently performed the in vitro primary rat hepatocyte unscheduled DNA synthesis (UDS ) assay and the in vivo test procedure with hepa tocyte treated with 2,4-D acid were investigated. For the Ames assay, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 were exposed to 5 to 7 dilutions of the test articles in the presence or absence of Aroclor 1254-induce rat liver homogenates. Note: TA 1538, which is considered a duplicate of TA98, was not used fo r all test articles. All bacterial assays were performed for each test agent with a hi gh concentration in excess of the limit dose for this test system (5000 μg/plate) or up to a cytotoxic con centration; negative (solvent) and the appropriate positive controls were included. A minimum of triplica te plates per dose per strain per condition was also included in each assay. Representative results are presented in Table 1. As shown none of the eight test articles induced a mutagenic response in any of the tester stra ins at any test article concentration. All negative and positive controls responded in the expected manner. Accordingly, all bacterial assays were considered negative and fulfill the regulatory requirements for acceptable bacterial gene mutation assays. In vitro UDS assays were conducted with isol ate primary rat hepatocytes exposed to the five to six test article dilutions using solubi lity or cytotoxicity as the starting concentration. Hepatocytes were doses for 18-20 hours and scored for net nucl ear grain (NNG) counts and the percentage of cells with ≥ 5 NNG. Data from these assays can be seen in Table 1 and clearly show that neither 2, 4-D acid nor its salts or esters induced a genotoxic effect. By contrast, the positive control

induced marked increases in NNG a nd the percentage of cells with ≥ 5 NNG. Accordingly, these assays are negative and fulfill the regulatory requirements for acceptable in vitro UDS assays.

Only 2, 4-D acid was tested in the in vivo UDS assay conducted with male Wistar Han rats administered single oral gavage doses of 400 and 1000 mg/kg. Five animals per dose received the selected test material doses, vehicle (1% methylcellulose) or positive controls and were sacrificed at 2-4 and 12-14 hours after dosing. Hepatocytes were recovered from three animals per group, cultured with [ 3 

### H] thymidine and scored for NNG. Dose selection was based on the findings of a preliminary range finding study with 0, 600, 800 or 1000 mg/kg. Clinical signs of toxicity in the range fi nding study were loss of

balance and red stains on the nose and mouth “shor tly after dosing” with 600 mg/kg. At 800 mg/kg, lethargy, abnormal breathing, unsteady ga it and rigid body tone were seen. Similar signs were observed at 1000 mg/kg within 5 hours and two rats were killed in extremis after 3 days (Bus, 2009). Accordingly, 1000 mg/kg was selected as the maximum tolerated dose (MTD). Findings from the main test indicated that 2, 4- D did not increase the frequency of NNG at any dose or treatment time.

### Page 43 of 186 Charles et al (1999b) In Vivo Micronucleus Assays on 2,4-Dichloro phenoxyacetic Acid and its Derivatives. [Bin: Mutagenicity] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] The final publication in th is series of Industry Task Force II Ge netic Toxicology investigations on 2, 4-

D details the in vivo mouse bone marrow micronucleus assays conducted with 2, 4-D (96.1%), 2, 4-D DEA (73.8%) 2, 4-D DMA (66.2%), 2, 4-D IPA (50.15 %), 2, 4-D TIPA (70.9%), 2, 4-D BEE (95.6%), 2, 4-D EHE (98%), and 2, 4-D IPE (97.1%). Doses se lected for the assays were based on the findings of mortality in groups of three male and three female administered 3 to 5 oral gavage doses of each agent. In the main study, groups of 5 male and 5 female mice/dose/sa crifice intervals received a single administration of three selected doses of each test agen t via oral gavage. Animals were sacrificed at 24, 48 and 72 hours postdosing and bone marrow cells we re harvested, processed, spread on to slides, stained and scored; there was no indi cation whether slides were coded prior to analysis. Appropriate vehicle and positive controls were included in each assay. The incide nce of micronucleated polychromatic erythrocytes (MPCE) and the PCE to normochromatic (NCE) ratio were determined by analyzing 1000 PCEs/animal.

All test agents were examined up to the maxi mum tolerated dose (MTD), based on mortality; the generally comparable MTDs estimate for each test agent were:

• 400 mg/kg 2, 4-D 400 mg/kg 2, 4-D IPE

• 443 mg/kg 2, 4-D DEA 376 mg/kg 2, 4-D IPA

• 397 mg/kg 2, 4-D DMA 542 mg/kg 2, 4-D TIPA

• 500 mg/kg 2, 4-D EHE 375 mg/kg 2, 4-D BEE

Results of the individual micronuclei analysis indicated that neither 2, 4-D nor its seven derivatives were cytotoxic to the bone marrow since all PCE: NCE ra tios were generally comparable to the vehicle control values. Similarly, none of the test treatments with 2, 4-D or its seven derivatives induced an aneugenic or clastogenic effect at any dose or sacrifice time in m ouse bone marrow. Accordingly, the test agents were adequately tested and found to be negative in these acceptable assays that fulfill the regulatory requirements for in vivo mammalian erythrocyte micronucleus assays. Figg et al., 2000 Increased lymphocyte replicative index followi ng 2,4-dichlorophenoxyacetic acid herbicide

exposure. [Bin: Mutagenicity] [Cited: NRDC Petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] Figg et al. (2000) found no evidence of micronuclei (MN) inducti on in the peripheral lymphocytes of 12 applicators spraying only 2, 4-D during a 6-month peri od. In this study, 12 applicators were compared to a control group consisting of 9 c ontrols (matched for gender, age, alcohol and tobacco uses, general health and geographical location). Blood and urine samples were coll ected prior to spraying and at every other week after a typical da y of 2,4-D application. The concentr ation of 2, 4-D in the collected urinary samples was determined. Lymphocyte cultu res were grown for 44 hours, and Cytochalasin B was added to prevent complete cyt okinesis. At 72 hours, lymphocytes were separated; slides were prepared, stained with antikinet ochore antibody and coated with fluorescent anti-goat human IgG, counterstained with the DNA dye, 4, 6-diamidino-2-phenylindole, refrigerated for 1 week, coded and

### Page 44 of 186 scored. At least 1000 binucleated lymphocytes were scored for micronuclei; cell division kinetics was calculated for the count of 400 cells per sample ( duplicate cultures). The replicative index (RI) was determined by calculating the percenta ge of cells with one, two, three or more nuclei per individual. Hematological end points such as hemoglobin, hemato crit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red cell c ount, white blood cell count and leu kocyte differential examinations were also conducted. The 2, 4-D c oncentration ‘grand mean among all applicators after spraying 2, 4-D was 240 ± 100 (±SE) ppb. The authors stated, “Urinary concentrations of 2, 4-D increased as hours spent spraying the herbicide increased.” Conseque ntly, the 12 individuals were grouped according to hours spent spraying 2, 4-D according to the following urinary concentrations: <20 ppb (4 subjects), 20- 40 ppb (3 subjects) or > 40 ppb (5 subjects). Results of the lymphocyte analysis revealed that there was no significant increase in the incidence of micronuclei in the exposed workers. However, the replicative index (RI) for lymphocytes was significantly (p<0.05) higher in applicators co mpared to non-applicants and in applicators after spraying compared to before spraying (p<0.016). It was stated that the increase in RI suggests “stimulated cell grow th that could contribute to ca rcinogenesis.” Nevertheless, the

authors concluded that their findi ngs did not support a “gen otoxic pathway” for 2, 4-D at mean urinary levels of 12 to 1285 ppb. Filkowski et al. (2003) Genotoxicity of 2,4-D and dicamba revealed by tran sgenic Arabidopsis thailiana plants harboring recombination and point mutation markers

. [Bin: Mutagenicity] [Cited: Beyond Pesticides; NYS DOH] [Human Studies Considerations Apply: No] Filkowski et al. (2003) expos ed transgenic seedlings of Arabidopsis thaliana to 0, 3, 10, 30, 100, 200 and 300 μg/L 2, 4- D (>98%) for 3 weeks under standard growth conditions; the number treated was not reported. Plants were collected, stained with 5-br omo-4-chloro-3-indolyl gluc uronide, incubated for 48

hours and bleached with ethanol. Intrachromosomal homologous recombinations and point mutations

were scored as blue sectors on white plants. It was indicated that “1000 ‘mutation’- and 200 ‘recombination’-monitoring plants were scored”. The experiment was repeated three times. Data were only presented in graphs; thus variab ility in the data could not be asce rtained. The mid-to low-doses of 2, 4-D (3, 10 and 30 μg/L) but not the high doses (100 and 300 μg/L) induced significant (p<0.05)

increases in A → G point mutations; no explanation was provided for the lack of an effect at the high

doses. Results also show that significant (p<0.05) increases in the induction of homologous recombinations (suggestive of DNA double strand breaks) occurred at all tested concentrations of 2, 4- D; a clear dose response was, ho wever, not evident. The author s stated, “...the increase in the recombination frequency at low concentrations of 2, 4-D correlated with the increase in the levels of A

→ G point mutations”.

### Page 45 of 186 Gollapudi et al., 1999 Evaluation of the genotoxicity of 2,4-dichloroph enoxyacetic acid and its derivatives in mammalian

cell cultures. [Bin: Mutagenicity] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Gollapudi et al.(1999) continued the I ndustry Task Force II efforts on 2, 4-D and investigated several 2, 4-D derivatives [2, 4-D BEE ( 95.6%), 2, 4-D IPA (50.15%) and 2, 4- D TIPA (70.9%)] in mammalian cells performing the Chinese hamster ovary (CHO) cell forward gene mutati on assay at the HGPRT locus and the rat lymphocyte in vitro cytogenetic assay. For the CHO/HGPRT assay, concentrations ranges of 150 to 1400μg/mL +/-S9 (2, 4-D BEE), 500 to 3000 μg/mL +/-S9 (2, 4-D IPA), or 800 to 5000 μg/mL +/-S9 (2, 4-D TIPA) were tested. Duplicate cultures per concentration were exposed to the selected test material doses and the appropriate solvent and positive controls for 4-5 hours, allowed a 7-9 day expression time, selected and plated for mutants and survivors. Each expe riment was independently repeated at least once. The highest concentration of each test agents was selected to represent either to the limit dose of 5000 μg/mL for the te st system (2, 4-D TIPA), the lim it of solubility (2, 4-D BEE) or severe cytotoxicity (2, 4-D IPA +/-S9). None of the 2, 4-D derivatives induced a mutagenic effect in CHO cells in either the absence or presence of S9 activation at any concentration tested. Gonzalez et al., 2005 Genotoxicity of the herbicide 2,4-dichloroph enoxyacetic and a commercial formulation, 2, 4- dichlorophenoxyacetic acid dimethylamine salt. [Bin: Mutagenicity] [Cited: NRDC Petition ] [Human Studies Considerations Apply: No] Gonzalez et al. (2005) evaluated 2,4-D (purity not reported) and commercial grade 2,4-D DMA (containing 60.2% 2,4-D as the active ingredient) for the ability to cause DNA damage in CHO cells using the SCE and Comet tests. Concentration ranges of 2- 10 μg/mL were used in both assays in the absence of S9 activation; treatment times for the SCE assay were 24 and 36 hours and 90 minutes and

36 hours for the comet test. For both the SCE assa y and the Comet test, duplicate cultures were prepared for each dose; three independent experime nts were conducted; and cultures treated with the

solvent acetone were included. The percentage of cells in M 1, M2 and M3 division in 200 metaphases/sample, the proliferative rate and the MIs/2000 cells were determ ined. There were no significant differences for cell-cycle progression or alterations in the rate of cell proliferation. By contrast, 2, 4-D at 24 hours caused 25 and 35% reductions in the MI at 6 and 10 μg/mL; respectively, and 42 and 46% reductions at 6 and 10 μg/mL, resp ectively (36 hours). For 2, 4-D DMA, reduction of ~38 % (24 hours) and ~28% (36 h ours) were recorded for both 6 and 10 μg/mL. SCE data were presented in graphs and showed si gnificant (p<0.01) and dose-related in creases in SCE/cell at all levels (2, 6 and10 μg/mL) for both test mate rials, regardless of time. The fo ld increase at the high dose was 1.5 and 1.8 X for 2,4-D at 24 and 36 hours, respectiv ely and 1.6 and 2.1 X for 2,4-D DMA, at 24 and 36 hours, respectively. Similar evidence of DNA damage was seen in the comet test with significantly (p<0.01) increased and dose-related pe rcentages of cells with tail comets, indicative of DNA damage, at 2.0, 6.0 and 10.0 μg/mL in the absence of severe cytotoxicity ( ≥ 85% viability) at 90 minutes. At 36 hours, regardless of the test materi al, all cells had tail comets. The Ag ency concludes in agreement with the investigators that these data provide clear and reproducible evidence of the DNA-damaging activity of 2, 4-D and the DMA derivative. However, DNA damage alone, whether in vitro or in vivo cannot be

### Page 46 of 186 interpreted as causal event leading to mutagenicity or other adve rse outcomes in rodents or humans because of the ability of cells or w hole animals to repair damage to DNA. Holland et al., 2002 Micronucleus frequency and proliferation in hu man lymphocytes after exposure to herbicide 2,4- dichlorophenoxyacetic acid in vitro and in vivo. [Bin: Mutagenicity] [Cited: NRDC Petition] [Human Studies Considerations Apply: Yes]

In the in vitro micronucleus assays of Holland et al. (2002), two experiments were conducted with either 2 or 5 volunteers (total of 3 females and 2 males, nonsmokers, 26- 45 years). Note: one of the subjects from the first test participated in the second test . Whole blood cultures as we ll as isolated lymphocyte assays were examined in the first test; the second test only used isolated lymphocytes. Collection of blood samples, culture preparation, treatment cond itions and scoring of binucleated lymphocytes was conduced as described above in Figg et al. (2000) . The only procedural exceptions were that lymphocytes smears were not stained with antikineto chore antibody or coated with anti-goat human IgG

and MIs were determined from the count of 1000 cells /culture. In the first te st, samples of whole blood and isolated lymphocytes were exposed in vitro to pure (98%) 2, 4-D at c oncentrations of 0.001-1.0 mM and analyzed for micronucleus inducti on. In the second test, only isolat ed lymphocytes were exposed to both pure 2, 4-D or commercial 2, 4-D (9.4%2, 4-D but adjust ed to 98% pure 2, 4-D) at concentrations

of 0.005 and 0.3 mM.

Cultures exposed to concentratio ns ranging from 0.001- 1.0 mM of pure 2, 4-D (98%) for 4 hours showed only “a minimal increase in MN...at doses appr oaching cytotoxic levels” in either whole blood or isolated lymphocyt es. Similar findings were obtained for the commercial product at 0.3 mM 2, 4-D in isolated lymphocytes. However, the study authors conced ed that these increases were within the “lower range of normal MN for healthy adults”. RIs were significantly (p<0.001) d ecreased (~19%) at 0.3 mM of either pure or commercial 2, 4- D, regardless of the solvent (etha nol or culture medium) used. These data suggest a cytotoxic effect . Although Holland et al. (2002) claimed that 0.005 mM of commercial grade 2, 4-D induced an increase (nonsignificant) in th e RI of isolated lymphocytes, an examination of the data in Table 1 of this paper revealed that the pooled average RI for both commercial 2,-4 –D groups (10 samples) was 1.65 vs. 1.62 for the pooled solv ent control groups. This “increase” cannot be considered evidence of a prolifer ative response. Similarly, MIs we re unaffected by treatment with 0.005 or 0.3 mM 2, 4-D (commercial or pure). Thus, the lack of a meaningful increased RI at this low dose is supported by data showing that MIs were unchange d. The overall conclusions are that 2, 4 –D, regardless of the formulation, did not induce mi cronuclei in human lymphocytes up to levels approaching cytotoxicity. Madrigal-Bujaidar et al., (2001) Induction of sister chromatid exchanges by 2,4dic hlorophenoxyacetic acid in somatic and germ cells of mice exposed in vivo. [Bin: Mutagenicity] [Cited: NRDC Petition; 2, 4-D Task Force] [Human Studies Considerations Apply: No] In the study of Madrigal-Buj aidar et al. (2001), SCE induc tion in mouse bone marrow and spermatogonia were investigated. Groups of five male mice received oral doses of 0, 50, 100 or 200 mg/kg 2,4-D (98.95%) in alkalinized H 2 

### O (pH 10.5) or the vehicle, followed at 2 hours by an intraperitoneal (ip) inject ion of BrdU/charcoal and at 21 hours by an ip injection of colchicine. Bone marrow cells were harvested, stained by the fluores cent-plus-Giemsa technique , and scored; it was not

### Page 47 of 186 reported whether slides were code d prior to scoring. Thirty sec ond-division metaphases/animal were scored for SCE induction; the MIs, cell cycle delay a nd average generation time were also determined. For the examination of spermatogonial cells, groups of five male mice received the test material and BrdU/charcoal as described; cell di vision was halted by the subcutaneous injection of colchicine at 51

hours after BrdU administrati on and testes were harvested 4 hours later. Prepared slides were stained as

described and 25 mitosis /animal were scored for SCEs . The presented data s how that there were no adverse effects on the MI, cell cy cle progression on the average ge neration time in the bone marrow cells. By contrast, si gnificant (p<0.05) and dose-related incr eases in SCE induction (2-fold or approaching 2-fold increases) at 100 and 200 mg/kg were recorded for both cell types. It is also of note that the authors speculate that the fate of the ge notoxic damage on the sperma togonial cells is unclear since “teratogenic and embryotoxic studies on rodents treated with 2,4- D have given conflicting results and the dominant lethal mutation assay on mouse and rats has produced negative data. Thus cell repair, cell lethality, and zygote damage remain as three possi ble fates of the observed genotoxic alterations in spermatogonia.” However, these results should be vi ewed with caution since the extended 1-generation reproductive toxicity study in rats, designed to detect defects in sper m or poor reproductive performance in males, was negative. The Agency notes that simila r speculations can be rais ed for the fate of DNA damage in bone marrow cells since chronic and oncogenic studies in rats and mice were negative. It is

noted that this in vivo study only looked at a genot oxic, as opposed to a mutagenic, endpoint (sister chromatid exchange). Maire et al. (2007) 2, 4-Dichlorophenoxyacetic acid: Effects on Syrian hamster embryo (SHE) cell transformation, cMyc expression, DNA damage and apoptosis. [Bin: Mutagenicity] [Cited: NYS DOH ] [Human Studies Considerations Apply: No]

In a series of in vitro investigations, Maire et al. (2007) exposed Syrian ha mster embryo (SHE) cells to 2, 4-D (98%) at concentrations of 0, 4.5, 11.5, 23 or 34 μM (equivalen t to 0, 1, 2.5, 5 or 7.5 μg/mL, respectively) and evaluated the cultures for malig nant cell transformation, a poptosis, expression of the cmyc gene and gene product, and DNA damage (Comet test). For the cell transformation assay, Xirradiated SHE feeder cells were seeded and grown fo r 25 hours. Target cells we re seeded on top of the feeder cells, allowed a 24-hours period of quiescence a nd 10 dishes /concentration were treated with the selected test article levels of 2, 4-D for 7 days. At the end of exposure, dishes were washed, fixed and stained. Appropr iate solvent (H 2 

### O) and positive controls were incl uded in each assay. One thousand

colonies/concentration were counted and examined for morphology; cloning efficiency (CE) and the transformation frequency (TF) were determined.

For apoptosis, cells were also sampled 48 hours after s eeding, trypinized, reseeded and treated with of 0, 4.5, 11.5 and 23 μM 2, 4-D for 5 and 24 hours. After treatment, cells were suspended in culture medium deprived of growth factors for 3 hours, harveste d and centrifuged. Cell pelle ts were stained with fluorescent acridine orange and ethidium bromide, mi xed, placed on slides, and scored for the number of apoptotic cells/100 cells/slide. Three slides/concentration were examined.

For the RT-PCR determination of gene expression, cells were again sample d 48 hours after seeding, trypinized, reseeded and treated with of 0, 4.5, 11.5 and 23 μM 2, 4-D for 5 hours. After treatment, total RNA was extracted and complementary DNA (c-DNA) was synthesized from 1μg of total RNA using oligo d (T) as a primer and reverse transcriptase. PCR amplification of the c-DNA was performed with c-myc, bcl-2 and bax primers specific to SHE cells. Prepara tions were denatured and PCR products

### Page 48 of 186 were sequenced, cloned and electr ophoresed. Signals were analyzed by densitometry and data were expressed as density ratios of th e transcript corresponding to the c-myc gene. Three different experiments were conducted with different batches of SHE cells. Western bl ot analysis was conducted in parallel with RT-PCR to assess effects on c-myc protein.

For the comet assay, SHE cells were trypsinized 48 hour s after seeding, reseeded and treated with 0, 4.5, 11.5, or 23μM 2, 4-D for 5 and 24 hours. At least three experiments we re performed and included in each assay were the appropriate solvent and positive control. Cells were recovered and electrophoresed;

nuclear DNA was stained with ethidium bromide and sc ored blind. Cells (75/slide/concentration) were scored randomly and the percentage of DNA in the tail was determined as a measure of DNA damage.

Results indicate that 2, 4-D indu ced a dose-dependent and sign ificant (p<0.01) increases in morphological cell transformation of SHE cells at 11.5 (2.4%) and 23μM (3.8%); relative cloning efficiency at 11.5 and 23μM was 14%. No significant increase in transformed cells was observed at the high dose of 34 μM 2, 4-D. There were also no signi ficant increases in apopt otic cells 5 or 24 hours after exposure to the selected dose s of 2,4-D. These data are support ed by the lack of an effect on expression of bcl-2 and bax genes, which are associated with regu lation of the apoptotic pathways. By

contrast

, a significant (p<0.05) increase in the up-regulation of c-myc RNA at 11.5 and 23 μM was reported; the increases represented 2.3-fold at these levels and imm unoblotting confirmed the increase in

the c-myc protein

. Thus, 2, 4-D induced over expression of c-myc at both the RNA and the protein level; this finding is consistent with the transforming activ ity of 2, 4-D at comparable doses. Data from the

comet test revealed that treatm ent with 11.5 and 23 μM for 5 or 24 hours also induced a dose-dependent and significant (p<0.01) increase in the number of DNA-damaged cells. No dead cells were found at any dose or harvest time.

Overall, the investigators conclude d that 2, 4-D induces morphologic ce ll transformation in SHE cells at doses that extend into the cytotoxic range and caused overexpression of c-myc (at both the mRNA and the protein levels) and DNA damage. The Agency notes that the SHE morphologic transformation assay is an

in vitro test for carcinogenesis, not for mutagenicity. Further, all of the findings reported by Maire et al., (2007) were generated from in vitro studies. It is, therefore, not likely that these effects will be expressed in vivo because of the negative findings in acceptable/guideline chroni c/carcinogenic bioassays in rats and mice for cancer

detection. 

### Soloneski et al., 2007 Herbicide 2,4-dichlorophenoxyacetic acid (2, 4-D induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes [Bin: Mutagenicity] [Cited: NRDC Petition ] [Human Studies Considerations Apply: No] In another study of SCE induction, Soloneski et al. (2007) assayed 2, 4-D (purity not reported) and commercial grade 2, 4-D DMA (purity not listed) at levels of 10 to 100 μg/mL in human whole blood (WBC) and plasma leukocyte cultures (PLC). Treatment time was 72 hours. Du plicate cultures were prepared for each dose or the solvent control, acetone and three independent experiments were conducted. Smears were prepared and stained as previo usly described and metapha ses were scored blind for cell cycle progression, MIs and SCE induction. Resu lts show that both test materials significantly (generally p<0.05) affected cell cycl e progression in WBCs at doses ≥ 25 μg/mL (note: the 100-μg/mL group was not scored for 2, 4-D) and only at 100 μg /mL (2, 4-D) for PLCs. Mean SCE frequencies were also significantly (p ≤ 0.05) increased at 10, 25 and 50 μg/mL (2, 4-D) and at 25, 50 and 100 μg/mL

### Page 49 of 186 (2, 4-D DMA) when WBCs were test ed. No activity was seen at any dose when PLCs were examined. Significant reductions in the proliferative rates a nd the MIs were found in WBCs and PLCs after treatment with 50 or 100 μg/mL 2, 4-D, respectivel y. For 2, 4-D DMA reduced proliferative rates and MIs were noted at the high dose bu t only in WBC. The discrepancie s in the data between WBCs and PLCs were considered by the authors to be related to the detoxificati ng properties of erythrocytes, which are present in the PLC cultures but not the purified leukocyte cultures. The authors, citing several investigators, further speculated th at the cellular response caused by both test agents in this study could be related to oxidative damage since lipid peroxidation has been reported for 2,4-D. Tripathy et al. (1993) Genotoxicity of 2,4-dichlorophenoxyacetic acid test ed in somatic and germ- line cells of Drosophila [Bin: Mutagenicity] [Cited: NRDC Petition ] [Human Studies Considerations Apply: No]

In a Drosophila melanogaster wing spot test and sex-linked reces sive lethal assay, Tripathy et al. (1993), exposed second- and third- instar mwh + / + fl 3 

### transheterozygous larvae to 2.5, 5.0, 10.0 mM 2, 4-D (98%) prepared in 3% ethanol + 1% Tween 80 a nd incorporated into the feeding medium for 48 and 72 hours (i.e., “the rest of their larval li fe”); the high dose repr esents 50% of the LD 50. 

### Following exposure, 40 wings/treatment or control group of “eclos ed” flies (i.e., flies that have wings that are highly folded and compact; within an hour, each wing e xpands) were examined for the type and number of spots. Duplicate experiments were performed; the data were pooled because there were no statistically significant diffe rences among experiments.

In the sex-linked recessive lethal assay, Oregon-R la rvae were fed diets contai ning comparable doses of 2, 4-D as described for 3 days; male flies were mated to 3 Basc virgin females for 3 days to obtain the F 1 

### Basc/ Y siblings (Brood 1). Brood 1- males and females were mated (1:1) to produce Brood 2 (Brood 2 represents the sampling of germ ce lls that were primarily late or middle spermatids). X chromosomes from 60-65 males/test group (~1230-1330 X chromosome s/test group) and 150 pooled vehicle controls (3130 X chromosomes) were scored; the X-linked recessive lethals were calculated. Duplicate experiments were performed; the data were pool ed because there were no statistically significant differences among experiments.

Results indicated that after 48 hour s of exposure to 5.0 and 10.0 mM 2, 4-D, a significant (p<0.05) and dose-related increase in the number of spots/per wing (small single and large single wing spots) as well as the total spots was induced. Following 72 hours of exposure, significant in creases were obtained only at the high dose for small single and large single and twin wing spots and total spots. The low dose (2.5 mM) was not active. Based on thes e data, it was concluded that 2, 4-D induced somatic cell genotoxic damage in the primordial wing cells of Drosophila

.

Significant (p<0.05) and dose-related increases in the lethal frequencies were calculated at 5.0 and 10.0 mM 2, 4-D (48-hour exposure) and at all doses (72- hour exposure). Accordi ngly, the authors concluded that in addition to somatic cell damage, 2, 4-D is also genotoxic in the male germinal cells of Drosophila

. The Agency notes that positive findings with D. melanogaster studies have not received the same degree of confidence as assays that are part of the regulator y test battery for mutagenicity. It is further doubtful that these data would contribute much to a weight-of-t he-evidence approach for the mutagenicity of 2-4-D

because their relevance to human health is not clear.

### Page 50 of 186 Tuschl and Schwab (2003) Cytotoxic effects of the herbicide 2, 4-di chlorophenoxyacetic acid in HepG2 cells. [Bin: Mutagenicity] [Cited: NRDC Petition ] [Human Studies Considerations Apply: No] Tuschl and Schwab (2003) found that exposure of the human hepatoma cell line, HepG2 to 2, 4-D (purity no stated) in 1% ethanol i nduces apoptosis but only at high doses (4, 8 or 16 mM; equivalent to 0.9, 1.8 or 3.6 mg/mL) that induced severe effects on ce ll cycling, increased necro tic cells and increased

breakdown of mitochondrial membranes. The data in this article were on ly presented in graphic form. It is of note, however, that these doses are ≈ 1000X higher than the doses re ported by Maire et al. (2007). The relevance of the findings is unclear because of severe cytot oxicity at all levels. Studies on cytotoxicity alone do not provi de evidence of genotoxicity. Venkov et al. (1993) Genotoxicity of 2,4-dichlorophenoxyacetic acid tested in somatic and germ-line cells of Drosophila. [Bin: Mutagenicity] [Cited: NRDC petition ] [Human Studies Considerations Apply: No] In this assay, Venkov et al. (2000) exposed Saccharomyces cerevisiae D7 ts1 to 2, 4-D (100%) test concentrations at 4, 8, or 16 mM (~900, 1800 or 3600 μg/m L, respectively) for 1 to 8 hours; cells were recovered and plated for survival and the reversion a nd mitotic gene conversion rates were calculated. Additionally, 100% pure phenoxyacetic acid, 4-chlorophenoxyacetic acid and 2, 4, 5- trichlorophenoxyacetic acid (2, 4, 5-T) were tested in this yeast assay following a 4-hour treatment. Results show that 2, 4-D at 8 mM induced gene muta tions and gene conversions at levels causing ~40%

reduction in cell survival. The effect was time a nd dose dependent with the peak response occurring

after 8-hours of treatment with 8 mM. No evidence of genetic activity was se en for phenoxyacetic acid or 2,4, 5-T; 4-chlorophenoxyacetic acid at 16 mM i nduced mitotic gene convers ion but did not increase

reversion to isoleu cine protrotrophy.

These investigators also examined the cytotoxic e ffects of 2, 4-D (16, 160 and 1600 μM) and 2, 4, 5-T (16, 160 and 1600 μM) in human transformed hematopoi etic cells lines (LAMA- 84 cells, derived from chronic myelogenous leukemia cells and U-937 cells, deri ved from histocytic lymphoma cells). Both test agents were 100% lethal at the high concentr ation. At 160 μM 2, 4-D, cell survival was 13.4 and 52.5 % for the LAMA-84 and U-937 cell lines, respectively. For the lowest dose of 2, 4-D, survival for

both cell lines was ≥ 89%. The data indicate that 2, 4-D was slightly more cytotoxic than 2, 4, 5-T since cell survival at 160 μM 2, 4, 5-T was 41 and 53% fo r the LAMA-84 and U-937 cell lines, respectively. Based on these findings, the investigators concl uded that 2, 4-D is cyto toxic, mutagenic and recombinogenic in yeast and cytotoxi c in transformed human cells. Da ta on the cytogenetic analysis of

C57b1 male and female mice is discussed under the section on chromosome aberrations and micronucleus induction. The Agency notes that this is an in vitro test for carcinogenic ity, not a test for mutagenicity. The results are not consiste nt with the tumor profile for 2, 4-D.

### Page 51 of 186 Venkov et al. (2000) Genotoxicity of 2,4-dichlorophenoxyacetic acid tested in somatic and germ-line cells of Drosophila. [Bin: Mutagenicity] [Cited: 2, 4-D Task Force ] [Human Studies Considerations Apply: No] As part of the analysis of 2, 4- D and other substituted phenoxyacetic acids in yeast and cytotoxicity assays in human transformed hemat opoietic cells (see Section, Assays in Yeast, Plants and Insects), Venkov et al. (2000) also investigate 100% pure 2, 4- D and 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5- T) for the induction of structural chromosome abe rrations following single ip administrations of both agents at 3.5 and 4.0 mg/kg, resp ectively. Accordingly, bone marrow cel ls were harvested from groups

of four male and four female mice at 12 and 24 hours postdosing. The appropriate vehicle (0.9% NaCl) and positive controls were include d. At least 100 metaphases per an imal were scored for aberrant structural morphology and the MIs were determined from the count of 1000 cells per animal. It was not stated whether slides were coded prior to analys is. 2, 4-D at 3.5 mg/kg induced significant (p<0.0.5) increases in the frequency of cells with aberrations at both sacrific e times (3.4 and 5.5% vs. 1.5 % for the vehicle control at 12 and 24 hours, respectively) ; the predominant type of aberration induced by 2,4- D was chromatid breaks, suggesting cytotoxicity, and exchanges suggesting clasto genicity. 2, 4-D also reduced the MIs by 69% at 12 hours and 35% at 24 hours. By cont rast, 2, 4, 5-T was neither cytotoxic nor clastogenic in bone marrow cells harvested 12 postexposure to 4.0 mg/kg. Based on the findings from this investigation, the authors concluded th at 2, 4-D was clastogenic in mouse bone marrow cells when administered via ip injection. Zeljezic and Garaj-Vrhovac, (2004 ) Chromosomal aberrations, micronuclei and nuclear buds induced in human lymphocytes by 2, 4- dichlorophenoxyacetic acid pesticide formulation. [Bin: Mutagenicity] [Cited : NRDC Petition; 2, 4-D Task Force ] [Human Studies Considerations Apply: No] Zeljezic and Garaj-Vrhovac (2004) exposed human lymphocytes, harvested from two young, healthy non-smoking volunteers, to Deherban A®, a commercial formulation of 2, 4-D adjusted to contain 0.4 or 4 μg/mL 2, 4-D in the absence and the presence of S9. The S9 fraction was derived from the livers of male Sprague-Dawley rats treated w ith intraperitoneal (ip) injections of Deherban A® adjusted to contain 500 mg/mL 2, 4-D for 3 consecutive days. Lymphocytes were treated for 24 hours, recovered, processed, stained with Giemsa and scored (1000 metaphases/co ncentration) for chromosome

aberrations. Similar cultures were treated for 24 hours with 0.4 or 4 μg/mL 2, 4- D +/-S9; cytochalasin B

was added and cells were harves ted, processed, and stained as previously described for the in vitro micronucleus assay. Cells were scored (1000 binuc leated lymphocytes/concen tration) for micronuclei and nuclear buds. Appropriate solvent and positive c ontrol cultures were included. The article did not

indicate whether slides were coded to eliminate bias prior to scoring.

Both concentrations of 2, 4-D induced statistically significant (p<0.05) increases in the percentage of cells with aberrant chromosome aberrations (p rimarily chromatid and chromosome breaks) in lymphocytes from both donors. The number of micr onuclei and nuclear buds at both nonactivated and

S9-activated test material concentrations was al so significantly increased (p<0.05) at 0.4 and 4 μg/mL

+/-S9 in cells also harvested from two different donors. The cytogenetic, micronuclei and nuclear bud responses were uniformly and significantly (p<0.05) hi gher in cells treated in the presence of S9 at both test levels. Hence, it can be concluded that S9 activation was not required to demonstrate the

### Page 52 of 186 clastogenic effect induced by 2, 4-D in this study.

Hazard Data Summary: Studies of several types and designs were cited in the NRDC petition on 2, 4- D and various related documents (see attached for de tailed review of each citation). These included subcutaneous and intraperitoneal exposures to maternal animals w ith resulting gestational and/or lactational exposure of the offspring; subcutaneous exposure of offspring, and oral exposures to the maternal rat and/or offspring. Potential effects id entified in these studies included effects on the endocrine system, including the thyroid, estrogenic a nd androgenic effects, deve lopmental neurotoxicity and effects on neurotransmitters (dopamine and serotoni n), myelination, and mate rnal behavior. Overall, these studies do not identify any hazard not alrea dy identified by the Agency in its assessment. Additionally, the studies cited by NR DC were performed at dose levels above the satu ration level for renal clearance (i.e., doses that overwhelm the body’s abil ity to excrete 2, 4-D). Further, in many cases, the studies were performed at only one dose level, which does not provide dos e-response data; important in determining cause and effect. A se ries of doses provides information s howing that with an increase in dose, there’s an increase in response (magnitude of eff ect and/or severity of effe ct). Other citations were

in vitro endocrine screening assays, ma ny of which have been include d in the Endocrine Disruptor Review Team assessment of 2, 4-D (see TXR No. 0055461). These cita tions were submitted in support of concern for the potential of 2, 4-D to interact with the estrogen, andr ogen and thyroid hormone systems. All totaled, the results of the cited studies do not yield information that would cause the Agency to modify its conclusions from the 2005 Reregistration Eligibility Document (RED) for 2,4-D (EPA 738-R-05-002) that pertain to hazard.

As required in the RED, and in response to the da ta call-in, the 2,4-D Task Force has submitted an F1- extended 1-generation reproductive toxicity study on 2,4-D (MRID 47417901), which provides a comprehensive evaluation of the adult rat repro ductive systems, including an evaluation of gonadal function, the estrous cycle, sperm parameters, mating perform ance, conception, gestation, parturition and lactation, and selected systemic toxicity parameters, as well as su rvival, growth and development of the offspring. A separate satellite group of dams was included for assessments of gross pathology, clinical pathology, thyroid hormones, toxicokinetics, and selected repr oductive and systemic parameters during gestation. F1 offspring were evaluated for potential effects on the developing nervous system, immune system, reproductive and e ndocrine systems, thyroid functio n, and other systemic toxicity parameters. This study has b een evaluated (TXR Nos. 0055341 and 0050697) and is classified acceptable/non-guideline.

All of the effects identified in the cited st udies have been considered in conjunction with the results from the rat extended 1-generation re productive toxicity study on 2, 4-D (MRID 47972101), which evaluated the potential of 2, 4-D to affect the various endpoi nts identified by NRDC as potential adverse effects following 2, 4-D exposure. Regarding concerns expressed in the petition with resp ect to endocrine disrupting potential, the F1 extended 1-generati on reproductive toxicity study on 2, 4-D evaluated thyroid hormones (T3, T4 and TSH), thyroid weight and hi stopathology across life stages, and also evaluated auditory startle and brain morphometry data, which have been reported to be sensitive to thyroid disruption, in the DNT component of the st udy. Parameters sensitive to anti-androgenicity or androgenicity (balano-preputial separation, anogenital distance, qua ntitative F1 nipple retention, spermiology, organ weights and detail ed histopathological evaluations of male reproductive organs) and estrogen sensitive parameters (estrous cyclicity assessm ent in parental females and F1 offspring, time to vaginal opening including assessmen t of vaginal thread retention, anogenital distance, quantitative ovarian follicle count in F1 females, reproductive or gan weight evaluations a nd detailed histopathology

### Page 53 of 186 of female reproductive organs) were evaluated. Th ere were no adverse effects observed on any of the numerous thyroid parameters, estrogen-sensitive pa rameters, and anti-androgenicity or androgenicity parameters evaluated in the ex tended 1-generation reproductive toxicity study, and the NOAEL was established at 45 mg/kg/day in male s and 40 mg/kg/day in females. Both NOAELs are greater than the PODs utilized for the human health risk assessment supporting the 2, 4-D RED.

Regarding concerns expressed in th e petition with respect to developmental neurotoxicity, in the extended 1-generation reproductive t oxicity study on 2, 4-D, offspri ng were exposed during gestation, lactation ( via the milk), and directly (via the diet) follo wing weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, whic h included functional obser vational battery (FOB), motor activity, and acoustic startle response (ASR). An evaluation of myelin was performed on the PND 22 and PND 60 offspring, and the young rats were pe rfused for central nervous system (CNS) and peripheral nerve neuropathology eval uation and brain morphometry. At the highest dose tested (82 mg/kg bw/day in males/59 mg/kg bw/day in fema les), there was no evidence of developmental neurotoxicity and no adverse effect on myelination at any dose level. Bo th NOAELs are greater than the PODs utilized for the human health risk assessment supporting the 2, 4-D RED.

Regarding concerns expressed in the petition with respect to mutagenicity/genotoxicity, based on a review of the cited studies on mutagenicity and the rather extensive Genetic Toxicology database on 2, 4-D, the only conclusion that can be reached with assurance is that 2, 4-D induces DNA damage both in

vitro and in vivo and gene mutations and/or chromosome muta tions in yeast, plants and insects. However, DNA damage alone, whether in vitro or in vivo, cannot be interprete d as a causal event leading to mutagenicity or other a dverse outcomes in rodents or humans because the ability of cells or whole animals to repair damage to DNA cannot be overlooked. Between the in vitro and in vivo tests, the latter carry the greater weight in assessing mutagenic potential because in vitro tests do not capture how a living body responds to a toxi c insult, including its ability to detoxify putative mutagens and genotoxicants.

The F1-extended one-generation reproductive toxici ty study evaluated thyroid hormones (T3, T4 and TSH), thyroid weight, and histopathol ogy across life stages, and also eval uated auditory startle and brain morphometry data. Exposure relate d effects in females in the F1-e xtended one-generat ion study were limited to non-statistically significa nt decreases in T3 and T4 and increased TSH, associated with adaptive changes (depleted colloid) in the thyroid follicles of GD 17 females at a dose that clearly

exceeded the threshold for saturation of renal cleara nce. No robust effects on auditory startle or statistically significant brain mor phometric changes were seen in th e DNT component of the extended one-generation reproductive toxicity study. Myelin deposition was also characterized at PND 22 and 60, and no exposure-related changes were observed. Overall, there are no findings s uggesting an effect on the thyroid at doses below the satu ration threshold for renal clearance. There were no adverse effects observed on any of the numerous thyroid para meters evaluated in the extended 1-generation reproductive study, and the NOAEL was established at 45 mg/kg/day in males and 40 mg/kg/day in females. Both NOAELs are greater than the PODs utilized for the human health risk assessment supporting the 2, 4-D RED. In summary, several studies were cited in the NRDC petition and the associated documents. Most were cited correctly as reported by the investigators, but the overall results have little or no impact on the hazard assessment c onclusions that have been developed by the Agency for 2,4-D. None of the issues of concern iden tified in the NRDC petition identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. Additionally, the

### Page 54 of 186 results do not yield information that would cause th e Agency to modify its conclusions from the 2005 RED for 2, 4-D. The results of the extended 1-generation reprod uctive toxicity study support the previously selected PODs utilized for the human health risk assessment s upporting the 2, 4-D RED.

An exposure pathway identified in the NRDC petiti on is lactational exposure to 2, 4-D. The Agency currently is addressing the issue of a lack of informa tion regarding the levels of 2, 4-D that can occur in

human breast milk. The studies cited by NRDC established effects that occurred at very high dose levels that exceed renal clearance capabilities. This allowe d investigators to examine effects associated with 2,4-D exposure in order to postulate mechanisms of ac tion and to guide future research. In fact, the

information contained in these studies informed the design of the recent extended 1-generation reproduction study in rats. The extended 1-generati on reproductive toxicity study more definitively evaluated the impacts of l actational exposure of treated offspring and, as such, is considered a more definitive source for evaluating these phenomena. Along with being accounted for in the hazard characterization of 2, 4-D, the potential for dietary consumption of 2, 4-D from milk has been adequately considered. Residues in all manner of milk containing food forms (e.g., milk fat, nonfat milk solids, etc.) have been accounted for in the diet ary exposure assessment based on monitoring data from the USDA Pesticide Data Program (PDP). There were no detections of 2,4-D in any samples so one half

of the detection limit was used as the basis for al l analyses and the most highly exposed group (children 1-2 years old) in the analysis acco unted for only 33 and 8.5 percent of the acute and chronic levels of concern, respectively, when all dietary consumption wa s considered. This finding indicates that residues in the actual food supply would have to be much gr eater to be of concern. Further, exposures from human breast milk are expected to be lower than fr om cow’s milk because re sidues in cow’s milk are expected to greatly exceed the values that would be present in human breast milk because exposure of dairy cattle to pesticides are generally significantl y higher than humans (i.e., to lerances for grass (hay) and grass (forage) for 2,4-D are 300 and 360 ppm, respectively, while the 2,4-D tolerances for various human foods are all much lower in the single digits or less than 1 ppm as desc ribed in 40 CFR 180.142). There are uncertainties associated with attempting to characterize exposures from breastmilk. In order to address these EPA is seeking monitoring data for 2, 4-D in human breastmilk (e.g., EPA’s National Children’s Study).

NRDC also discussed factors that can impact derm al absorption and cited se veral studies, which EPA reviewed as well as those provided by other commenters. These studies examined the effects of alcohol consumption in conjunction with topical sunscreenand/or DEET application on dermal absorption of 2,4-D. They also discussed the impact of occl usion on absorption. Several human dermal absorption studies were considered in the Agency assessment as part of the weight of evidence for determining the dermal absorption factor. A value of 10% was used by the Agency based on th ese data and given the variability noted in the results. Additionally, a 10X un certainty factor applied fo r potential variation in sensitivity among members of the human population (UF H 

### interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A 

### ; intraspecies) were applied. Therefore, the Agency is confident that the dermal absorpti on value used does not under estimate dermal ri sk and is adequately protective. It is to be noted that although a dermal absorption value from human data was used, the full

10X UF H 

### was retained to account for uncertainties associat ed with the relative sensitivity to a pesticide within a population. The UF A 

### is typically used to account for the uncertainty associated with the extrapolation of animal testing re sults to humans for risk assessment, including dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has

been eliminated. Therefore, the Agency is confident that the dermal absorption value used does not

underestimate dermal risk and is adequately protective.

### Page 55 of 186 4.0 Overview of Exposure/Epidemiological Data

A summary of the available exposur e data is included below. In some cases specific papers were utilized in the NRDC peti tion itself and in one or more subseque nt, related comments. In other cases citations were utilized only in single comments. For clarity, this section summarizes the actions associated with each citation and the results are pres ented in alphabetical order based on the name of the primary author. Additionally, it is noted where each citation has been used.

Alexander et al (2007) Biomonitoring of 2,4-dichlorophenoxyacetic acid exposure and dose in farm families [Bin: Population Based Monitoring] [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] This study is part of the indus try FFES (Farm Family Exposure St udy) conducted by industry to compare with the Agricultural Health Study conducted by the NIH. Families were evaluated in South Carolina and Minnesota using similar methods to the Ag Health Study. The use of the results of this study in the 2,4-D Task Force Commen ts on the NRDC Petition appears reasonable and consistent with other studies of this type. The investigators report ed urine samples were collected for 3 days after 2,4-D application in both applicators and their family memb ers. Median urine 2,4-D co ncentrations at baseline and day after application were 2.1 and 73.1 μ g/L for applicators, below the limit of detection, and 1.2

μ g/L for spouses, and 1.5 and 2.9 μ g/L for children. The geometric mean systemic doses (micrograms per kilogram body weight) were 2.46 (applicators), 0.8 (spouses), 0.22 (all children), 0.32 (children 4– 11 years of age), and 0.12 (children ≥ 12 years of age). The 2,4-D task force indicated EPA estimates of exposure were reasonable and cited the systemic dose values reported by the investigators. Arbuckle et al (2002) Predictors of herbicide exposure in farm applicators. [Bin: Population Based Monitoring] [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] This study monitored a subset of pa rticipants in a retrospective cohor t study of Ontario farm families. Immediately prior to and after ha ndling phenoxy herbicides (including 2,4-D) for the first time during the season, 126 applicators provided pre-exposure spot urine samples and a subsequent 24 hour urine sample. They also completed a questionnaire on pesticide use and handling practices for the 1 st 

### day of application. The validity of the questionnaire as a predic tor of exposure was exam ined. The arithmetic means for 2,4-D in urine were 27.6 and 2.6 μg/L for a pplicators and those not i nvolved in application, respectively. The maximum reported 2,4-D concentr ations were 410.0 and 66.0 μg/L for applicators and those not involved in applicatio n, respectively. Assuming that the presence of 2,4-D in the urine was a measure of true exposure and that questi onnaire indications of 2,4-D use were the exposure classification subject to e rror, then the questi onnaire’s prediction of exposure had a sensitivity of 56.7% and specificity of 86.4%. Investigators also indicated the only protective factor identified in the 2,4-D multivariate model was the wearing of rubber gloves during mixing or lo ading of the herbicide. The 2,4-D task force indicated the use of gloves dramatica lly reduces exposure which is consistent with the conclusions of the investigators even when no precautions were taken regarding glove condition. Figgs et al (2000) Increased lymphocyte replicative index following 2,4-dichlorophenoxy acetic acid herbicide exposure [Bin: Epidemiology & Population Based Monitoring]

### Page 56 of 186 [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] The objective of this study was to evaluate peripheral bl ood lymphocyte proliferatio n (replicative index: RI) and micronuclei frequency (MF) among 2,4-D herb icide applicators. Twel ve applicators (one 17 years old) spraying only 2,4-D provided a blood and urine specimen upon enrollment, several urine samples during the spraying season, and a blood specime n at the end of the study. Nine controls also provided blood and urine specimens. Standard me thods were used to quantify impacts and 2,4-D residues. Applicator RI and MF we re compared before and after sprayi ng and with controls. Applicators contributed 45 urine specimens and concentrati ons ranged from 1.0 to 1700 μg 2,4-D/g creatinine/L urine. Applicator RI increased after spraying (p =0.016), independent of tobacco and alcohol use, and demonstrated a weak dose-response with increas ing urinary 2,4-D levels (p=0.15). Among 2,4-D applicators pre-exposure comp lete blood counts and lymphocyt e immunophenotypes were not significantly different from post-exposure measurem ents. Investigators concluded urinary 2,4-D concentration may be associated with lymphocyte re plicative index, a cell pro liferation biomarker. Investigators also indicate d the results of this st udy do not support a genotoxi c pathway (see additional review under Mutagenicity above, page 43) . In the NRDC petition, this study is cited in a manner that reflects the conclusions drawn by the investigators excep t it was not explicitly st ated by the investigators

that there is an increased risk of chronic toxi city due to the apparent exceedance of clearance mechanisms. The 2,4-D task force al so cited this study corre ctly but included char acterization elements which were not raised by NRDC in cluding indicating the authors descri bed the study as preliminary, pilot investigation, and the first to show a relationshi p between RI and urine leve ls. The task force also cited the investigator indication that the link betw een 2,4-D function and lymphocyte replicative index is unclear and that the mechanism, our reports and ot her recent investigations do not support a genotoxic pathway. Garabrant et al (2002) Review of 2,4-dichlorophenoxyacetic acid (2,4-D) Epidemiology and Toxicology [Bin: Epidemiology] [Cited: 2,4-D Task Force Comments] [Human Studies Considerations Apply: Yes] A literature review was completed using a variety of animal data and epidemiological evidence to determine if 2,4-D or its salts or esters damage DNA under physiologic conditi ons or induce oncogenic or carcinogenic effects. Investig ators concluded overall the availa ble evidence from epidemiologic studies is not adequate to conclude that any form of cancer is causally associ ated with 2,4-D exposure.

There is no human eviden ce of adverse reproductive out comes related to 2,4-D. The available data from animal studies of acute, subchronic, and chronic e xposure to 2,4-D, its salts, and esters show an unequivocal lack of systemic toxicity at doses that do not exceed renal clearance mechanisms. There is no evidence that 2,4-D in any of its forms activates or transforms the immune system in animals at any dose. At high doses, 2,4-D damages the liver and ki dney and irritates mucous membranes. Although myotonia and alterations in gait and behavioral indices are obse rved after overwhelm ing doses of 2,4-D in the microgram/kg/day range, it is unlikely that 2,4-D has any neurotoxic potential at doses below those required to induce systemic toxicity. This study was conducted for the 2,4-D Industry Task Force II on Research Data to provide an update of the ev idence on human toxicity and cancer risks related to 2,4-D on research completed between 1995 and 2001. The use of the results of this study in the Task Force Comments on the Petition appears reasonable. No environmental media sampling or direct exposure monitoring from the cons idered studies were reported. Garry et al (2002) Birth Defects, Season of Conception, and Sex of Ch ildren Born to Pesticide Applicators Living in the

### Page 57 of 186 Red River Valley of Minnesota, USA [Bin: Epidemiology] [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] This study was a cross-sectional study of 695 fa milies and 1,532 children conducted during 1997 to 1998 in the Red River Valley of Minnesota. This study was a follow up to a previous study conducted in 1989-1991. Birth defects, season of conception and the sex of child ren born to pesticide applicators were evaluated. The investigators concluded that the present and previous studies point out that a ) herbicides applied in the spring may be a factor in the birth defects observed and b ) fungicides can be a significant factor in the determination of sex of th e children of the families of the RRV. They also indicated use of the phosphonamino herbicides (glyphosate, Roundup) was overrepresented in the adverse birth and developmental effect group. Forty- three percent of the children (6 of 14) who had parent-reported ADD/ADHD used phosphonamino herbicides (OR = 3.6; CI, 1.35–9.65). No other commonly used pesticide compared by major organ an d/or functional system was uniquely associated with specific adverse birth or developmental effect s including the phenoxy acids. The 2,4-D task force indicated the results of this study showed no statistically significan t correlations between 2,4-D use and excess adverse birth or neurodevelopmental effects whic h is consistent with the conclusions made by the investigators. Garry et al (1996) Pesticide Appliers, Biocides, and Birth Defects in Rural Minnesota [Bin: Epidemiology] [Cited: NRDC Petition; Beyond Pesticides; 2,4-D Task Force] [Human Studies Considerations Apply: Yes] This study evaluated the hypothesis that offspring of pesticide appliers might have increased risks of birth anomalies. This was investigated by linking 4,935 births to 34,772 state-lic ensed, private pesticide appliers in Minnesota occurring between 1989 and 1992 to the Minnesota state birt h registry containing 210,723 live births in the same timeframe. Western Minnesota, a major wheat, sugar beet, and potato growing region, showed the highest ra te of birth anomalies per 1000 live births: 30.0 for pr ivate appliers versus 26.9 for the general popula tion of the same region. The highest frequency of use of chlorophenoxy herbicides and fungici des also occurred in western Mi nnesota. Births in the general population of western Minnesota showed a significant increase in birth an omalies in the same three birth anomaly categories as appliers and for central ner vous system anomalies. This increase was most pronounced for infants conceived in th e spring. The seasonal e ffect did not occur in other regions. The

male/female sex ratio for the four birth an omaly categories of interest in high phenoxy herbicide/fungicide use is 2.8 fo r appliers versus 1.5 for the gene ral population of th e same region (p=0.05). The use of the results of this study in the NRDC Petition and in the comments from Beyond Pesticides reflect the specific conclusions described in the st udy but NRDC and Beyond Pesticides ignored the comments provide d in later publications by the same investigator which were cited by the 2,4-D task force comments on the petition which gene rally indicate lower incidence rates. The 2,4-D task force commented the study of Garry and co-wor kers (1996) assessing poten tial associations of

herbicide use and birth de fect outcomes in an agricultural region of Minnesota was later described by

this same research team as “a first effort” that suffered from a “limited reporting timeframe” (Garry et.al., 2002). In the latter report , the 2,4-D task force indicated th e authors concluded that a more detailed cross-sectional analysis of this area showed no statistically significant correlations between 2,4-

D use and excess adverse birth or neurodevelopmental effects. Garry et al (2001) Biomarker Correlations of Urinary 2,4-D Levels in Foresters: Genomic Instability and Endocrine

### Page 58 of 186 Disruption [Bin: Population Based Monitoring & Epidemiology] [Cited: Beyond Pesticides] [Human Studies Considerations Apply: Yes] Forest pesticide applicators were selected for consideration in this study of chlorophenoxy herbicides.

Aberrations in G-banded chromoso mes, reproductive hormone levels , and polymerase chain reactionbased V(D)J rearrangement frequencies were examin ed in these applicators whose exposures were mostly limited to chlorophenoxy herbicides. Data from appliers where chlorophenoxy use was less frequent were also examined. The biomarker outcome data were compared to urinary levels of 2,4- dichlorophenoxyacetic acid (2,4-D) obtained at the tim e of maximum 2,4-D use. Further comparisons

of outcome data were made to the total volume of herbicides applied during the entire pesticide-use season. Twenty-four applicators an d 15 minimally exposed foresters (control) subjects were studied. Categorized by applicator method, men who used a ha nd-held, backpack sprayer in their ap plications showed the highest average level (453.6 ppb) of 2,4-D in urine. Investigators concluded that herbicide applicators with high urinary levels of 2,4-D (backpack and boo m spray applications) exhibited elevated LH levels. They also indicated workers exhibite d altered genomic stability as measured by V(D)J rearrangement frequency, which appears reversible m onths after peak exposure. Finally investigators commented though highly detailed, the limited sample size warrants cautious inte rpretation of the data. The use of the results of this study in the comme nts from Beyond Pesticides appears to be reasonable and it reflects the specific conc lusions described by the investig ators (i.e., Garry “found a direct correlation of urinary levels of 2,4-D with serum levels of lute inizing hormone (LH) and high

testosterone levels at th e time of highest exposure suggest a di rect effect on horm onal levels by the chlorophenoxy herbicide). Grover et al (1986) Factors Affecting the Exposure of Ground-r ig Applicators to 2,4-D Dimethylamine Salt [Bin: Planned Exposure Monitoring] [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] A total of 30 individual spray operations using 2,4-D dimethylamine salt were monitored. These operations involved farmers carryi ng out spray operations with trac tor-drawn ground rigs, involving handling, transferring, mixing, and applying to wheat. Air sampling using a typical personal sampling pump method was used. Dermal exposure was m easured using patches and a handwash method.

Patches were worn under 2 work clot hing and on the exterior. Some a pplicators wore protective gloves. Most applicators in the study used tractors with no cab s but 13 of 30 had some sort of enclosure. Urine was also collected and analyzed for 2,4-D residues. A total of nine subj ects participated in the research and all but one were monitored multiple times w ith one worker being monitored during 7 spray operations. Hand exposures accounted for 80 to 90 percent of the poten tial cumulative exposure. The 2,4-D on the rest of the body ranged from 10 to 20 percent of the potential cumulative exposure. Urinary 2,4-D excretion accounted for 1 to 2 percent of the potential cumulative exposure. The median air concentration was 2.1 μg/m 3 

### with a range of <0.01 to 101 μg/m 3 

### . Dermal (non-hand) dose values had a median of 27.9 μg/kg BW with a minimum of 7.5 μg/kg BW and a maximum of 278 μg/kg BW.

Hand dose values had a median of 120 μg/kg BW with a minimum of 0.1 μg/kg BW and a maximum of 552 μg/kg BW. There is no mention in the study of consideration by an institutional review board. This study was conducted prior to the ini tiation of the Pesticide Handlers Exposure Database (PHED). The

use of the results of this study in the comments from the 2,4-D task force appear s to be reasonable and it reflects the specific conclusions desc ribed by the investigator s. Direct exposure monitoring was reported and could be considered for quantitativ e use. However, it should be not ed that PHED is currently used

### Page 59 of 186 for routine assessments of this type rather than individual studies. Hardell, et al (1999) A Case–Control Study of Non-Hodgkin Lymphoma and Exposure to Pesticides [Bin: Epidemiology] [Cited: Beyond Pesticides] [Human Studies Considerations Apply: Yes] A population-based case–control st udy in northern and middle Sweden encompassing 442 cases and twice as many controls was performed. Expos ure data were ascert ained by comprehensive questionnaires, and the questionnaires were supple mented by telephone intervie ws. In total, 404 cases and 741 controls answered the questionnaire. Increas ed risk for NHL was found for subjects exposed to herbicides (odds ratio [O R], 1.6; 95% confidence interval [CI] , 1.0 –2.5) and fungicides (OR, 3.7; 95% CI, 1.1–13.0). Among herbicides, the phenoxyacetic acid s dominated (OR, 1.5; 95% CI, 0.9 –2.4); and, when subclassified, one of these, 4-chloro-2-m ethyl phenoxyacetic acid (MCPA), turned out to be significantly associated with NHL (OR, 2.7; 95% CI, 1.0–6.9). For severa l categories of herbicides, it was noted that only exposure during the most recen t decades before diagnosis of NHL was associated

with an increased risk of NHL. Investigators concluded exposure to herbicides in total, including phenoxyacetic acids, during the decades before NHL dia gnosis resulted in increased risk for NHL. Thus, the risk following exposure was related to the la tency period. The combination of 2,4-D and 2,4,5-T, which constituted Agent Orange in U.S. warfare in Vietnam, was the most predominantly used herbicides in Swedish forestry. In all cases the results presented by the investigators included both 2,4- D and 2,4,5-T concurrently which ma y confound results based on current cultural practices which have eliminated 2,4,5-T concurrent use. Also, TCDD contamination has been eliminated by improved manufacturing processes for 2,4-D. Finally, improved requirements for personal protective equipment are used in current practices which would also impact the OR (Odds Ratio) values. The citation of this study by Beyond Pesticides does not account fo r the confounding factors of 2,4,5-T and TCDD exposures in their attribu tion of enhanced rates of non-Hodgkins Ly mphoma associated with 2,4-D use. Hines et al (2001)

Distributions and Determinants of Pre-emergent Herbicide Expos ures Among Custom Applicators [Bin: Planned Exposure Monitoring] [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] The primary objective of this stud y was to characterize the exposure distributions of the herbicides alachlor, atrazine, 2,4-D 2-ethylhexy l ester (2,4-D EH), and metolach lor among a group of applicators during the spring pre-emergent spra y season. A secondary objective was to evaluate determinants of exposure and to estimate within- and between-worker vari ance components. Fifteen applicators were sampled using a systematic design that included spray and non-spra y days and multiple measurements (five to seven) on each applicator. Air, patch, a nd handwash samples were collected on 89 applicatordays. Applicator-days were classified into three categories: target herbic ide sprayed, non-target herbicide sprayed, and no herbicide sprayed. Mixe d-model regression analysis was used. For all exposure metrics, adjusted mean herbicide exposure s were significantly highe r on days when target herbicides were sprayed as compared to non-spra y days. For 2,4-D EH only, adjusted mean exposures on non-target herbicide spray days were significantly hi gher than on non-spray days. For all exposure metrics, the within-worker variability (GSDW 2.1–5.6) was greater than the be tween-worker variability (GSDB 1.2–2.7). Investigators concl uded wearing gloves significantly reduced adjusted mean hand exposure for all herbicides (4–20 fo ld). This study evaluated several determinants of worker exposure and also produced distributions of exposure under varied field use circumstances. The use of the results of this study in the comments from the 2,4-D task fo rce appears to be reasonab le as they reflect the

### Page 60 of 186 specific conclusions described by the investigators in that the study illustrated that protective gloves significantly reduce exposures. It should be noted th at PHED is currently used for routine assessments of this type rather than individua l studies but the values generated in this study could be used to

calculate risks if desired. Lavy et al (1987) Exposure of Forestry Ground Workers To 2,4-D, Picloram, and Dichlorprop [Bin: Planned Exposure Monitoring] [Cited: 2,4-D Task Force] [Human Studies Considerations Apply: Yes] Urine samples from 80 forest workers applying 2,4-D pl us dichlorprop or picloram were included. Four crews of 20 workers each were studied during applic ation by backpack, injection bar, hypohatchet or a hack and squirt methods. Each worker was evalua ted under two operational modes. In the first (T1), the workers wore their usual clothing and followed the work habits they ordinarily used on their job. In the second (T2), conducted 6 days after the first, wo rkers were given new leather gloves and boots and asked to take all feasible precautions (e.g., wash hands , bathe, wash clothes, avoid sprayed areas) to prevent herbicide exposure and to wash immediately if they come in contact with the chemical. For all application methods except backpacks the T-2 treatment decreased the absorbed dose. During both T-1 and T-2 the clothing of backpack sprayers often became saturated w ith spray, dew and perspiration, and these workers received a higher absorbed dose of 2,4-D (0.04 – 0.24 mg/kg/bw) than did workers in other crews. Hypohatchet workers re ceived a greater dose than did injection ba r or hack-and-squirt workers. Investigators evaluated the utility of using improved hygiene practices with fresh gloves and boots as a means of reducing exposures. In case s where exposures impinge mainly on the feet and hands the T-2 conditions described by the authors coupled with enhanced hygiene appear to reduce exposures. As noted by the authors, T-2 conditions and enhanced hygiene were not as effective with backpack applicators where much of the exposure occurs around the neck a nd upper chest/back area and they were noted to have been working in dense vege tation areas which had been previously treated (i.e., 2 to 7 meters high). Investigators also calculated various margins of safety with the lowest one being 272 for the highest exposed backpack applicators in T-2 conditions (based on geometric mean and NOEL of 24 mg/kg/d). It was conc luded that having a calculated MO S less than the level of concern was attributable to very low dermal absorption sin ce the backpack applicators had very high levels of contact with the 2,4-D spray solutio n as evidenced by their wet clothi ng and observed activities This study evaluated several determinants of worker expos ure and also produced distributions of exposure

under varied field use circumstances. The use of the results of this study in the comments from the 2,4-D task force appears to be reasonable in that they reflect the specific c onclusions described by the investigators (e.g., T2 conditions tend to reduce expos ures and 2,4-D has a low dermal absorption level).

It should be noted that PH ED is currently used for routine assessments of this type rather than individual studies but the values generated in this study co uld be used to calcula te risks if desired. Lerda, et al (1991) Study of Reproductive Function In Persons Occupationally Exposed to 2,4-D [Bin: Population Based Monitoring & Epidemiology] [Cited: NRDC Petition & Beyond Pesticides] [Human Studies Considerations Apply: Yes] The reproductive function of 32 male farm sprayers who were exposed to 2,4-D was evaluated. These evaluations were completed in March through July 1989. The e xposure periods were August to September 1988 with effects being determined in Ma rch 1989 and possible recove ry being evaluated in July 1989. Controls were evaluated in April 1989. Sperm morphology, numbers, volume and other

factors were evaluated. Th e levels of 2,4-D in urine were also dete rmined. It is not clear when the urine

### Page 61 of 186 samples were collected relative to exposure events. The results indicate a mean urinary le vel of 2,4-D of 9.02 mg/liter in the 32 farmers selected for this study. The sperm of these 32 farmers show a significant increase in the incidence of asthenospermia, necros permia, and teratospermia compared to the measured control group. Over time, asthenospermia and necr ospermia diminished but the abnormal spermatozoa (teratospermia) continued. Inve stigators concluded that expos ure to 2,4-D at the measured concentrations produces harmful effects on germinal epithelium, causing alterations of spermatogenesis. In the petition NRDC cited the results of this st udy in a manner reflective of the conclusions of the investigators. Beyond Pesticides al so cited the results of this study in a ma nner reflective of the conclusions of the investigators. No environmental media samp ling was completed. The direct exposure monitoring could be considered for quantitati ve use. However, PHED is currently used for routine assessments of this type rather than individua l studies. No explanation of the circumstances of how exposures occurred was included in the discussions in the paper so those as pects of the study could not be considered. Additionally, the locale of this study was not described so it is not clear that the practices evaluated in this study ar e reflective of current cultural pr actices in the United States. McDuffie, et al (2005) Insect Repellents, Phenoxyherbicide Exposure, and Non-Hodgkin’s Lymphoma [Bin: Epidemiology] [Cited: NRDC Petition] [Human Studies Considerations Apply: Yes] A hypothetical explanation was tested of contradict ory results in studies of phenoxyherbicides and nonHodgkins lymphoma (NHL), that the exposure of rubber gloves recommended for use by farmers when mixing or applying pesticides simultaneously to 2,4-D (2,4-dichlorophenoxya cetic acid), DEET (N,Ndiethyl-m-toluamide), and ultraviolet rays incr eased their permeability to 2,4-D. A case (NHL n _513)/control (n _ 1506) study among me n was conducted using age; provin ce of residence; exposure to insect repellents containing DEET, phenoxyherbici des, or dicamba; and gloves when handling pesticides. The stratum with reported exposure to mecoprop, to DEET and the use of rubber gloves had higher odds ratios (3.86; 95% confidence interval _ 1.57–9.49) compared with strata with other combinations (e.g., 2,4-D with OR = 1.77 and range fr om 0.90 –3.45). The investigators acknowledged that they did not determine the ex act type of rubber gloves used, whic h may vary in their permeability or whether gloves were specifically used while mixi ng or applying 2,4-D or other phenoxyherbicides of interest. The temporal relationship between the mixing or application of pesticid es and the use of DEET

is unknown. Lack of information in each of these inst ances would tend to bias the results toward the null. They also acknowledged the case–control de sign which they used has inherent limitations, including recall bias and selection bias. Investigators concluded the etiologic complexity of NHL was demonstrated. The investigators in dicated in future research sample sizes must be large enough to statistically address the issues of exposure to multiple pesticides, either simultaneously or concurrently

and other effect modifiers or confounders, which include personal hygiene habits during and after handling pesticides and other agricu ltural chemicals, personal and fam ily medical history, and cigarettesmoking practices. NRDC indicates in the petiti on that there is a rela tionship between NHL and phenoxyacid herbicide use, DEET use and the use rubber gloves. NRDC also indicated that this study suggests that herbicides could be penetrating the skin even when gl oves are worn and this could be leading to development of NHL in humans. While increased OR values were calculated by the investigators there are several critic al uncertainties which have not b een addressed (e.g., glove type and timing of DEET use). The investigators also did not indicate if enhanced dermal absorption was the mechanism or glove permeability failure contributed to higher exposures and subsequent higher OR

values. Moody, et al (1992)

### Page 62 of 186 Effect of the Mosquito Repellent DEET and Long wave Ultraviolet Radiation On Permeation of the Herbicide 2,4-D and the Insecticide DDT in Natural Rubber Gloves [Bin: Planned Exposure Monitoring] [Cited: NRDC Petition & 2,4-D Task Force Comments] [Human Studies Considerations Apply: No] A laboratory based permeation cell test was conducted to determine the effect of the commonly used insecticide DEET on the pe rmeability of 100 percent latex rubber gloves (Becton Dickson N36-124). These gloves could be worn by applicators as part of a chemical protective clothing ensemble. Glove permeation analysis was conducted with an automa ted in vitro diffusion analysis (AIDA) method employing an in-house, flow throug h permeation cell design. Permeati on of 14C ring labeled 2,4-D in natural rubber glove material was 2.4±1.81 percent at 48 hours after treatment of the glove with 2,4-D applied with DEET; this was not significantly diffe rent (Student’s t-test; p<0.05) from 3.2±3.46 percent permeation of 2,4-D observed without DEET. Investigators concluded th at because chemical protective clothing is commonly exposed to solar UVA this study ra ises concerns about the efficacy and safety of protective equipment in general. This study describes the permeability changes in a single glove material after exposure to UVA and also DEET coupled with 2, 4-D. The manner in which this study is cited by NRDC is not appropriate because they indi cate that the gloves are highly permeable when the glove is exposed to DEET and UVA. The investigator s reported that with DEET the permeation rate of the glove material was similar with 2,4-D whether or not DEET was presen t. The latex material tested did indicate increased permeability to UVA. NRDC indicates it then follows that enhanced exposures

would occur. This conclusion is not warranted becau se it is not clear the application of UVA in this study mimics real world conditions (e.g., intensity of light beam). Also, only one type of latex rubber glove material was evaluated and its composition was not clear (e.g., UV stabilization or not?). Current production methods likely use UVA and UVB stabilizers in the manufacturing process. It is also easy to recommend other types of glove materials, which are UVA/UVB impervious. Finally, it should be noted that in PHED, exposures under real world cond itions with actual glove use are measured and used to develop risk estimates. While chamber permeability tests of glove material s are useful they do not necessarily accurately predict prot ection factors for gloves, which can also be impacted by design and seam sealing methods. The Agency concurs with the comments of the 2,4-D task force on the NRDC use of this study. Morgan, et al (2008) Adult and children’s exposure to 2,4-D from multiple sources and pathways [Bin: Population Based Monitoring & Planned Exposure Monitoring] [Cited: NRDC Petition Supplement, 2,4-D Task Force Comment & New York State Comment] [Human Studies Considerations Apply: Yes] This study investigated the 2,4-dichlorophenoxyacet ic acid (2,4-D) herbicide exposures of 135 preschool-aged children and their ad ult caregivers at 135 homes in No rth Carolina (NC) and Ohio (OH). Participants were randomly recrui ted from six NC and six OH countie s. Monitoring was performed over a 48-h period at the participants’ homes. Environmenta l samples included soil, ou tdoor air, indoor air, and carpet dust. Personal samples collected by the adult caregivers concerni ng themselves and their children consisted of solid food, liquid food, handwipe , and spot urine samples. 2,4-D was detected in all types of environmental samples but most often in carpet dust sample s, with detection frequencies of 83% and 98% in NC and OH, respectively. The median level of 2,4-D in the carpet dust samples was about three times higher in OH homes compar ed to NC homes (156 vs. 47.5 ng/g, P<0.0002). For personal samples, 2,4-D was more frequently detected in the handwipe samples from OH participants (>48%) than from NC participants (<9%). Handwipe levels at the 95t h percentile were about five times higher for OH children (0.1 ng/cm 2 

### ) and adults (0.03 ng/cm 2 

### ) than for the NC children (0.02 ng/cm 2 

### ) and

### Page 63 of 186 adults (<0.005 ng/cm 2 

### ). 2,4-D was detected in more than 85% of the child and adult urine samples in both states. The median urinary 2,4-D concentrati on was more than twice as high for OH children compared to NC children (1.2 vs. 0.5 ng/ml, P< 0.0001); however, the median concentration was identical at 0.7 ng/ml for both NC a nd OH adults. The intraclass correlati on coefficient of reliability for

an individual’s urinar y 2,4-D measurements, estimated from the unadjusted (0.31–0.62) and specific gravity-adjusted (0.37–0.73) values, were somewhat lo w for each group in this study. The variability in urinary 2,4-D measurements over the 48-h period fo r both children and adults in NC and OH suggests that several spot samples were needed to adequately assess these participants’ exposures to 2,4-D in residential settings. Investigators concluded results from this study show ed that children and their adult caregivers in NC and OH were likely exposed to 2,4 -D through several pathways at their homes. In addition, our findings suggest that the OH children might have been exposed to higher levels of 2,4-D through the dermal and nondietary routes of exposure than the NC children and the NC and OH adults. This study was designed using the CTEPP (Children’ s Total Exposure To Persistent Pesticides and Other Persistent Organic Pollutants) multi-media approach. The key difference is was that in this case potential exposures were quantified for children who did no t attend daycare and th eir adult caregiver’s exposure was also evaluated. The investigators conc luded 2,4-D urinary levels were low compared to

established reference doses estab lished by IRIS. This study was cite d appropriately by the 2,4-D task force including in its description of possible body burden values that c ould be experienced in households (i.e., NC and OH families were sampled). Additionally, the reported house dust levels were compared by the 2,4-D task force to previous values define d by Nishioka (1996) cite d by NRDC. Additionally, investigators indicated levels did not indicate a health concern compared to IRIS and existing BE values. The New York Departments of Environmental Conser vation and Health both in dicated that EPA should consider this study in its 2,4-D risk assessment. The values could be considered quantitatively in an assessment but the results would always be compared to those calculated using applicable existing methods (e.g., SOPs For Residential Exposure Assessment). Finally, th is study was cited in the NRDC supplement to the petition. It wa s cited properly and NRDC indicat ed residues can be tracked into

indoor environments. NRDC also indicated the Agency did not acc ount for this exposure pathway. NRDC indicates the data from this study could be us ed quantitatively for risk assessment purposes. In fact, in its updated SOPs for Residential Exposure Assessment the Agency has proposed a method for completing such assessments. It coul d be incorporated as appropriate. Nishioka, et al (1996) Transport of Lawn-Applied Herbicide Acids from Turf to Home: Correla tion of Dislodgeable 2,4-D Turf Residues with Carpet Dust and Carpet Surface Residues [Bin: Population Based Monitoring & Planned Exposure Monitoring] [Cited: NRDC Petition, 2,4-D Task Fo rce & Beyond Pesticides Comments] [Human Studies Considerations Apply: Yes] Transport of lawn-applied herbicides into the home via walking over treated turf, defined here as trackin, was measured at five different times out to 1 week af ter application. At five discrete times after herbicide application, study participants walked thr ough a treated turf plot a nd then over a section of carpet to simulate the transfer that might occur with a homeowne r walking across turf (e.g., to a mailbox). Dislodgeable turf residues were collected prior to walking; disl odgeable carpet surface residues and carpet dust were collect ed after walking. Residues of turf-applied 2,4-D and dicamba were measured in carpet dust and on the carpet surface af ter track-in. Both carpet dust levels and carpet surface dislodgeable residue levels were highly correlated with turf dislodgeable residue levels. Turf dislodgeable residues were 0.1

− 0.2% of turf application le vels. Transfer of herbicid es from turf to carpet dust was 3% of the turf dislodgeable residues. Transf er from turf to carpet surface was 0.3% of the turf dislodgeable residues. Herbicide 2,4- D was measured in the carpet dust of suburban homes at the 0.1

− 5

### Page 64 of 186 μ g/g levels that are predicted by this efficiency of mass transfer. New tools used to collect these samples included the polyurethane foam roller dislodgeable re sidue sampler and the high-volume solid surface sampler. Investigators concluded th ey expect to find 2,4-D in residential carpet du st up to 1 year after turf application at concentrations of approximately 0.5 μ g/g and at carpet loadi ngs of approximately 0.3

μ g/m 2 

### . These estimates are in agreement with the range of 2,4- D found in nine residential dust samples:

0.1-5 μ g/g and 0.02

− 1.1 μ g/m 2 

### . These 2,4-D levels suggest track -in as a contributing transport mechanism, with the number of children correlated with both carpet loading (r 2 

### = 0.49; p = 0.04) and with dust concentration (r 2 

### = 0.35; p = 0.09) as measured in regressi on analyses. The investigators also indicated in a recently reported study, the urine from matched populations of children in two Arkansas towns was analyzed for 2,4-D and 2,4-D was detected in urine of 18% of the children from the town containing a 2,4-D manuf acturing plant and was present in the urin e of 23% of the children in the town with no manufacturing plant. The similarity of exposur e rates and detection leve ls indicated an exposure source other than the manufacturing plant, thou gh the source was unknown. Both NRDC and Beyond Pesticides cited the conclusions by th e investigators that 2,4-D persists indoors for up to one year. This value was not empirically derived through meas urements but instead it was concluded by the investigators based on a series of assumptions and sampling which only o ccurred out to 1 week after an application of 2,4-D. The Agency does not concur with these conclu sions by the investigators. The 2,4- D task force correctly indicates that the levels were calculated by the investigators and then compared them to empirically derived dus t concentrations by Morgan (2008) which were lower than those calculated by the investigators. The 2,4-D task force did not disc uss the temporal considerations associated with the interpretation in this compar ison. Also, the Agency does not concur with the conclusions by the investigators be cause the assumptions do not account for any degradation processes nor is the short sampling timeframe of 1 week discussed as an un certainty in the conclusions. Yeary, et al (1986) Urinary Excretion of 2,4-D In Commercial Lawn Specialists [Bin: Planned Exposure Monitoring] [Cited: 2,4-D Task Force Comments] [Human Studies Considerations Apply: Yes] Occupational exposure to the herbicide 2,4-D was meas ured in 45 commercial lawn care specialists who had been spraying the herbicide on lawns on a daily ba sis, excluding Sundays, for a period of at least 3 weeks. Exposure was determined by collecting all urine voided during a 24 hour period, and analysis of 2,4-D was made by gas chromatography. The 24 hour ur inary excretion of 2,4-D wa s considered to be a reasonable estimate of the daily ab sorbed dose. Workers wore latex gloves, an apron or coveralls, goggles or faceshield, and rubber boots when handling concentrated product. When spraying dilute mixtures workers wore rubber boots, long pants, and short-sleeved shir ts. Glove use was optional. The urinary concentrations of 2,4-D ranged from non-de tectable at a sensitivity of 0.01 ppm to 2.29 ppm with a median of 0.18 ppm. The quantity excr eted on a body weight basis (mg/kg/day) was 0.0032 ± 0.0010, 0.0063 ± 0.0018, 0.00035 ± 0.0005, and 0.00138 ± 0.0005 mg/kg respectively at 4 work locations. Investigators concluded these values are considerably less than the WHO/FAO acceptable daily dietary intake. The use of the results of th is study in the comments from the 2,4-D task force appears to be reasonable in that they reflect the specific conclusions descri bed by the investigators. However, it should be noted that , as written, the 2,4-D task for ce summary of the study could be misleading because they indicate e xposures did not exceed a level of 6 μ g/kg/day which was really the maximum mean value from particular site with the highest average exposures. Exposures on a individual basis actually ranged about 10 times higher that this value as noted in the highest urinary concentration reported of 2.29 ppm. The direct exposure monitoring could be considered for quantitative use. However, PHED is currently used for routine assessments of this type rather than

### Page 65 of 186 individual studies.

Exposure and Epidemiological Data Summary: Several types of studies were cited in the NRDC petition on 2,4-D and various related documents. These included exposure monitoring in several forms, epidemiological analyses, environmental measuremen ts, and methodological anal yses. All totaled, the results of these efforts do not yiel d information which would cause the Agency to modify its conclusions from the 2005 Reregistration Eligibility Document (RED) for 2,4-D (EPA 738-R-05-002) that pertain to occupational and residential risks. In summary, some risks were identified and the Agency implemented appropriate risk management measures to address them.

Exposure Studies: A number of exposure monitoring studies cited in the NRDC petition and associated comments included biological monitoring results, a m easurement of exposure using passive dosimetry methods, or some measure of environmental cont amination of 2,4-D (e.g., house dust levels cited by Nishioka 1996). None of these values as reported seem to indicate exposures at levels which are of concern using the current endpoints for short- and inte rmediate term exposures as referenced in the 2005 Agency RED human health risk assessment. In fact, many of these values repres ent risk levels that are far below the Agency level of concern and they are mo stly below or in more limited cases similar to the levels used to characterize the po tential risks associated with 2,4- D use in the Agency RED. The process used in Agency risk assessments is to evalua te current cultural practices but with the stipulation that the upper range of an ticipated exposures (i.e., maximum appli cation rate and exte nsive acres treated coupled with central tendency exposure rates compared to subchronic endpoints) be considered in order to determine if all proposed label uses should be allo wable. It could be argued that some of the cited studies could provide additional insight into the type s of job tasks that could be associated with 2,4-D use but this research does not repr esent current agricultural practices which would add uncertainty to the analysis (e.g., Lavy et al describes different tasks in forestry management but was completed in 1986 before current requirements for worker protection were established). Also, some studies investigated the factors which impact exposure (e.g., Hines et al 2001). Many of the re ferenced studies provide similar information on exposure factors but this issue is much broader than the studies considered herein and for consistency the Agency uses the Pesticide Handlers Exposure Database (PHED) to predict occupational handler exposures to alleviate su ch concerns. In addition to the studies mentioned by NRDC and petition comments, similar trends are noted in more current research. Aylward et al (2009) 4 

### concluded that “current use patterns and ri sk management efforts” are keep ing levels “well below current noncancer reference values” established by the Agenc y. This paper used biomonitoring equivalents as the basis for the analysis defined by Aylward and Hays (2008). 5 

### It should be noted that when compared to the studies cited by NRDC and pe tition comments that the values al so do not indicate a concern. Finally, Thomas et al (2009) 6 

### reported biomarker urine concentrations from the Agricultural Health Study cohort ( www.Aghealth.org ) lower than current Agency levels of concern which is consistent with

the results of the cited studies as well. In summ ary, the exposure monitoring studies cited by NRDC do not indicate risks of concern and the values presente d are similar to or lower than those used for risk

4 Aylward LL, Morgan MK, Arbuckle TE, Barr DB, Burns CJ, Alexander BH, et al. 2010. Biomonitoring Data for 2,4- Dichlorophenoxyacetic Acid in the United States and Canada: Interpretation in a Public Health Risk Assessment Context Using Biomonitoring Equivalents . Environ Health Perspect 118:177-181. doi:10.1289/ehp.0900970 5 Aylward LL, Hays, SM (2008) Biomonitoring Equivalents (BE) dossier for 2,4-dichlorophenoxyacetic acid (2,4-D) (CAS No. 94-75-7) , Regulatory Toxicology and Pharmacology 51 (2008) S37–S48 6 Thomas, K.W., Dosemeci, M., Hoppin, J., Sheldon, L.S., Cr oghan L.S., Gordon, S.M., Jones, M.L., Reynolds, S.J., Raymer, J.H., Akland, G.G., Lynch, C.F., Knott, C.E., Sa ndler, D.P., Blair, A.E., and Alavanj, M.C. (2009) Urinary biomarker, dermal, and air measurement results for 2,4-D and chlorpyrifos farm applicators in the Agricultural Health Study , Journal of Exposure Science and Environmental Epidemiology (2009), 1–16

### Page 66 of 186 assessment purposes by the Agency.

Consideration of Dust as Exposure Source: To examine whether 2,4-D contamination of indoor dust might significantly alter the findings of the risk assessment, EPA considered how the indoor residue

values in the Nishioka studies w ould affect the risk assessment. EPA assumed for screening purposes that toddlers consume 100 mg/day of dust containi ng the highest dust concentr ation found in Nishioka studies (67 μg/g). The 2,4-D levels in dust in the Nishioka studies were generally much lower than 67 μg/g (e.g., 1996 maximum is 4.85 μg/g, and 2001 median is 10 μg/g). The value of 100 mg/day for dust consumption is drawn from the EPA’s Child Specifi c Exposure Factors Handbook and is the same value assumed for soil consumption. This value was al so used in the Nishioka studies. Additional conservatisms in this screening assessment are the a ssumption that (1) that exposures from dust occur assumed despite the uncertainties noted in the 2009 FIFRA Scientific Advisory Panel Report where the recently revised SOPs For Residential Risk Assessment were reviewed; and (2) 2,4 -D residues do not to decline over time even though 2,4-D is known to dissipate quickly

. Based on these assumptions, margins of exposure range from approximately 32,000 to 150,000 depending upon whether the duration of exposure considered is acute, short- or intermediate term in duration. 7



### As such, use of this highest dust concentration valu e would not impact the findings of the current risk assessment. If it is further assumed that dusts persist in impacted residences in such a way that ingestion of the highest concentration would occur in a ch ronic exposure pattern and that the highest noted concentration in dust would never dissipate, which is counter-intuitive given how 2,4-D is used and its known rapid dissipation characteristics, risks are still not of concern. In such si tuations, dust would be the predominant source for chronic exposures but margins of exposure still would exceed 11,000 based on the chronic dietary point of departure (5 mg/kg/d). 7



### Epidemiological Research: The appropriate consideration of epidemiological research into regulatory decision making is difficult due to the inherent uncertainty associated with interpreting such data. 8 

### In fact the Agency has been undertakin g a process to develop a more ri gorous approach for incorporating epidemiological research into regulatory decision making. 9 

### With regards to specific pesticides such as 2,4-D the Agency has been actively considering epidem iology in an extensive way because of the nature

of the available research and the potential concerns which can be identified based on this research. In fact, the FIFRA Scientific Advisory Panel evaluated this type inform ation applicable to 2,4-D in 1994 and the panel concluded that “data are not sufficien t to conclude that there is a cause and effect relationship between exposure to 2,4-D and non-Hodgkin’s lymphoma.” In 1996 and then in 2004 further reviews were conducted of additional ep idemiological studies by Blondell (D311464, 12/8/04) both essentially with similar conclusions. In some cases studies completed by the same investigators

who were cited in Blonde ll (2004) were also cite d in the NRDC petition and the other pertinent documents (e.g., Garabrant et al (2002)). All the additional data identified in the NRDC petition do not indicate major differences from the Agency conclu sions based on epidemiological evidence outlined by Blondell (2004).

7 Calculated using: POD (mg/kg/d)/[(67μg 2,4-D/1000 mg soil*100 mg soil ingestion)/15 kg child body weight] where Acute POD = 67 mg/kg/d, Short-term POD = 25 mg/kg/d, Intermediate-term POD = 15 mg/kg/d, and chronic POD = 5 mg/kg/d 8 Similar conclusions are drawn in CA OEHHA (2009) Public Health Goals For Chemicals in Drinking Water: 2,4-D

available at http://oehha.ca.gov/water/phg/pdf/24dphg010209.pdf

9 http://www.epa.gov/scipoly/sap/meetings/2010/020210meeting.html

### Page 67 of 186 The cited epidemiological studies we re of different designs, focused on a variety of exposed groups, and also contained other types of in formation such as biological m onitoring results or environmental measurements. In some cases the NRDC analys is ignored additional re search completed by investigators which impacted the ov erall conclusions drawn by investig ators regarding th e public health implications of 2,4-D. One such example is the use of Garry et al (1996) by the NRDC which ignored the conclusions drawn in Garry et al (2002) which indicated less concerns than in the original paper.

In some cases investigators were concerned about po ssible causal links to certain effects. The studies where results are related to 2,4-D and that appear to present more likely results of concern are discussed

further below. Selected issues are described below along with a rati onale regarding how the findings of these specific studies should be considered.

Case-control studies : In these studies investigators trie d to determine if 2,4-D exposure can affect genomic stability, repr oductive hormone levels (lutei nizing hormone), non-Hodgkin’s lymphoma (NHL) and increased birth defects. Case-control studies identify factors that may contribute to a medical condition by comparing patie nts who have that condition (the cases) with subjects (the controls) who do not have the condition, but shar ed the common characters and experience within a defined time period (incl uding the exposure situation). The studies

discussed below do not de monstrate causation.

In 1998, Hardell and Eriksson conducted a case-control study to determine the importance of phenoxyacetic acids and other pesticides in the etiology of NHL. Although the study reported exposure to phenoxyacetic acids including 2,4-D (significantly with MCPA) and glyphosate resulted in increased risk NHL, authors admitted that the in-depth knowledge of active ingredient concentration was lacking, and r ecall bias in providing informa tion for pesticide might have occurred since the exposures occurred a few deca des ago (i.e., the exposu re assessment aspects of this study were uncertain).

Garry et.al. (2001)mentioned that cases with high urinar y 2,4-D levels had el evated LH levels, and exhibited altered genomic st ability; however, to what extent the cumulative chromosome

effect observed were related to adjuvant use was unsure, also th e sample size was small (only 24 exposed and 15 controls) making the results uncertai n. It should also be noted that the mean 2,4-D residue level in urine (454 ppb) is also no t a level where saturatio n of the LH mechanism would be anticipated and thus would not be of concern.

Literature Review: A review of the epidemiological and toxicological studies from 1995 to 2001 by Garabrant and Philbert, concluded that 2,4-D did not have any genotoxic potential; also it did not have carcinogenic and reproductive toxic effects. However, they suggested an association between exposure to 2,4-D and neurological effects su ch as myotonia, persistent peripheral polyneuropathy, demyelin ation and ganglion degeneration in CNS. They also indicate 2,4-D exposure may have an association with su icide, depression, anxiety, and post-traumatic stress syndrome. However, these results are uncer tain because the studies did not control for the effects of age, exposure to dioxin, and other confounding factors. Thes e studies also did not adequately quantify the exposure aspects of these suggested relationships. The reviewed articles were from 1995-2001, and this review was done at the request of the, “Industry task force II”. It should also be noted that the use patterns and possible use of adjuvants or dioxin contamination

### Page 68 of 186 differs in current practice from the timefr ame when these studie s were conducted.

Ecological Study: Garry et.al. (1996) c onducted ecological study. They found that rate of birth defects were increased in offspring born to pest icide applicators, infant s conceived in spring, and offspring born to the general population residing in pesticide (chloropheno xy herbicide) high use region. However, analysis was done at the group level, rather than i ndividual level. Thus individuals were assigned an e xposure classification that might not represent their true exposure making this aspect of the study highly uncertain . Although this study cons idered the pregnancy risk factors, it did not mention in detailed if important ma ternal factors (such as, infections, vaccines, drug use) that cause birt h defects were also considered.

A broad array of epidemiological research was cited in the NRDC petition and associated comments.

This review indicates that descri bed by Blondell (2004) are s till pertinent (i.e., 2,4- D does not appear to be carcinogenic). With regard to other possible eff ects investigators have yet to prove that there is a causal relationship between 2,4-D and these effects at exposure levels which w ould be encountered in user populations, especially give n current use practices and work er protection requirements (e.g., neurotoxicity). However, more recent research may necessitate further investigation into the possible effects of 2,4-D as illustrated by findings in the Agricultural Health Study (e.g., rhinitis) 10 

### and possible

links to cardiovascular disease. 11 

### Any issues pertaining to the valid ity of these studies will also be addressed (e.g., Schreinemachers use of data wh ich do not represent cu rrent use practices).

Exposure Methods: Some studies cited in the NRDC pe tition and associated documents provide methodological information pertinent to the risk assessment and risk management process (e.g., cell permeation test for breakthrough of latex rubber glove material conduc ted by Moody et al 1992). Such studies, while well conducted, may provide little impact on the health outcomes associated with 2, 4-D use because different glove materials could be re commended for use by applicators which could be less permeable to 2,4-D end use products or less susceptib le to environmental condi tions (e.g., because they are UV stabilized). Differences in glove materials are considered in the development of end-use product pesticide labels according to the nature of the cha llenge agent which should acc ount for issues noted in

these studies. 12 

### Addtionally, NRDC cited the Riviere et al. study to claim that occlusion can significantly enhance skin absorption of 2, 4-D. In normal pesticide expo sure situations, fabric occlusion would be anticipated under normal work clothing. Guidelines for evaluating dermal toxicity of pesticides require that a gauze dr essing be placed over the dosage site which closely simulates fabric occlusion. This method, coupled with the consideration of appropriate uncertainty factors accounts for any uncertainty raised by NRDC. Finally, a st udy NRDC was cited by Moody et al. regarding the permeability of worker gloves in conjunction with simultaneous exposure to DEET and sunlight. However, EPA does not concur with the conclusions made by NRDC because the conditions of this research do not represent likely ex posure conditions which would be ex pected currently in agriculture. This is because DEET would have to be present in all circumstances where workers use gloves at similar concentrations and new glove mate rials account for sunlight exposur e through UV stabilization. It should also be noted that exposures used to calcu late risks have been measured under real world

10 Slager RE, Poole JA, LeVa n TD, Sandler DP, Alavanja MC, Hoppin JA. Rhinitis associated with pesticide exposure among commercial pesticide applicators in the Agricultural Health Study. Occup Environ Med. 2009 Nov;66(11):718-24. Epub 2009 Mar 15. PubMed PMID: 19289390; PubMed Central PMCID: PMC2936571.

11 Schreinemachers DM (2010) Perturbation of lipids and glucose metabolism associated with previous 2,4-D exposure: a cross-sectional study of NHANES III data, 1988-1994. Environ Health. 2010 Feb 26;9:11.

12 http://www.epa.gov/oppfead1 /labeling/lrm/chap-10.pdf

### Page 69 of 186 conditions with actual glove use, which also account s for possible effects of field conditions on glove efficacy in risk assessment.

Summary: The Agency generally concurs with the analysis of this ev idence by the 2,4-D task force. Several studies were cited in the NRDC petition and associated comments. Most were cited correctly as reported by the investigators but, ov erall, the results do not impact th e risk conclusions which have been developed by the Agency for 2, 4-D. This is because of several critical considerations including some information is outdated. For example, 2, 4-D use patterns have changed in agriculture since the

timeframes where the cited data we re generated. Additionally, po ssible confounding aspects such as potential dioxin contamination is no longer a factor in the modern ma nufacturing processes for 2, 4-D. In many studies there are significant uncertaintie s that were identified, including preliminary conclusions were cited by NRDC not accounting for late r research by the same i nvestigators. Also, in many cases the exposure related aspects on the analyses were inade quately addressed.

The Agency is currently involved in processes to re fine many of its exposure assessment inputs and to establish better methods for the considering epidemio logical research into th e regulatory process (i.e., 2010 SAP review). The Agency is also re-evaluat ing pesticide risks on a cy clical basis under its registration review process. Given these two efforts, the Agency will further evaluate research related to 2,4-D under registration review. It has also been actively participating in epidemiological research efforts such as the Agricultural Health Study and, as part of this process, will pursue additional information related to 2,4-D and the potential fo r health effects in poten tially exposed populations.

5.0 Overview of Pet Incidents

A summary of the available pet incide nt data is included below. In some cases specific papers were utilized in the NRDC petition itself or in one or more subsequent, related comments. In other cases citations were utilized only in single comments. For clarity, this section summarizes the actions associated with each citation and the results are pres ented in alphabetical order based on the name of the primary author. Additionally, it is noted where each citation has been used.

Hayes, et al (1991) Case-Control Study of Canine Malignant Lymphoma : Positive Association With Dog Owner’s Use of 2,4-Dichlorophenoxyacetic acid herbicides [Bin: Pet Exposure Monitoring] [Cited: Beyond Pesticides] [Human Studies Considerations Apply: No] A hospital-based case control study of companion dogs which examined developing canine malignant lymphoma associated with the use of chemicals in and around the home. A se lf administered owner questionnaire and/or phone interviews were co mpleted for 491 cases, 466 nontumor controls, and 479 tumor controls. Dogs diagnosed between Janu ary 1984 and February 1988 were identified from 3 veterinary teaching hospitals (MI, IN, CO). Dogs living in house holds where 2,4-D was applied more frequently had significantly higher odds ratios that control situations (Odds Ratio=1.3). Risk of canine malignant lymphoma rose to twofol d excess with four or more yearly owner applications of 2,4-D. A positive trend (P<0.02) was observed for the yearly num ber of applications. Other possible factors (e.g., flea product use, cigarette use, carp et presence) were evaluated but fo und not to be a factor. Known susceptible breeds (e.g., boxers, mas tiffs) were also addressed. Th e investigators commented on how

### Page 70 of 186 recall biases were addressed in the study but they acknowledged a major weakness is a lack of precise exposure data. No environmental or personal samp ling was completed and the author s acknowledge significant uncertainty pertaining to a lack of exposure data. It is also not clear how germane the results of this study are given the timeframe over which this study was conducted compared to modern use patterns associated with 2,4-D. Th e citation of this study by Beyond Pes ticides reflects the conclusions of the investigators but it does not account for the uncertainty associat ed with the expos ure predictors acknowledged by the investigators in this study. It should also be noted that in the follow-up 1995 paper to this investigators note significant levels of cri ticisms that are addressed in the subsequent document described below. Please refer to that summary of Hayes et al 1995 for additional information. It should be noted that the NRDC petiti on does not make any mention of exposure to pets or via pets. Hayes, et al (1994) On the Association Between Canine Malignant Lymphoma and Opportunity for Exposure to 2,4- Dichlorophenoxyacetic Acid [Bin: Pet Exposure Monitoring] [Cited: Beyond Pesticides] [Human Studies Considerations Apply: No] This article was written in response to specific criticisms leveled at the Hayes et al 1991 article described above. Several specific considerations were addressed including how control groups were treated, whether responses were via mail or phone, potential hospital selec tion bias, difficulties associated with exposure classificat ion including frequency of use, a nd knowledge related to the specific use of 2,4-D products. The authors addr essed each of the app licable criticisms. The results of Hayes et al 1991 were supported in their analysis. The auth ors clarified the conclu sions to better address uncertainty issues. The investigator s also acknowledged that the report ed values could be explained by bias or confounding factors. It was also indicated th at the results should be interpreted with caution because of low exposures. The investigators also c oncur with several commenters that the study does not prove that 2,4-D exposure in a home environment is a cause of malignant lymphoma in companion dogs. They again indicate it does warrant further resear ch on the issue. The cita tion of this study in the Beyond Pesticides comments to the NRDC petition is correct. However, it is somewhat misleading because they do not reiterat e the level of uncertainty voiced by the investigators in their comments about the applicability of the result s and the call in both Hayes 1991 and 1995 for additional research to investigate this relatively weak relationship be tween 2,4-D exposure and cani ne malignant lymphoma. Reynolds, et al (1994) Canine Exposure To Herbicide Treated Lawns and Urinary Excretion of 2,4-Dichlorophenoxyacetic

Acid [Bin: Pet Exposure Monitoring] [Cited: Beyond Pesticides] [Human Studies Considerations Apply: No] A recent study by Hayes et al (J NatI. Cancer. Inst., 83: 1 226-i 231 , 1991) found an increased risk of malignant Iymphoma associated with exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) in pet dogs. A study was conducted to determine the extent to wh ich dogs absorb and excr ete 2,4-D in urine after

contact with treated lawns under natural conditions. Among 44 dogs potentially exposed to 2,4-Dtreated lawns an average of 10.9 days after application, 2,4-D concentrations greater than or equal to 10.0 μg/l were found in 33 dogs (7 5%) and concentrations of ≥ 50 μg/l were found in 17 (39%). Among 15 dogs with no known exposure to a 2,4-D-treated lawn in the previous 42 days , 4 (27%) had evidence of 2,4-D in urine, 1 at a concentration of ≥ 50 μg/l . The odds ratio for the association between exposure to a 2,4-D-treated lawn and the detection of ≥ 50 μg/l 2,4-D in urine was 8 .8 (95% confidence interval, 1 .4-56.2). Dogs exposed to lawns treated within 7 days before urine collection were more than 50 times

### Page 71 of 186 as likely to have 2,4- D at concentrations ≥ 50 μg/l than dogs with exposure to a lawn treated more than 1 week previously (odds ratio = 56.0; 95% c onfidence interval, 10.0- 312.2). The highest mean concentration of 2,4-D in urine (21 3 mg/l) was found in dogs sampled w ithin 2 days after application of the herbicide. Investigators indicated these fi ndings demonstrate that dogs living in and around residences with recent 2,4-D lawn treatment absorb measurable amounts of the herbicide for several days after application and thus may constitute a useful animal model for evaluation of the effects of herbicides on the induction of ly mphoid cancer. This study was c onsidered in the comments of Beyond Pesticides and was cited appropriate ly except that the comparison made to previous data could not be

verified. It should be noted that the NRDC petition does not make a ny mention of exposure to pets or

via pets.

Pet Exposure Summary:

EPA evaluated the studies cited by Beyond Pest icides and the studies do not change the Agency’s current conclusions. The two studies conducted by Hayes et al. in 1991 and 1995 focused on an epidemiological evaluation (i.e., case control st udy) of the relationship between 2,4-D use on lawns and the incidence of malignant lymphoma in comp anion dogs. This evaluati on used a hospital-based recall questionnaire approach. There were several cr iticisms of this paper which were addressed in 1995 by Hayes et al., including issues associated with the exposure assessment process and the uncertainties

associated with low odds ratios. The authors ackn owledged a major weakness w ith the studies were a lack of environmental or personal sampling and possi ble recall bias. Further, significant uncertainty exists due to the lack of exposure data. In addition, the 2005 RED require d that, in order for a product to be eligible for reregistration, label language needed to be added restri cting pet access to treatment areas during and immediately after applica tion. All labels now have this language, which changes the use patterns that existed dur ing the time of the Hayes studies in the early 1990s because now pets should not be in the treatment area during or immediately after application. Furt her, the authors concurred with

several commenters that the studie s do not prove that 2,4-D exposure in a home environment is a cause of malignant lymphoma in comp anion dogs. Therefore, Hayes et al does not impact the Agency’s previous assessment.

Reynolds et al. 1994 attempted to supplement Hayes et al. by attempting to refine the ability to predict exposures from lawn herbicides like 2,4- D by collecting exposure ch aracteristics using an interview/questionnaire approach and by monitoring of 2,4-D in pet urine. They concluded that gathering data via owner questionnaires could be a good surrogate predictor of exposure. Among 44 dogs potentially exposed to 2,4-D- treated lawns an average of 10.9 days after application, 2,4-D concentrations greater than or equal to 10.0 μg/l were found in 33 dogs (75%) and concentrations of ≥ 50 μg/l were found in 17 (39%). Among 15 dogs with no known exposure to a 2,4-D-treated lawn in the previous 42 days, 4 (27%) had evidence of 2,4-D in urine, one at a concentration of ≥ 50 μg/l . The odds ratio for the association between exposure to a 2,4-D-treated lawn and the detection of ≥ 50 μg/l 2,4-D in urine was 8.8 (95% confidence in terval, 1 .4-56.2). Dogs exposed to lawns treated within 7 days

before urine collection were more than 50 times as likely to have 2,4-D at concentrations ≥ 50 μg/l than dogs with exposure to a lawn treate d more than one week previously (odds ratio = 56.0; 95% confidence interval, 10.0-312.2). The highest mean concentrati on of 2,4-D in urine (21 3 mg/l) was found in dogs sampled within two days after appl ication of the herbicide. The au thors indicated that these findings demonstrate that dogs living in and around residences with recent 2,4-D lawn treatment, absorb measurable amounts of the herbicide for several days after application and, thus, may constitute a useful animal model for evaluating the e ffects of herbicides on the inducti on of lymphoid cancer. EPA believes

### Page 72 of 186 the use of interviews/questionnaires may have some utility as a surrogate means of predicting exposures in epidemiological research. However, with regard to the specific issue of establishing a model specific to 2,4-D exposure and cancer induction the results ar e incomplete and have c onsiderable uncertainty, which is consistent with the findings of the investigators who concluded that additional research on this

matter is needed.

In summary, the studies cited by Beyond Pesticides, in its comment s to support its assertion that 2,4-D has been shown to induce lymphoma, did not a ddress key uncertainties (lack of sampling, timing of the studies, and recall bias) in the study results. In particular, Beyond Pesticides failed to note the relatively weak association between exposure and lymphoma onset desc ribed by Hayes et al. Further,

because these studies were conducted prior to the risk mitigation measure identified in the RED, and now reflected in product label language, limiti ng pet access during and immediately following application, the resulting new use pa tterns should result in reduced exposure. As such, while the Agency concurs with Hayes et al that perhaps additio nal research is warranted, it does not believe that

there is evidence of critical animal health issues which warrant changes to its current conclusions.

6.0 Summary

Several types of studies were ci ted in the NRDC petition on 2,4-D and comments from other sources. These included a variety of toxicity, exposure, epid emiological, and pet incide nt data. The position set forth in the petition is that ther e is sufficient scientific evidence to justify the can cellation of 2,4-D containing products because it is not possible to ma ke a safety finding. Comments from other sources both support (i.e., Beyond Pesticides) and counter mand the conclusions made by NRDC (e.g., 2, 4-D Task Force). After considering the analyses and conclusions included in these documents the Agency does not believe the petition or ot her related comments provide any in formation that would cause the Agency to modify its conclusions on endpoints used in risk assessment from the 2005 Reregistration Eligibility Document (RED) for 2, 4-D (EPA 738-R-05-002).

The toxicity data that were cite d included subcutaneous and intrap eritoneal exposures to maternal animals with resulting gestational an d/or lactational exposure of the offs pring; subcutaneous exposure of offspring, and oral exposures to the maternal rat a nd/or offspring. These studies included evaluations of the endocrine system, including the thyroid, es trogenic and androgenic effects, developmental neurotoxicity and effects on neurotransmitters (dopa mine and serotonin), myelination, and maternal

behavior. Some invest igations were conducted in vivo and others in vitro . Overall, these studies do not

alter the POD used in the 2005 RED and human health risk assessment. Additionally, the studies cited by NRDC and others were performed at dose levels above the satura tion level for renal clearance ( i.e., at levels that overwhelm the body’s ability to excrete 2, 4-D) and in many cases were performed at only one dose, which does not provide dose-response data; i.e., a no-effect dose a nd another dose where the

effects are observed but at a di fferent magnitude, were not iden tified. Other citations were in vitro endocrine screening assays, many of which have been included in the Endocrine Disruptor Review Team assessment of 2, 4-D. The Tier 1 battery assays were designed as screens. Their intended purpose is to evaluate whether a substance is capable of in teracting with the endocrin e system The EDSP Tier 1 data is due to the Agency in the fall of 2011. On co mpletion of the review of Tier 1 data, the Agency will conduct a Weight-of-Evidence (WoE) to consider the potential of the chemical to disrupt either the estrogen, androgen or thyroid (EAT ) hormone systems. The WoE a pproach that is been drafted provides general guidance in which basic principles a nd criteria are described fo r an interpretive process for evaluating the EDSP Tier 1 screening results toge ther with other considera tions and supportive data

### Page 73 of 186 sources to identify candidate chemicals for Tier 2 testing. Chemicals with de monstrated evidence of a potential to interact with EAT hor mone systems will be considered as candidates for Tier 2 testing. Currently, the Agency is developing Tier 2 EDSP tests for ecological species, which may include the following: Amphibian 2-Generation Study, Avian 2- Generation Study, Fish Lifecycle Study and the Invertebrate Lifecycle Study. The Tier 2 mammalia n study is currently the 2-Generation Reproduction and Fertility Effects Study (Note: the OECD Guideline for the Extended F1 One Generation Reproduction Test Guideline is also under considera tion). It should be noted that the fact that a substance may interact with a horm one system does not mean that when the substance is used, it will

cause adverse effects in huma ns or ecological systems.

In addition to the studies considered in the NRDC and associated analyses, the 2, 4-D Task Force submitted an F1-Extended 1-Generation Reproductive t oxicity study in response to the Data Call-In for a 2-generation reproduction study an d a developmental neurotoxicity study. The Extended 1-Generation Reproduction study included an assessment of several key endpoints: an evalua tion of potential effects on parental male and female reproductive function, offspring survival and growth. Additional endpoints included endocrine and system ic toxicity parameters such as estrous cyclicity (female adult rats and offspring), sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and balano-preputial separation), and t hyroid gland weight, hor mone and histopathology assessment. The test guideline and the 2, 4-D study al so included options that evaluated development of the immune and nervous systems.

The results of the 2, 4-D extended 1-generati on reproductive toxicity study support the current conclusions of the Agency outlined in the RED. In summary, several studies were cited in the NRDC petition and the associated documents . Most were cited correctly as reported by the investigators, but

the overall results have little or no impact on the endpoints selected for human health risk assessment by the Agency for 2,4-D. This is because none of the studies identified an effect of concern at a lower dose than previously identified by the Agency in the 2005 RED, and the majority of the cited studies were performed at dose levels that exceed the renal clearance mechanism for 2, 4-D.

With regard to the exposure and epidemiological information presented by NRDC and in the related comments, conclusions are similar in that most studies were cited corr ectly as reported by the investigators but, overall, the results do not impact the risk conclusions that have been developed by the Agency for 2,4-D. This is because of several criti cal considerations. For ex ample, 2, 4-D use patterns have changed in agriculture since the timeframes wh ere the cited data were generated. Additionally, possible confounding aspects such as potential dioxin contamination are no longer a factor in the modern manufacturing processes for 2, 4-D. In many studies there are significant uncertainties, which were identified including preliminary conclusion s that were cited by NRDC not accounting for later research by the same inve stigators. Also, in ma ny cases the exposure relate d aspects on the analyses were inadequately addressed.

No studies were cited related to the issue of pet exposure except in the Beyond Pesticides comments.

The results of these thre e studies were cited corr ectly in the Beyond Pesticid es comments (i.e., 2, 4-D can be shown to induce lymphoma) but the key uncer tainties in the results were not addressed. In particular, a discussion of the re latively weak association between exposure and lymphoma onset was not included and the impact of changing use practices as well as differences in formulations was not

addressed.

### Page 74 of 186 In summary, the Agency did not conclude that the information provided by NRDC or in the associated

comments warrants alteration of the c onclusions described in the RED for 2,4-D. It should be noted that the Agency is re-evaluating pesticide risks on a cyc lical basis under its regist ration review process. Concurrent to this effort, the Agen cy is also involved in many efforts to refine its risk assessment policies including the development of updated expos ure assessment inputs and methods; establishing better methods for the considering ep idemiological research in the regul atory process; and consideration of the issues identified in recent National Academy of Science reviews related to risk assessment (e.g., Toxicity Testing in the 21 st 

### Century); and more active participati on with several epid emiological cohorts focused on agriculture and the use of pesticides (e.g., Agricultural H ealth Study). More current use information and knowledge of current cultural practices will also be av ailable for the registration review process. Given this construct, the Agency will further evaluate research related to 2, 4-D under registration review and will modify the conc lusions from the RED as appropriate.Page75of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,20091Alexander, B.H., Mandel, J.S.,Baker, B.A., Burns,C.J., Bartels,M.J., Acquavella,J.F., Gustin, C.Biomonitoring of 2,4-dichlorophenoxyacetic acid exposureand dose in farm families.2007 X PB2Arbuckle, T.E., Burnett, R.,Cole, D., Teschke, K.,Dosemeci, M., Bancej, C.,Zhang, J.Predictors of herbicide exposure in farmapplicators.2002 X PL3Arias E.Cytogenetic Effects of Short- and Longterm Exposure of Chick Embryos to thePhenoxyherbicide 2,4-D.2007 X MUT4Arias E.Sister chromatid exchange induction bythe hervicide 2, 4-dichlorophenoxyacetic acid in chickembryos.2003 X MUT5Blair, R.M., Fang, H., Branham,W.S., Hass,, B.S., Dial, S.L.,Moland, C.L., Tong, W., Shi, L.,Perkins, R., Sheehan, D.M.The estrogen receptor relative bindingaffinities of 188 natural andxenochemicals: Structural diversity ofligands.2000 X EN6Bon, E., Barbe, U., NunezRodriguez, J., Cuisset, B.,Pellissero, C, Sumpter, J.P.,LeMenn, F.Plasma vitellogenin levels during theannua reprroductive cycle of the femalerainbow trout (Onchorhynchus mykiss):Establishment and validation of anELISA.1997 X ECO7Bortolozzi A et al.Asymmetrical Development of theMonoamine Systems in 2,4-dicholorophenoxyacetic Acid TreatedRats2003 X MET8Bortolozzi A, Evangelista deDuffard AM, Dajas F, DuffardR, Silveira R.Intercerebral administration of 2, 4-dichlorophenoxyacetic acid inducesbehavioral and neurochemicalalterations in the rat brain.2001 X X IP/SUBQ9Bortolozzi AA et al.Effects of 2,4-dichlorophenoxyaceticacid exposure on dopamine D2-likereceptors in rat brain.2004 X IP/SUBQPage76of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200910Bortolozzi AA et al.Behavioral Alterations Induced in Ratsby a Pre- and Postnatal Exposure to 2,4-Dichlorophenoxyacetic Acid1999 X IP/SUBQ11Brand and JendrzejewskiThe Effect of Chronic Ethanol Ingestionon skin Penetration of 2,4-D.2008 X DA12Brand RM, Charron AR, duttonL, Gavlic TL, et al.Effects of chronic alcohol consumptionon dermal penetration of pesticides inrats.2004 X DA13Brand RM, McMahon L, et al.Transdermal absorption of the herbicide2,4-dichlorophenoxyacetic acid isenhanced by both ethanol consuptionand sunscreen application.2007 X DA14Brand RM, Spalding M, MuellerC.Sunscreens can increase dermalpenetration fof 2, 4-dichlorophenoxyacetic acid2002 X DA15Brusco A et al.2,4-Dichlorophenoxyacetic AcidThrough Lactation Induces Astrogliosisin Rat Brain.1997 X IP/SUBQ16Brusick DL. et al.Genetic Toxicology. In: Principles andmethods of Toxicology, ed. AW HaX.2008 X NR17Bukowska B.Effects of 2,4-D and its metabolite 2,4-dichlorophenol on antioxidant enzymesand level of glutathione in humanerythrocytes.2003 X MET18CalamariD, and Vighi M.A proposal to define quality objectivesfor aquatic life for mixtures of chemicalsubstances.1992 X ECO19Charles JM, Cunny HC, WilsonRD, Bus JSComparative subchronic studies on 2,4-dichlorophenoxyacetic acid, amine, andester in rats.1996 X X X X NCPage77of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200920Charles JM, Cunny HC, WilsonRD, Bus JS, Ivett Jl, Murli H,and Gollapudi B.In Vivo Micronucleus Assays on 2,4-Dichlorophenoxyacetic Acid and itsDerivatives.1999 X MUT21Charles JM, Cunny HC, WilsonRD, Bus JS, Lawlor TE, CifoneMA, Fellows M, and GollapudiB.Ames Assays and Unscheduled DNASynthesis Assays on 2,4-Dichlorophenoxyacetic Acid and itsDerivatives1999 X MUT22Charles JM, Hanley TR Jr,Wilson RD, van Ravenzwaay B.,Bus JS.Developmental toxicity studies in ratsand rabbits in 2,4-dichlorophenoxyacetic acid and itsforms.2001 X NR23Cox, C. 2,4-D Herbicide Factsheet 2005 X NR24Duffard R, Bortolozzi A, FerriA, Garcia G, Evangelista deDuffard AM.Developmental neurotoxicity of theherbicide 2,4-dichlorophenoxyaceticacid1995 X X NEU25Duffard R, Garcia G, rosso S,Bortolozzi A, Madariaga M, diPaolo O, Evangelista de DuffardAM.Central nervous system myelin deficit inrats exposed to 2,4-dichlorophenoxyacetic acid throughoutlactation.1996 X X X NEU26Evangelista de Duffard AM,Bortolozzi A, Duffard RO.Altered behavioral responses in 2, 4-dichlorophenoxyacetic acid treated andamphetamine challenged rats.1995 X X NEU27Feldmann R.G., Mailbach, H.I..Percutaneous penetration of somepesticides and herbicides in man.1974 X DA28Ferri A, Duffard R, Evangelistade Duffard Am.Selective oxidative stress in brain areasof neonate rats exposed to 2,4-dichlorophenoxyacetic acid throughtmother's milk.2007 X X NEU29Figgs LW, Holland NT,Rothmann N, Zahm SH, et al.Increased lymphocyte replicative indexfollowing 2,4-dichlorophenoxyaceticacid herbicide exposure.2000 X X EPIPage78of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200930Filkowski J, Besplug J, Burke P,Kovalchuk I, and Kovalchuk O.Genotoxicity of 2,4-D and dicambarevealed by transgenic Arabidopsisthailiana plants harboring recombinationand point mutation markers.2003 X X ECO31Garabrant D.H., Phibert M.A.Review of 2,4-dichlorophenoxyaceticacid (2,4-D) Epidemiology andToxicology.2002 X X EPI32Garcia G, Taliaferro P, et al.Study of tyrosine hydroxylaseimmunoreative neurons in neonate ratslactationally exposed to 2,4-dichlorophenoxyacetic acid2004 X X NEU33Garcia GB, Konjuh C, DuffardRO, Evangelista de Duffard AM.Dopamine-beta-hydroxylaseimmunohistochemical study in the locuscoeruleus of neonate rats exposed to2,4-dichlorophenoxyacetic acid throughmother's milk.2006 X X NEU34Garry VF, Hawkins ME,Erickson LL, Long-Simpson LK,Holland SE, Burroughs BL.Birth defects, season of conception, andsex of children born to pesticideapplicators living in the Red RiverValley of Minnesota, USA.2002 XEPIPB35Garry VF, Schreinemachers D,Harkins ME, et al.Pesticide appliers, biocides, and birthdefects in rural Minnesota.1996 X X XEPIPB36Garry VF, Tarone RE, KirschIR, Abdallah JM, Lombardi DP,Long LK, Burroughs BL, BarrDB, and Kesner JS.Biomarker correlations of urinary 2,4-Dlevels in foresters: genomic instabilityand endocrine disruption.2001 XEPIPB37Giasson BI, Lee VM.A new link between pesticides andParkinson's disease.2000 XMUTNEUPage79of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200938Glickman LT, Raghavan M,Knapp DW, Bonney PL, andDawson MH.Hervicide exposure and the risk oftransitional cell carcinoma of theurinary bladder in Scottish Terriers.2004 X PI39Gollapudi BB, Charles JM,Linscombe VA, Day SJ, and BusJS.Evaluation of the genotoxicity of 2,4-dichlorophenoxyacetic acid and itsderivatives in mammalian cell cultures.1999 X MUT40Gonzalez M, Soloneski S,Reigosa MA, Larramendy ML.Genotoxicity of the herbicide 2,4-dichlorophenoxyacetic and acommercial formulation, 2, 4-dichlorophenoxyacetic aciddimethylamine salt.2005 X MUT41Grover Cessna AJ, Muir NI,Reidel S, Franklin C, Yoshida K.Factors affecting the exposure ofground-rig applicators to 2,4-dimethylamine salt.1986 X PL42Haddow JE, Palomaki GE, AllanWC, Williams JR, Knight GJ,Gagnon J, O'Heir CD, MitchellML, Hermos RJ, Waisbren SE,Faix JD, Klein RZ.Maternal thyroid deficiency duringpregnancy and subsequentneuropsychological development of thechild.1999 X NEU43Hardell, L. and Ericsson, M.A case-control study of non-Hodgkinlymphoma and exposure to pesticides.1999 XEPIPB44Harris SA, Solomon KR.Percutaneous penetration of 2, 4-dichlorophenoxyacetic acid and 2, 4-ddimethylamine salt in humanvolunteers.1992 X DA (H)45Hayes, HM, Tarone RE, andCantor KP.On the Association between CanineMalignant Lymphoma and Opportunityfor Exposure to 2,4-Dichlorophenoxyacetic Acid.1995 X PI46Hayes, HM, Tarone RE, CantorKP, Jessen CR, MrCurnin DM,and Richardson RC.Case- Control Study of CanineMalignant Lymphoma: PositiveAssociatiion With Dog Owner's Use of2,4-Dichlorophenoxyacetic Acid1991 XPIPLPage80of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,2009Herbicides.47Hines CJ, Deddens JA, TuckerSP, Hornung RW.Distributions and Determinants of PreEmergent Herbicide Exposures amongCustom Applicators.2001 X PL48Holland NT, Duramad P,Rothman N, Figgs LW, et al.Micronucleus frequency andproliferation in human lymphocytesafter exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro andin vivo.2002 X ???Howard, Phillip H.Handbook of Environmental Fate andExposure Data for Organic Chemicals.1991 X NR49Hurst MR, Sheahan DA.The potential for oestrogenic effects ofpesticides in headwater streams in theUK.2003 X ECO50Hutchinson TH. 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Dakeyama, K.Saito, M. Imagawa, S. Takatori,Y. Kitagawa, S. Hori, h. Utsumi.Estrogenic activites of 517 chemicals byyeast two-hybrid assay.2000 X EN80Nishioka, MG Burkholder HM,Brinkman MC, Gordon SM.Measuring lawn transport of lawnapplied herbicide acids from turf tohome: Correlation of dislodgeable 2, 4-D turf residues with carpet dust andcarpet surface residues.1996 X X X PB81NRDCNatural Resources Defense Council'spetition to revoke all tolerances andcancel all registrations for the pesticide2, 4-D.2008 X NRPage84of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200982Oakes DJ, and Pollak JK.The in vitro evaluation of the toxicitiesof three related herbicide formulationscontaining ester derivative of 2,4,5-Tand 2,4-D using sub-mitochondrrialparticles.2000 X NC83Oakes DJ, Webster WS, BrownWoodman PDC, Ritchie HE.A study of the potential for a herbicideformulation containing 2,4-D andpicloram to cause male-mediateddevelpmental toxicity in rats.2002 X EN84Pelletier O, Ritter L, Caron J,Somers D.Disposition of 2,4-dichlorophenoxyacetic aciddimethylamine salt by Fischer 344 ratsdosed orally and dermally.1989 X MET85Petit F, Le Goff P, Cravedi J-P,Volataire Y, Pakdel F.Two complementary bioassays forscreening the estrogenic potency ofxenobiotics; recombinant yeast for troutestrogen receptor andtrout hepatocytecultures.1997 X ECO86Pont AR, Charron AR, BrandRM.Active ingredients in sunscreens act astopical penetration enhancers for theherbicide ,4-dichlorophenoxyaceticacid.2004 X DA87Rawlings NC, Cook SJ,Waldbilling D.Effects of the pesticides carbofuran,chlorpyrifos, dimethoate, lindane,triallate, trifluralin, 2,4-D andpentachlorophenol on the methbolicendocrine and reproductive endocrinesystem in ewes.1998 X X ECO88Reynolds PM, Reif JS, RamsdellHS, and Tessari JD.Canine exposure to herbicide-treatedlawns and urinary excretion of 2,4-dichlorophenoxycetic acid.1994 X PIPage85of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200989Riviere JE, Baynes RE, BrooksJD, Yeatts JL, Monteiro-RiviereNA.Percutaneous absorption of topical N,N-diethyl-m-toluamide (DEET): effectsof exposure variables andcoadministered toxicants.2003 X DA90Rodwell DE, Brown WR.A dietary two-Generation ReproductionStudy in Fischer 344 Rats with 2,4-dichlorphenoxyacetic acid.1986 X NR91Ross RH, Driver JH, Harris SA,Maibach HI.Dermal absorption of 2,4-D: a review ofspecies differences.2005 X DA92Rosso B et al.2,4-Dichlorophenoxyacetic AcidDisrupts the Cytoskeleton andDisorganizes the Golgi Apparatus ofCultured Neurons.2000 X NEU93Rosso SB et al.Effects of 2,4-dichlorophenoxyaceticacid on central nervous system ofdevelopmental rats.1997 X NEU94Rosso SB, Garcia GB,Madariaga MJ, Evangelista deDuffard AM, Duffard RO.2, 4-Dichlorophenoxyacetic acid indeveloping rats alters behaviour,myelination and the regions braingangliosides pattern2000 X X X NEU95Sachs PD.Handbook of Successful EcologicalLawn Care.1996 X NR96Saghir SA, Marty MS, Clark AJ,Zablotny CL, Bus JS, Peral AW,Yano BL, neal BH.A dietary dose range-finding andtoxicokinetic (tk) study of 2, 4-dichlorophenoxyacetic acid (2,4-D) inadult crl:cd(sd) rats and their offspring:I. Toxicokinetics.2009 X EN97Saghir SA, Mendrala AL,Bartels MJ, Day SJ, Hansen SC,Sushynski JM, and Bus JS.Strategies to assess systemic exposureof chemicals in subchronic/chronic dietand drinking water studies.2006 X MET98Schubert S. et al.Sensitivity of Brown Trout Reproductioto Long-Term Estrogenic Exposure.2008 X ECOPage86of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,200999Soloneski S, Gonzalez NV,Reigosa MA, Larramendy ML.Herbicide 2,4-dichlorophenoxyaceticacid (2, 4-D induced cytogeneticdamage in human lymphocytes in vitroin presence of erythrocytes2007 X MUT100Soto AM, Sonnenschein C,Chung KL, Fernandez MF, OleaN, Serrano FO.The E-SCREEN assay as a tool toidentify estrogens: An update onestrogenic environmental pollutants.1995 X EN101Spiteri ID, Guillette LJ Jr, CrainDA.The functional and structuralobservations of the neonatalreproductive system of alligatorsexposed in ovo to atrazine, 2,4-D, orestradiol.1999 X ECO102Sturtz N, Bongiovanni B, et al.Detection of 2, 4-dichlorophenoxyaceticacid in rat milk of dams exposed duringlactation and milk analysis of theirmajor components.2006 X X NEU103Sturtz N, Evangelista de DuffardAM, Duffard R.Detection of 2,4-dichlorophenoxyaceticacid (2,4-D) residues in neonates breastfed by 2,4-D exposed dams.2000 X X NEU104 Sturz N, Deis RP, Jahn GA,Duffard R, and Evangelista deDuffard AM.Effect of 2,4-Dichlorophenoxyaceticacid on rat maternal behavior.2008 X X X NEU105The Herbicide Resistance ActionCommittee (HRAC)Classification of Herbicides Accordingto Mode of Action.2005 X NR106Ton C, Lin Y, Willett C.Zebrafish as a model for developmentalneurotoxicity testing.2006 X NEU107Tripathy NK, Routray PK, SahuGP, Kumar AA,Genotoxicity of 2,4-dichlorophenoxyacetic acid tested insomatic and germ-line cells ofDrosophila1993 X MUTTuschl h, Schwab C.Cytotoxic effects of the herbicide 2, 4-dichlorophenoxyacetic acid in HepG2cells.2003 X MUTPage87of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,2009US Federal RegisterOrder Denying Objections to Issuanceof Tolerances.2005 X NRUS Federal RegisterOrder Denying Petition To RevokeSubstances2006 XNRUS Federal Register Proposed Tolerance Actions. 2007a XNRUS Federal Register 2,4-D Tolerance Actions 2007b XNRUS Federal Register 2,4-D Tolerance Actions 2007c XNRUS Federal RegisterOrder Denying NRDC's Petition toRevoke All DDVP Tolerances.2007d XNRUSEPA2, 4-D. HED's Human Health RiskAssessment for the ReregistrationEligibility Decision (RED) Revised toReflect Error-only comments fromResgistrants.2004 XNRUSEPA2, 4-D. HED's Human Health RiskAssessment for the ReregistrationEligibility Decision (RED) Revised toReflect Public Comments2005 XNRUSEPA 2,4-D RED Facts. 2005 XNRUSEPAAcssessing Pesticide Cumulative RiskIn: Pesticides: Health and Safety.2008 XNRUSEPAFact Sheet: Protecting Children fromPesticides2002 XNRUSEPAGeneric and Product Specific Data Call-in Notice2005 XNRUSEPAReregistration Eligibility Decision for2,4-D2005 X X XNRUSEPAReregistration Eligibility Determinationfor 2, 4-DXNRUSEPA Response to comments 2005 XNRUSEPAThe National Pesticide Retrieval System(NPIRS)1998-2009XNRPage88of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,2009USEPA MemorandumOffice of Prevention, Pesiticides, andToxic Substances, Updated HumanHealth Risk Assessment.2002 XNRUSEPA Memorandum 2,4-D: Response to Public comments 2004 X XNRUSEPA MemorandumHED's Human Health Risk Assessmentfor the Reregistration EligibilityDecision (RED).2004 XNRUSEPA Memorandum2,4-D: Response to Phase 5 Publiccomments.2005 XNRUSEPA MemorandumReview of Recent 2,4-D CancerEpidemiology Studies2004 XNRvan Ravenzwaay B, hardwickTD, Needham D, Pethen S, andLappin GJ.Comparative metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) inrat and dog.2003 X METVenkov P, Topashka-AnchevaM, Georgieva M, Alexieva V,Karanov E.Genotoxicity of 2,4-dichlorophenoxyacetic acid tested insomatic and germ-line cells ofDrosophila.1993 X MUTVighi m, Altenburger R,Arrhenius A, Backhaus T,Bodeker W, Blanck H,Consolaro F, Faust M, Finizio A,Froehner K, Gramatica P,Grimme LH, Gronvall F, HamerV, Scholze M, and Walter H.Water quality objectives for mixtures oftoxic chemicals: problems andperspectives.2003 X ECOVonier PM, CD Andrew, JAMclachlan, LJ Guillete Jr; SFArnold.Interaction of environmental chemicalswith the estrogen and progesteronereceptors from the oviduct of theAmerican alligator.1996 X ECOPage89of186Appendix A: Data SummaryStudy IdentificationSummary of Citation UseBinStudy Authors*Study NameStudyYearOriginalNRDCPetition,Nov 6,2008Supplementto NRDCPetition, Feb23, 2009Task ForceCommentson Petition,Feb 23, 2009BeyondPesticidesComment,Feb 23, 2009NewtonComment,Feb 15,2009NY StateComment,Feb 23,2009NCASIComment,Feb 20,2009Wester RC, J Melendres, LSedic, H Maibach, and JERiviere.Percutaneous absorption of salicylicAcid, Theophylline, 2,4-DDimethylamine, Diethyl Hexyl PhthalicAcid, and p-Aminobenzoic Acid in theisolated perfused procine skin flapcompared to man in vivo.1998 X DAWSSA. Herbicides: Chemical Structures 2007 X NRXie L, Thrippleton k, Irwin MA,et al.Evaluation of estrogenic activities ofaquatic herbicides and surfactants usingan rainbow trout vitellogenin assay.2005 X X X X ECOYeary RA.Urinary excretion of 2,4-D incommersial lawn specialists.1986 X PLZeljezic D, Garaj-Vrhovac V.Chromosomal abberrations, micronucleiand nuclear buds induced in humanlymphocytes by 2, 4-dichlorophenoxyacetic acid pesticideformulation.2004 X X MUT*studies numbered alphabetically

### Page 90 of 186 

# Appendix B:

Hazard Data Summaries*

*(Study #) refers to the number liste d in Appendix A Data Summary table

### Page 91 of 186 BIN: NEUROTOXICITY

*(Study 7) Citation : Bortolozzi, A.; Duffard, R; and Evangelista de Duffard A (2003). Asymmetrical Development of the Monoamine Systems in 2, 4-Dichlorophenoxyacetic acid (2,4-D) Treated Rats. NeuroToxicology 24: 149-

157. [Human Studies Considerations Apply: No]
Principal Endpoint: [Bin: Neurotoxicity] Methods Summary: Pregnant Wistar rats administered 2,4-D via diet (0, 70 mg 2,4-D/kg/day) from gestation day (GD) 16 to post partum day (PND) 23; litters culled to 8 /litter on PND 0; after weaning, 2,4-D pups were administered untreated diet (T1) or maintained on 2,4-D diet up to PND 90 (T2). Neonates sacrificed by decapitation; brain areas removed were prefrontal cortex (PFc), striatum (St), nucleus accumbens (NAc), substantia nigra (SN), ventral tegmental area (VTA), midbrain (Mb, whic h did not involve separate analysis of SN and VTA) and cerebellum (Cer), which was separated from the pons medulla. After the cereb ellum had been removed from the brainstem, the midbrain was separated by a cut at the level of the inferior coll iculus; levels of endogenous monoamines and their metabolites were measured [noradrenaline (NA), dopamine (DA), 3,4- dihyrdoxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyi ndolacetic acid (5-HIAA). Results Summary: 5-HT was increased in several brain areas (both sexes) in both groups (PFc, striatum, midbrain) and in 2,4-D post weaning group (SN, cerebellum). Alterations ( ↑↓ ) in NA, DA, DOPAC, HVA were observed in various brain areas and in both sexes (with a nd/or without 2,4-D post weaning treatment).

Table 1. Effects of pre-/postnatal exposure on NA, DA, DOPAC, and HVA of 90-day-od rat brain areas

Group NA DA DOPAC HVA

male female male female Male Female male Female

PFc

T1

T2 -

- -

- ↓ 45% ↓ 54% ↓ 32%* ↓ 45%** ↓ 42%** ↓ 58%** ↓ 55%** ↓ 77%** - ↓ 43%** - ↓ 46%**

Striatum

T1

T2 -

- -

- -

- - ↑ 30%* -

- - ↑ 63%** -

- -

- Nac

T1

T2 -

- -

- -

- -

- - ↑ 30%* - ↑ 30%* - ↑ 71%** - ↑ 64%**

Mid

T1

T2 -

- -

- - ↓ 26%* ↓ 41%* ↓ 62%** ↓ 38%* ↓ 43%* ↓ 41%* ↓ 60%** -

- -

- SN

T1

T2 - ↑ 43%* - ↑ 50%* - ↑ 57%** - ↑ 30%* -

- -

- -

- -

- VTA

T1

T2 -

- -

- - ↓ 37%* - ↓ 58%** - ↓ 48%* ↓ 30%* ↓ 72%** - ↓ 59%** - ↓ 59%**

Cerebellum

T1

T2 - ↓ 29%* - ↓ 1%** - ↑ 0%** ↓ 46%** ↓ 69%** - ↓ 40%* - ↓ 47%** -

- -

- % ↑↓ ; * p<0.05; ** p<0.01

Table 2. Effect of pre- and postnatal exposure on 5-HT and 5-HIAA levels of 90-day-old rat brain

areas

5-HT -HIAA

male Female Male Female

PFc

T1

T2 ↑ 23%* ↑ 23%* ↑ 49%* ↑ 37%* - -

- Striatum

T1

T2 ↑ 24%* ↑ 35%* ↑ 34%* ↑ 51%* - ↑ 43%** - ↑ 37%**

Nac

T1

T2 -

- -

- -

- -

- Midbrain

### Page 92 of 186 T1

T2 ↑ 30%* ↑ 39%* - ↑ 29%* -

- - ↑ 29%*

SN

T1

T2 - ↑ 36%* - ↑ 69%** -

- -

- VTA

T1

T2 -

- -

- -

- -

- Cerebellum

T1

T2 - ↑ 36%* - ↑ 36%* -

- -

% ↑↓ ; * p<0.05; ** p<0.01

CONCLUSIONS: according to authors, results support view that during the critical period in which monoamines can influence cell development, neonatal exposure to 2,4-D produc es alterations on these neurotransmitter systems; equated to previous findings expressed in spontaneous circling act ivity towards the right side by adult offspring of dams exposed to 2,4-D, indicating a correla tion between monoamines brain level alte rations and this behavior. Subchronic 2,4-D exposures modify neurotransmitter levels in cerebral br ain areas that are involved in neurodegenerative diseases like Parkinson and schizophrenia.

COMMENT/ HED : The cited study is one of several research studi es from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Study does not provide any dose-response information, since only dose was tested (70 mg/kg/day) and it exceeds rat renal clear ance levels (overwhelms the body’s ability to excrete 2,4- D). It does not provide information regarding potential effect s of 2, 4-D on neurotransmitters at dose levels below the saturation level for renal clearance. An extended 1-genera tion reproductive toxicity study on 2,4-D was submitted to Agency in February of 2010, in response to the data requirements identified in the 2005 RED, included a developmental neurotoxicity a ssessment. The young rat was exposed during gestation, lactation via the milk, and directly via the diet following weaning. Offspr ing were subjected to a developmen tal neurotoxicity (DNT) assessment, which included functional observational battery (FOB), landing f oot splay, grip strength, mo tor activity, habituation, and acoustic startle response (ASR). The young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation and brain morphometry, and a special stain was used to ev aluate brain myelination.

Three dose levels were tested. At the hi ghest dose (82 mg/kg bw/day in males/59 mg/kg bw/day in females), there were no treatment-related adverse effects on any of the numer ous parameters evaluated to assess the developmental neurotoxicity potential of 2, 4-D. The parental rats were assessed for clinical signs of toxicity and maternal behavior, among other parameters. Maternal behavi or towards the offspring was comparab le among the groups, and there were no clinical signs, such as circling to the right, observed in the dams or offspring. Alt hough monoamine levels were not monitored in this study, neonatal exposure was similar to th at in the cited study, and th e three dose levels (intended dose levels of 5, 15, 30/40 mg/kg/day) provide dose-response information and were adequate (approaching saturation of renal clearance). The extended 1-generation reproduc tive toxicity study of 2, 4-D addresses the neurotoxicity/developmental neurotoxi city concerns raised by NRDC. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that w ould cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-genera tion reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY: NYS DOH – cited as additional studies that should be considered in any new assessment along with those identified in the NRDC petition.

(Study 10) Citation : Bortolozzi, A.; Duffard, R; and Evangelista de Duffard A (1999). Behavioral Alterations Induced in Rats by a Pre- and Postnatal Exposure to 2, 4-Dichlorophenoxyacetic Acid. NeuroToxicology and Teratology 21 (4): 451-465. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Pregnant Wistar rats administered 2,4-D via diet (0, 70 mg 2,4-D/kg/day) from gestation day (GD) 16 to post partum day (PND) 23; litters culled to 8/litter on PND 0; after weaning, 2,4- D pups were administered untreated diet (T1) or maintained on 2,4-D diet up to PND 90 (T2). Parameters procedures: physical/sexual development (fur growth , eye opening, testes descent, vaginal opening); surface righting (on PND 1, 3, 5, 6, 8, 10; considered a reflection of subcortical maturation); negative geotaxis (tests labyrinthine and cerebellar integration, from PND 2-16); hindlimb support (tests neuromuscular and locomotor development. PND 9-23); open field tests (measures activity and habituation, PND 12, 15, 19, 21; PND 30, 45, 60, 75, 90; # crosses, rearings, vertical head movements, grooming episodes); 5-HT syndrome behaviors [forepaw move ments (treading, excessive placement or tapping), hunching (back arching), sprawling of limbs, backing, hindlimb abduction, mobility (ability for locomotion

### Page 93 of 186 despite gait abnormalities, including paddling), abnormal head movements (vertical or lateral side-to-side repetitive movements), wet-dog shakes (including head shakes), lying on side, limb extension dystonic, and flat body posture]; circling behavior (PND 60, 75, 90); measurement of catalepsy (PND 30, 60, 90). Results Summary: ontogeny of righting reflex delayed (days 1, 3, 5, 6 only); negative geotaxis (longer mean latency of 2,4-D pups from PND 1-14); hindlimb support times significantly decreased (days 11-15 only); open field (exploratory activity) behavior: # of crosses, vertical head movements, rearings, groomings increased in 2,4-D pups compared to controls (PND 12-21); during PND 60-90, # crosses and rearings were decreased compared to control (both sexes), and males showed increased # grooming PND 60-90, compared to control. 5-HT syndrome behaviors: 2,4-D induced distinctive motor syndrome: increased forepaw tapping, stiff or elevated tail, mobility, and sprawling of limbs (PND 1-7; not observed in adults PND 60 and 90 except stiff tail);

Table 1. 5-HT Syndrome Behaviors

Male Female Flat body posture

PND 30

C

T1

T2 0

0

0 0

0

0

PND 60

C

T1

T2 0

0

1.10±0.25* 0

0

0.82±0.20*

PND 90

C

T1

T2 0.25±0.08

0.22±0.13

2.60±0.28* 0.15±0.09

0.12±0.09

1.58±0.58* Hunching

PND 30

C

T1

T2 0.50±0.12

0.46±0.10

1.12±0.33* 0.58±0.18

0.54±0.11

0.98±0.25*

PND 60

C

T1

T2 0

0

0.91±0.33* 0

0.75±0.19

1.57±0.22*

PND 90

C

T1

T2 0

0

1.15±0.54* 0

0

2.19±0.39*

Stiff tail

PND 30

C

T1

T2 0.32±0.10

0.39±0.08

2.32±0.65* 0.29±0.05

0.41±0.19

1.98±0.33*

PND 60

C

T1

T2 0.29±0.02

0.55±0.03

1.95±0.23* 0.33±0.01

0.45±0.12

2.06±0.42*

PND 90

C

T1

T2 0.30±0.09

0.41±0.15

2.10±0.25 0.28±0.05

0.55±0.10

2.22±0.39* Forepaw treading

PND 30

### Page 94 of 186 C

T1

T2 0.31±0.09

0.35±0.05

0.52±0.07 0.28±0.10

0.32±0.01

0.58±0.07

PND 60

C

T1

T2 0

0

0 0

0

0

PND 90

C

T1

T2 0

0

0 0

0

0

Head movement

PND 30

C

T1

T2 1.25±0.38

1.38±0.58

2.00±0.32* 0.95±0.15

1.52±0.60*

1.89±0.25*

PND 60

C

T1

T2 0

0

0 0

0

0

PND 90

C

T1

T2 0

0

0 0

0

0 * p<0.05; maximum score of 4; mean±SEM; data from Table 2 (page 459)

CONCLUSIONS: 2,4-D exposure at a dose producing no overt signs of toxicity may cause long-term alterations in functional state of rat CNS, detected through a neurobehavioral test battery; exposure during pre- and postnatal development induces behavioral abno rmalities that seem to disappear in adulthood after 2,4-D cessation ; mechanism of 2,4-D neurotoxicity not yet known

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Study does not provide any dose-response information. The only dose used ( 70 mg/kg/day) exceeds rat renal clear ance levels and does not provide information regarding potential effects of 2, 4-D on neurotransmitters at dose levels below the saturation level for renal clearance. An extended 1-generation reproductive toxicity study on 2,4-D was submitted to Agency in February of 2010, in response to the data requirements identified in the 2005 RED, which included a developmental neurotoxicity assessment. Offspring were exposed to 2, 4-D during gestation,

lactation via the milk, and directly via the diet through PND 22, 70, 139, and the developmental neurotoxicity component of the study included neurobehavioral tests at dose levels that do not saturate metabolic processes. Parameters evaluated in the study include an assessment of developmental neurotoxicity (including FOB observations, landing foot splay, grip strength, motor activity, acoustic startle response, habituation, and a neuropathological asse ssment). Also evaluated were potential effects in offspring on survival, growth, development of immune and reproductive systems; endocrine and selected systemic toxicity parameters, estrous cycle evaluation, evaluation of sperm parameters, thyroid hormone assessment; anogenital distance, nipple retention and puberty onset (vaginal opening and preputial separation. There were no treatment-related adverse e ffects on any of the numerous parameters evaluated to assess neurotoxicity potential, behavior, physical development and sexual development following 2, 4-D exposure. Most of the same parameters evaluated in the cited study were evaluated in the extended 1- generation reproductive toxicity study at dose levels (intended dose levels of 5, 15, 30/40 mg/kg/day) considered appropriate (app roaching saturation of renal clearance). Th e cited study does not identify a more sensitive endpoint for human risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NYS DOH

– cited as additional studies that should be considered in any new assessment along

### Page 95 of 186 with those identified in the NRDC petition.

(Study 9) Citation : Bortolozzi, AA; Evangelista de Duffard A.; Duffard, R; and Antonelli, MC (2004). Effects of 2, 4-Dichlorophenoxyacetic acid Exposure on Dopamine D 2 –Like Receptors in Rat Brain. Neurotoxicology and
Teratology 26: 599-605. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Pregnant Wistar rats administered 2,4-D via diet (0, 70 mg 2,4-D/kg/day) from gestation day (GD) 16 to post partum (PND 23); litte rs culled to 8/litter on PND 0; after weaning, 2,4-D pups were administered untreated di et or maintained on 2,4-D diet up to PND 90. Neonates sacrificed at PND 6, 15, 30, 45, or 90 for membrane receptor binding assays employing [ 3 H] nemonapride. Results Summary: subchronic oral exposure increased DA D 2 -type receptor 40% in Cpu; DA D 2 -type receptor levels increased in PFc (15 and 30 days) and Cer (30 and 90 days); fema les more affected than males. When 2,4-D exposure stopped (T1), DA D 2 –type receptor density was apparently recovered and stabilized to control level; suggest reversible vulnerability of D 2 –type receptors to 2,4-D exposure.

CONCLUSION: Regional increases in D 2 –type receptor density may explain certain behaviors, such as catalepsy and right-t urning preference in 2,4-D e xposed rats reported earlier.

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Study does not provide any dose-response information. The only dose used ( 70 mg/kg/day) exceeds rat renal clear ance levels and does not provide information regarding ontogeny of dopaminergic D 2 –type receptors in prefrontal cortex (PFc), striatum (CPc), hippocampus (H), and cerebellum (Cer) and potential effects of 2, 4-D on neurotransmitters at dose

levels below the saturation level for renal clearance. An extended 1-generation reproductive toxicity study on 2,4-D (MRID 47972101) was submitted to Agency in February of 2010, in response to the data requirements identified in the 2005 RED, which included a developmental neurotoxicity assessment. Offspring were exposed to 2, 4-D during gestation, lactation via milk, and directly via the diet through PND 22, 70, 139, and the developmental neurotoxicity component of the study included neurobehavioral tests at dose levels that do not saturate metabolic pr ocesses. Parameters evaluated in the study include an assessment of developmental neurotoxicity (including FOB observations, landing foot splay, grip strength, motor activity, acoustic startle response, habitu ation, and a neuropathological assessment). The neuropathology/histopathological assessment included cross-sections of the olfactory bulb, cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, pons, medulla oblongata, and cerebellum; sections were prepared from the trigeminal ganglion and nerve, pituitary gland, eyes with optic nerves, spinal cord (cervical and lumbar), olfactory epitheliu m, and skeletal muscles (gastrocnemius and anterior tibial); and sections containing representative sections of the cerebellum and corpus callosum were stained to asse ss myelin. Spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial (proximal and distal (muscular) and sural) were

examined also. There were no treatment-related adverse effects on any of the numerous parameters evaluated to assess neurotoxicity potential. The behaviors being addressed in the cited study were not replicated in the extended 1-generation reproductive toxicity study at dose levels (5, 15, 30/40 mg/kg/day) considered appropriate (approaching saturation of renal clearance). The cited stud y does not identify a more sensitive endpoint for human risk assessment than already identified by the Agency. Additionally, the results do not yield information that would cause th e Agency to modify its conclusions from the 2005 RED for 2, 4-D.

CITED BY NYS DOH – cited as additional studies that should be considered in any new assessment along with those identified in the NRDC petition.

(Study 8) Citation : Bortolozzi, A.; Evangelista de Duffard A.; Dajas, F.; Duffard, R; and Silveira, R. (2001). Intracerebral Administration of 2, 4-Dichlorophenoxyacetic acid Induces Behavioral and Neurochemical Alterations in the Rat Brain. NeuroToxicology 22: 221-232. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Intrastriatal injection : male Wistar rats received 100 μg of 2,4-D in 1 μL DMSO into one striatum; Accumbens (NAc) injection: 100 μg of 2,4-D in 0.5 μL DMSO into one NAc; Medial Forebrain Bundle (MFB) injection: 50 μg of 2,4-D in 0.5 μL DMSO into one MFB or 8 μg of 6-OHDA dissolved in 0.5 μL saline into one MFB; estimated 2, 4-D concentrations in each brain area (striatum, Nac,

### Page 96 of 186 and MFB) were 2-4 mM, 40-100 fold the concentration after systemic treatment (although concentration in each brain area seems high, 2,4-D penetr ation rate into the brain is low) Results Summary: 2,4-D injected into 1 striatum produced a marked depression in locomotor activity and elicited a moderate circling towards the ipsilateral side at 6 and 24 hours post dose; these changes were accompanied by a ↓ of 5-HT and an ↑ in homovanillic acid (HVA) levels; administration into the nucleus

accumbens induced similar behavioral and neurochemical patterns, although rats did not present notorious turning

CONCLUSION: neurotoxic effects of 2,4-D on basal ganglia by interacting with the monoaminergicsystem depended not only on the exact location of injection, but also on the dose and time period postinjection; toxicity produced by 2,4-D appears to be different in monoaminergic terminals, axonal fibers,

and cell bodies

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The route of exposure is not appropriate; human exposure would not be by direct injection into the brain; unknown relationship between the dose injected into various areas of the brain and that attained following oral/dermal/inhalation exposures; provides potential mode of action information (mechanistic assessment). An extended 1-generation reproductive toxicity study on 2,4-D was submitted to Agency in Febr uary of 2010, in response to the data requirements identified in the 2005 RED, which included a developmental neurotoxicity assessment. Offspring were exposed to 2, 4-D during gestation, lactation, and directly via the diet through PND 22, 70, 139, and the developmental neurotoxicity component of the study included neurobehavioral tests at dose levels that do not saturate metabolic processes. Parameters evaluated in the study include an assessment of developmental neurotoxicity (including FOB observations, landing foot splay, grip strength, motor activity, acoustic startle response, habituation, and a neuropathological asse ssment). Also evaluated were potential effects in offspring on survival, growth, development of immune and reproductive systems; endocrine and selected systemic toxicity parameters, estrous cycle evaluation, evaluation of sperm parameters, thyroid hormone assessment; anogenital distance, nipple retention and puberty onset. There were no treatment-related adverse effects on any of the numerous parameters evaluated to assess neurotoxicity potential, behavior, physical development, and sexual development following 2, 4-D exposure post-natally. Although monoamine levels were not monitored in this study, the route of exposure (oral) was appropriate for human health risk assessment and the dose levels (5, 15, 30/40 mg/kg/day) were adequate (approaching saturation of renal clearance). The circling behavior noted in th e cited study was not repli cated in the extended 1- generation reproductive toxicity study.

The cited study does not identify a more sensitive endpoint for human risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NRDC (35) – rodent studies have revealed a region-specific neurotoxic effect on basal ganglia of the brain, resulting in an array of effects on critical neurotransmitters and adverse effects on behavior. NRDC cited the study, among others, as evidence that postnatal exposures to 2, 4-D during the critical period of development of the infant brain raise serious scientific concerns.

CITED BY Task Force

– The Task Force states that this study cited by NRDC was not a developmental neurotoxicity study but a very high dose adult toxicity study where 2,4-D was directly administered into the rat’s brain, which is a route not relevant to human exposures. They also pointed out that the estimated concentrations of 2, 4-D in each brain area were 2-4 mM, which the author define d as 40- to100-fold the concentration in brain after systemic treatment.

(Study 15) Citation : Brusco, A.; Saavedra, JP; Garcia , G.; Tagliaferro, P.; Evangelista de Duffard A.; and Duffard, R (1997). 2, 4-Dichlorophenoxyacetic Acid Through L actation Induces Astrogliosis in Rat Brain. Molecular
and Chemical Neuropathology 30: 175-185. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Female Wistar rats (dams) administered 2,4-D i.p. (DMSO, 70 mg 2,4-D/kg/day) from PND 9-25; litters culled to 10/litter on PND 0; ne onates sacrificed at PND 25; brain and brainstem removed. Immunocytochemical staining was performed using an anti-GFAP and anti-S-100 protein as

### Page 97 of 186 primary antibodies. Results Summary: Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-day old pups exposed to 2,4-D through mother’s milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocam pus and cerebellum. Specific marker s were GFAP and S-100 protein. There was a significant increase in number, size, number of processes, and density of immunostaining in 2,4-D exposed pups.

► goal of study was to detect the effect of 2,4-D on astrocytes in different CNS regions of rats exposed to 2,4-D during lactation using 2 rat astroglial markers; paper indicates that one of the most widely documented cellular reactions following nervous system damage has been hypertrophy of astrocytes, often referred to as a reactive gliosis or astrogliosis.; it is ch aracterized by rapid synthesis of glial fibrillary acidic protein (GFAP) intermediate filament, which is a se nsitive and early biomarker of neurotoxicity; another rat astroglial marker, the S-100 protein, appeared co nfined to the cytosol of both normal and reactive

astrocytes

Mean Values of Area and F-Shape of Astrocytes Immunolabeled w/ Anti-GFAP or Anti-S-100 Protein Hippocampus Immunostaining for # astrocytes Area, μm 2 of each

astrocyte F-shape

GFAP

Control 2,4-D 14

31 39.9 ± 15.0

53.3 ± 21.5** 0.30 ± 0.14

0.43 ± 0.12**

S-100

Control 2,4-D 9

22 26.5 ± 8.4

29.8 ± 12.6 0.60 ± 0.12

0.68 ± 0.11* Mean Values of # of Astrocytes/Field (Ast/A), Maximum Diameter (MD) in Granular Layer and Distance Between Bergmn Fibers (DBf) in Molecular Layer - Cerebellum DBf, μm Ast/A MD

Control 2,4-D 15.12 ± 4.82 (n=25)

9.40 ± 4.09*** (n=20) 4 ± 1.41 (n=8)

12 ± 1.41** (n=21) 10.31 ± 2.94 (n=8)

13.47 ± 3.6 ♪ (n=21)

♪ p<0.025; * p<0.05; ** p0.001; *** p<0.0001;

CONCLUSION: exposure to 2,4-D during first days of life modifies the astroglial cytoarchitecture in parallel with previous neuronal changes observed

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The study does not provide any dose-response information (i.p. to dams, pups exposed via milk), and the dose to pups is unknown. The only dose tested (70 mg/kg/day) woul d be expected to exceed rat renal clear ance levels. The extended 1-generation reproductive toxicity study on 2,4-D, as described previously, in which offspring were exposed to 2, 4-D during gestation, lactation, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity (neurobehavioral tests) at dose levels up to those that saturate metabolic processes. The young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluations and brain morphometry, and a special stain was used to evaluate brain myelination. There were no treatment-related adverse effects on any of the numerous parameters evaluated to assess neurotoxicity and neuropathology potential following 2, 4-D. The cited study does not identify a more sensitive endpoint for human risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NYS DOH: identified as additional studies that should be considered in any new assessment along with those identified in the NRDC petition.

(Study 24) Citation : Duffard, R.; Bortolozzi, A; Ferri, A.; Garc ia, G.; and Evangelista de Duffard A. M. (1995). Developmental Neurotoxicity of the Herbicid e 2,4-Dichlorophenoxyacetic Acid. Neurotoxicology 16 (4): 764. [ Human Studies Considerations Apply: No ]

### Page 98 of 186 Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: offspring of dams treated (i.p) with 70 mg/kg/day 2,4-D/kg/day from GD 16 to weaning (PND 21). Offspring treated with control or 2,4-D via diet until adult life. Vaginal smears monitored, prolactin and progesterone measured. Citation is an ABSTRACT (few details of methods).

RESULTS: both pup groups exposed to 2,4-D via milk from GD 16-PND 21; then to control diet or to 2,4- D via diet. Seraprolactine and progesterone levels sligh tly increased in both grou ps; increased number and size of corpus luteum on the ovary of both 2,4-D treated groups, but only in the 2,4-D fed rats was the estrus cycle arrested in diestr ous, judged from the presence of an assortment of the cell types (predominantly leucocytes and nucleated cells) in the vaginal smears.

CONCLUSION: Authors stated that these preliminary results demonstrate a 2,4-D adverse effect on the endocrine system.

COMMENT/ HED: only an abstract . Study was binned according to c itation title; however, effects described relate to endocrine issues (see discussion under Endocrine BIN). Dose to pups via milk during this time interval unknown. Study does not provide any dose-response information. The only dose used (70 mg/kg/day) would be expected to exceed rat renal clearance levels and does not provide information regarding potential effects of 2, 4-D on estrus cycle, hormone levels at dose levels below the saturation level for renal clearance. The extended 1-generation reproduction study on 2, 4-D examined potential effects on female reproductive function, including endocrine and systemic toxicity parameters, such as estrous cyclicity (female adult rats and offspring), puberty onset, ovarian weight, histopathology of the ovary. No treatment-related effects were observed. The cited study does not identify a more sensitive endpoint for human risk assessment than already identified by the Ag ency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. The extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure

as to neurotoxic effects.

CITED BY NRDC (25) – one of several studies cited as demonstrating endocrine disrupting effects of 2,4-

D

CITED BY Task Force – cited as one of a series of studies that NRDC contends provides evidence of 2,4- D endocrine activity; TF does not consider study as providing compelling evidence of potential environmental or human endocrine health concerns; was conducted at high dose (well above saturation of renal clearance) and thus of limited value for risk assessment.

(Study 25) Citation : Duffard, R.; Garcia, G.; Rosso, S.; Bortolozzi, A; Madariaga, M.; diPaolo, O; and Evangelista de Duffard A. (1996). Central Nervous System Myelin Deficit in Rats Exposed to 2,4- Dichlorophenoxyacetic Acid Throughout Lactation. NeuroToxicology and Teratology 18 (6): 691-696. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: offspring (8 pups/dam) of dams treated with 100 mg/kg/day 2,4-D/kg/day once a day (i.p.) (1) for 16 days (PND 9-25); (2) for 6 days (P ND 9-15) then treated with DMSO until PND 25; (3) for 10 days (PND 15-25); from PND 9-15, dam treated with DMSO; (4) control treated with DMSO for 16 days (PND 9-25). Neonates were sacrificed on PND 25; brains were removed and weighed, and myelin markers (monohexosylceramide, phos pholipids, free fatty acids, cholesteryl esters) were assessed.

RESULTS: Brain weight was not affected but there was a decrease in body weight in groups exposed before PND 15. A decrease in total lipids was observed in PND 9-25 pups, and changes were noted in lipid brain composition (Tables 1 and 2); pups exposed via milk

Table 1. Body and Brain Weights; Lipids and Protein in Brain

Control PND 9-25 PND 9-15 PND 15-25 Body Weight (grams) 43.94±1.55 28.56±0.95 ( ↓ 35%) 34.02±1.09 ( ↓ 23%) 45.3±1.23 Brain (wet weigh,g )

1.26±0.01 1.18±0.02 1.25±0.02 Nd Total lipids (mg/g w.wt.) in Brain 0.07±0.01 0.03±0.02** ( ↓ 58%) 0.06±0.03 Nd Total proteins (mgg w.wt) n Brain

### Page 99 of 186 58.42±1.1 7.32±2.56 53.97±4.70 Nd *p <0.01; mean±SEM; nd no data provided

Tabe . Individual ipids Cont ent in Brin of 25-Day-old Pups

chemical assay ♪ Control PND 9-25 PND 9-15

Total cholesterol 9.58±1.01 11.60±088 7.56±.52

Cholesterol 93±0.79 8.30±0.88 7.58±.23 Cholesterol esters 0.65±0.013 2.36±0.32** ( ↑ 363%) 0.54±0.1 Ftty acids, phopholipids 13.10±0.3 9.87±0.69** ( ↓ 25%) 11.28±0.95

Phospholipids 314.09±18.92 192.67±36.45** ( ↓ 39%) 362.00±23.37 Free fatty acids 340.00±35.90 128.45±10.86** ( ↓ 62%) 148.13±61.21** ( ↓ 56%) *p <0.01; mean±SEM; ♪ mg/g wet weight;

Histochemical study demonstrated a decreased myelin staining in corpus callosum, cortical grey matter, and cerebellum of 25-day old rats exposed to 2,4-D from PND 15; myelin staining appeared normal in rats exposed before PND 15. Concentration of galactolip ids (cerebrosides and sulphatides, both sensitive and quantitative chemical markers for myelin sheaths in CNS development) was significantly reduced in PND15-25 pups compared to control (Table 3).

Table 3. Myelin Marker Lipid Content in Brain of 25-day-old Pups Treated PND 15-25 Group Monohexosylceramide Sulphatides

Control 2,4-D (15-25 days) 766.56±12.55 633.60±27.48* ( ↓ 17%) 433.17±15.20 195.64±6.17** ( ↓ 45%) * p<0.05; ** p<0.01; mean±SEM; μg/g wet weight

CONCLUSION: Myelin deficit in pups nursed from PND 9-25 or PND 15-25; in latter group, body and brain weight were not affected, so deficit in myelin cannot be attributed to undernourishment. In both groups, there was a decrease in brain total lipids, phospholipids, FFA content, and appearance of cholesteryl esters (presence of cholesteryl esters us ually indicates myelin breakdown); a clear deficit in myelin staining and mye lin chemical markers.

COMMENT/ HED: The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The only dose tested (100 mg/kg/day) was administered i.p. to dams, which is not relevant for human health pesticide risk assessment (dose via milk to pups), exceeds rat renal clearance levels and does not provide information regarding potential effects of 2, 4-D on myelination at dose levels below the saturation level for renal clearance. An extended 1- generation reproductive toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity and neuropathology (including an evaluation of myelin in the PND 22 and PND 60 offspring following exposure during gestation and lactation). The cited study was previously considered by the Agency in its rationale for additional toxicology data (DNT) in the RED. In the extended 1-generation reproductive toxicity study, there were no treatment-related adverse effects on myelination following 2, 4-D exposure at dose levels (5, 15, 30/40 mg/kg/day) considered appropriate (approaching saturation of renal cl earance). A NOAEL of 59 mg/kg/day (actual intake) was identified for effects on myelination, which is 2-11 times greater than the toxicity endpoints utilized for the human health risk assessment supporting the 2, 4-D RED. The changes in myelination in the cited study were not replicated in the extended 1-generation reproductive toxicity study. The cited study does not identify a more sensitive endpoint for human risk assessment than already identified by the Agency, and the results of the cited study do not yield information that would cause theAgency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended onegeneration reproduction study confirms the protectiveness of the existing Points of Departure as to

neurotoxic effects.

CITED BY NRDC (37) – cited as showing potential effects on the brain of neonatal rats exposed postnatally; 2,4-D appears to impair normal depositi on of myelin in developing brain; cites additional studies that identify adverse effects in offspring afte r lactational exposure and states that EPA failed to include any lactational exposure in its aggregate risk assessment; concludes that effects of 2,4-D make it

### Page 100 of 186 likely that fetuses, infants, and children will be more susceptible to long-term adverse health effects from exposure to 2, 4-D.

CITED BY Task Force – study conducted at high doses (w ell above pharmacokine tic nonlinearity) and included use of an unrepresentative route and mode of administration (intraperitoneal), which would further amplify nonlinear pharmacokinetic performance. The Task Force also considered this paper and other NRDC cited papers from the same group of investigat ors to be methodologically flawed [litter was not used as the unit for statistical analyses (to avoid bias du e to litter effects); the reporting of histopathological evaluation limited, etc.].

CITED BY NCASI – referenced as one of the studies cited in the petition as supporting claim that infants are at risk; argues that doses used in study are extremely high and lik ely exceed renal clearance levels and are in range known to produce fr ank maternal toxicity, which is inappropriate dose selection for developmental toxicity studies; considers that fact that high doses are needed to elicit the reported effects supports previous EPA conclusion that 2,4-D use acco rding to label direction does not pose a risk to

humans

(Study 26) Citation : Evangelista de Duffard A.; Bortolozzi, A.; and Duffard RO (1995). Altered Behavioral Responses in 2,4-Dichlorophenoxyacetic Acid Treated and Amphetamine Challenged Rats. NeuroToxicology 16 (3): 479-488. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Wistar rats (i.p.); doses: 2,4-D: 0, 50, and 100 mg/kg; 0, 5, 10 mg/kg amphetamine (acute); 0.9% saline solution; each 2,4-D dose group challenged with both levels of amphetamine and groups of both dose levels of each compound alone

RESULTS: various components of serotonin syndrome (beh avioral response induced by stimulation of 5- HT receptors) observed in rats after acute dose of 2,4-D plus amphetamine; not with 2,4-D alone

. CONCLUSION: 2,4-D appears to act through serotonergic and dopaminergic mechanisms

COMMENT/ HED: route of exposure not relevant for human health pesticide risk assessment; dose levels used (50 and 100 mg/kg/day) are at or exceed rat renal clearance levels following oral exposure. The study is one among several studies cited from the same researchers that were performed to assess the mechanism of 2, 4-D neurotoxicity. An extended 1-generation reproductive toxicity study on 2, 4-D, in which the parental rats and/or offspring were exposed to 2, 4-D during the pre-mating period, gestation, lactation via the milk, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity (including neurobehavioral tests) and neuropathology at dose levels that do not saturate metabolic processes (as described previously). No treatment-related adverse effects were observed on parental behavior or on any of the numerous parameters evaluated to assess developmental neurotoxicity potential following oral exposure to 2, 4-D.The cited study does not identify a more sensitive endpoint for

human health risk assessment than already identified by the Agency, an d the results of the cited study do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4- D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NRDC (34) – cited as evidence that 2,4-D results in delays in brain development and abnormal behavior patterns, including apathy, decreased so cial interactions, repetitive movements, tremors, immobility; females more sensitive

CITED BY TF – study does not assess brain development; finding could be non-specific effects rather than any selective neurological effect

CITED BY NCASI - petition fails to mention that dose le vels likely exceed renal clearance levels and route of exposure is not appropriate

(Study 28) Citation : Ferri A, Duffard R, Evangelista de Duffard A. (2 007). Selective Oxidative Stress in Brain Areas of Neonate Rats Exposed to 2,4-Dichlorophenoxyacetic Acid Through Mother’s Milk. Drug and Chemical Toxicology 30: 17-30. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: PND 0 pups (8 pups/dam); exposed pups of (1) dams treated with 100 mg 2,4-D/kg/day once a day (i.p.) for 16 days (PND 9-25); (2) dams treated with DMSO once a day (i.p.) for 16 days (PND 9-25); (3) dams treated with DMSO and saline once a day (i.p.) for 16 days (PND 9-25).

### Page 101 of 186 Neonates sacrificed by decapitation; br ain areas were removed and weighed (pre frontal cortex, hypo thalamus, hippocampus, striatum, and midbrain, which involved neither separate anal ysis of the substantia nigra not ventral tegmental area and cerebellum, which was separated from the pons medulla). After the cerebellum had been removed from the brain stem, the midbrain was separated by a cut at the level of the inferior colliculus).

Enzyme and Other Assays : superoxide dismutase (SOD) and glutathione pe roxidase (GSH-Px) activities; catalase activity; glutathione content; assay for r eactive oxygen species (ROS).

RESULTS: after 16 days of exposure via mother’s milk, ROS levels increased in th e prefrontal cortex (15.7%), striatum (11.1%), and midbrain (17.2%); GSH content decreased in striatum (37.4%) and midbrain (34.0%) of 2,4-D pups compared with basal levels of 25-day-old pups.

Table 1. Reactive Oxygen Species/Gl utathione Content/Protective Enzymes Group Prefrontal

cortex Striatum Cerebellum Hippocampus Midbrain hypothalamus

Reactive oxygen species levels (Fl/mg protein) in brain areas of 25-day-old pups lactationally exposed to 2,4-D

C

VC 2,4-D 18.1±0.6

17.8 ±0.7 20.6±.5*( ↑ 16) 21.8±0.9

22.6±0.8 25.1±0.7* ( ↑ 11) 24.3±1.2

24.0±0.9

23.7±1.1 19.3±1.4

18.0±1.0

18.1±1.1 19.9±1.1

20.3±1.0 23.8±0.7*( ↑ 17) 20.9±1.3

22.6±1.2

21.1±.3 Glutathione content (μg GSH/mg protein) in brain areas of 25-day-old pups lactationally exposed t 2,4-D

C

VC 2,4-D 1.25±0.35

1.23±0.29

1.29±0.20 1.23±0.33

1.31±0.24 0.82±0.18*( ↓ 37) 0.74±0.24

0.79±0.19

0.80±0.22 0.86±0.24

0.88±0.19

0.94±0.24 0.98±0.16

1.06±0.12 0.70±0.15*(34) 0.99±0.36

1.07±0.41

1.08±0.30 Protective enzymes (mU/mg protein) in brain areas of 25-day-old pups lactationally exposed to 2,4-D (Zn,Cu)SOD

C

VC 2,4-D 2000±180

1950±200 2450±3108( ↑ 26) 2430±175

2460±150

2540±220 2690±310

2730±330

2610±240 2290±180

2330±200 2980±320*( ↑ 28) 2100±80

1950±120

2140±90 2410±150

2320±160

2400±120

Catalase

C

VC 2,4-D 3100±190

2950±250 2200±200*( ↓ 25) 2800±130

2740±200 2250±150*( ↓ 18) 2440±165

2300±130

2530±170 2480±180

2580±160

2690±210 2400±180

2360±200 1850±120*( ↓ 22) 2680±160

2740±150

2810±210 (Se)GSh-Px

C

VC 2,4-D 30.5±1.6

30.0±1.4 24.2±1.9*( ↓ 19) 28.8±2.3

28.2±2.1 22.3±2.9*( ↓ 21) 25.3±1.5

25.6±9.1 29.5±2.1*( ↑ 15) 28.3±0.9

29.1±1.1 32.3±1.0*( ↑ 11) 32.3±1.0

31.9±1.1 27.6±1.0*( ↓ 14) 28.4±1.3

28.9±1.9

28.0±1.6(noSe)GSHPx

C

VC 2,4-D 20.5±0.9

20.8±1.1

19.0±1.0 18.6±0.7

18.6±0.6 15.7±0.5*( ↓ 16) 18.3±1.3

18.2±1.0

18.0±1.0 20.9±0.7

20.9±0.7

19.9±0.9 22.6±0.9

22.7±0.8 19.4±0.8*( ↓ 15) 15.6±1.0

15.8±0.7

17.0±0.7 Mean±SEM; VC vehicle control; * p<0.05; data from Tables 2 and 3 (pages 24-25)

CONCLUSION: 2,4-D induces disparate alterations on enzymatic activitie s of defensive mechanisms and/or modifications of the reactive oxygen species levels in specific neonate brain regions. The midbrain, striatum, and pref rontal cortex were the areas where the alterations were more remarkable and with similar tendency. The hippocampus did not suffer many alterations, and the hypothalamus was the area where no changes were observed. Results suggest that the developing brain areas have different susceptibilities to the adverse effect of 2,4-D, especially thos e areas related to the dopaminergic system, and that oxidative stress is one 2,4-D mechanism of toxicity.

**COMMENT/** HED: The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Dams exposed i.p., which is not a relevant mode of administration for human health pesticide risk assessment; pups were exposed via milk; do not know dose to the pup. 100 mg/kg/day via the oral route exceeds renal clearance levels; identifies possible oxidative stress in pup brain areas following lactational exposure. The extended 1- generation reproductive toxicity study on 2,4-D, as described previously (MRID 47972101), in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity [functional opera tional battery (FOB), motor activity, and acoustic startle response (ASR)] at dose

### Page 102 of 186 levels up to those that saturate metabolic processes. The young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluations and brain morphometry, and a special stain was used to evaluate brain myelination. There were no treatment-related adverse effects on any of the numerous parameters evaluated to assess neurotoxicity and neuropathology potential following 2, 4-D exposure at dose levels (5, 15, 30/40 mg/kg/day) considered appropriate (approaching saturation of renal clearance). The no observed adverse effect level (NOAEL) for developmental neurotoxicity [(calculated intake: 82 mg/kg/day (males)/59 mg/kg/day (females)] is greater than the PODs utilized for the human health risk assessment supporting the 2,4-D RED. The PODs used in the RED risk assessment are protective of chronic effects identified in the extended 1-generation reproductive toxicity study and of those identified in the cited study.

CITED BY NRDC (61)– cited as support for need to consider lact ational exposure to 2,4-D in aggregate RA; lactational exposure of neonatal rats to 2,4-D in maternal milk showed indications of oxidative stress in certain regions of brain (midbrai n, striatum, prefrontal cortex); one mechanism of toxicity

CITED BY TF – study conducted at high dose (well above pharmacokinetic nonlinearity) and included use of an unrepresentative route and mode of administration (intraperitoneal), which would further amplify nonlinear pharmacokinetic performance.

(Study 32) Citation : Garcia, G.; Tagliaferro, P.; Ferri A, Evan gelista de Duffard A.; Duffard, R.; and Brusco, A. (2004). Study of Tyrosine Hydroxylase Immunoreactive Neurons in Neonate Rats Lactationally Exposed to 2,4-Dichlorophenoxyacetic Acid. NeuroToxicology 25: 951-957. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: PND 0 pups (8 pups/dam); exposed pups of (1) dams treated with 70 mg 2,4- D/kg/day once a day (i.p.) for 16 days (PND 9-25); (2) dams treated with 100 mg 2,4-D/kg/day once a day (i.p.) for 16 days (PND 9-25); (3) dams treated with DMSO once a day (i.p.) for 16 days (PND 9-25). Neonates were anaesthetized and sacrificed by perf usion-fixation; brains were removed and weighed; immunohistochemical staining.

RESULTS: Brain and body weight were not affected in the 25-day-old pups nursed by the 70 mg/kg/day dams; decreased body ( ↓ 27.5%) and brain ( ↓ 13.8%) weights were observed in 25-day-old pups nursed by the 100 mg/kg/day dams. A similar decrease (25%) in TH-IR (tyrosine hydroxylase immunoreactivity) was observed in neurons of the SN (substantia nigra) at both dose levels; VTA (ventral tegmental area) TH neurons showed a decreased immunoreactivity only in 100 mg/kg/day pups (33%). Decreased 5-HT fiber density in SN and VTA was observed at both dose levels

Relative Optical Density of TH neurons in SN and VTA Control 70 mg/kg/day 100 mg/kg/day Relative optical density (ROD) of TH neurons in substatia nigra 3.21±0.75 2.40±0.71** ( ↓ 25%) 2.40±0.62** ( ↓ 25%) Relative optical density (ROD) of TH neurons in VTA 1.95±0.28 - 1.31±0.25 ( ↓ 33%)

Relative Area of 5-HTT-Immunolabeled Fibers in SN and VTA

Relative area of 5-HTT-immunolabeled fibers in substantia nigra 0.48±0.012 0.31±0.021 ( ↓ 36%) 0.33±0.023 ( ↓ 32%)

Relative area of 5-HTT-immunolabeled fibers in VTA 0.50±0.006 0.36±0.014 ( ↓ 28%) 0.39±0.023 ( ↓ 22%) Body weight (g)

49.3±2.8 A 45.6±2.6 A 35.7±3.0* A ( ↓ 27.5%) Brain weight (g)

1.38±0.05 A 1.34±0.06 A 1.19±0.05* A ( ↓ 13.78%)

mean ± s.d. or A SEM; ** p<0.01

CONCLUSION: TH-IR is significantly decreas ed, probably as a consequence of an inhibitory 5-HT

effect. This 2,4-D inhibitory effect on the dopaminergic neurons probably mediated by 5-HT may lead to biochemical and functional consequence like alterations in DA biosynthesis, storage, release, feedback control systems or receptor sensitivity.

COMMENT/ HED: The cited study is one of several research studies from the same group of investigators

### Page 103 of 186 looking into possible mechanism of 2, 4-D neurotoxicity. In substantia nigra and in ventral tegmental area, a decreased neuron tyrosine hydroxylase immunostaining along with diminution in serotonin (5-HT) fiber density were reported in 2,4-D (lactationally-expose d) pups. Dams were exposed i.p., which is not a relevant mode of administration for human health pesticide risk assessment; pups were exposed via milk; do not know dose to the pup. Both dose levels (70 and 100 mg/kg/day) via the oral route exceed renal clearance levels. Study identifies effects on pup brain following lactational exposure. The extended 1- generation reproductive toxicity study/DNT study on 2,4-D provides information on 2, 4-D levels in the milk and plasma of the offspring exposed during gestation and lactation via the milk, then directly via thediet up to PND 139 at dose levels up to those that saturate metabolic processes. There were no treatmentrelated adverse effects on any of the numerous parameters evaluated to assess neurotoxicity and neuropathology potential following 2, 4-D exposure. The no observed adverse effect level (NOAEL) for developmental neurotoxicity was established at 59 mg/kg/day. This NOAEL is greater than the PODs utilized for the human health risk assessment supporting the 2,4-D RED. The PODs used in the RED risk assessment are protective of chronic effects identified in the extended 1-generation study and of those identified in the cited study. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. An exposure pathway identified in the NRDC petition is lactational exposure to 2, 4-D. The Agency currently is addressing the issue of a lack of information regarding the levels of 2, 4-D that can occur in human breast milk. Preliminary studies performed to determine the doses to be used in the extended 1-generation reproductive toxicity study provide data on the levels of 2, 4-D in the milk of the dams and plasma levels of both the dams and offspring. Currently, it cannot be determine what the actual dose ( via milk) to each pup was due to the lack of information on the amount of milk ingested by the pups.

CITED BY NRDC (62) – study cited as support for need to consider lactational exposure to 2,4-D in aggregate RA; cited as showing pote ntial effects on the brain of neonatal rats exposed lactationally

CITED BY TF – study conducted at high doses (well above pharmacokinetic nonlinearity) and included use of an unrepresentative route and mode of administration (intraperitoneal), which would further amplify nonlinear pharmacokinetic performance.

(Study 33) Citation : Garcia, G.; Konjuh, C.; Duffard RO, Eva ngelista de Duffard A. (2006). Dopamine- β

Hydroxylase immunohistochemical Study in the Locus Coeruleus of Neonate Rats Exposed to 2,4- Dichlorophenoxyacetic Acid Through Mother’s Milk. Drug and Chemical Toxicology 29, 435-442. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: PND 0 pups (8 pups/dam); exposed pups of (1) dams treated with 70 mg 2,4- D/kg/day once a day (i.p.) for 14 days (PND 9-22); (2) dams treated with 100 mg 2,4-D/kg/day once a day (i.p.) for 14 days (PND 9-22); (3) dams treated with DMSO once a day (i.p.) for 14 days (PND 9-22). On PND 22, neonates were anaesthetized and transcardially perfused-fixed; brains were removed and weighed; relative optical density (ROD) of the D β H stained LC (locus coeruleus) sections in all rats was measured.

RESULTS: Brain and body weight were not affected in the 22-day-old pups nursed by the 70 mg/kg/day dams; decreased body (20%) was observed in 25-day- old pups nursed by the 100 mg/kg/day dams (brain weight not affected). There was a si gnificant decrease (73% to 75%) in D- β -H immunoreactivity (D- β -Hir) in exposed neonates at both dose levels. Morphological data confirmed these observations: ROD of D- β -H immunoreactivity was 0.200 ±0.025 an d 0.172 ± 0.029 at 70 and 100 mg/ kg/day, respectively compared to control (0.752 ±0.387).

CONCLUSION: TH-IR is significantly decreas ed, probably as a consequence of an inhibitory 5-HT

effect. This 2,4-D inhibitory effect on the dopaminergic neurons probably mediated by 5-HT may lead to biochemical and functional consequence like alterations in DA biosynthesis, storage, release, feedback control systems or receptor sensitiv ity; both doses produced similar e ffect implies that it was maximal

COMMENT/ HED: The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The dams were exposed i.p., which is not a relevant mode of administration for human health pesticide risk assessment; pups were exposed via milk; do not know dose to the pup. 70 mg/kg/day and 100 mg/kg/day via the oral route exceed renal clearance

### Page 104 of 186 levels; identifies effects on pup brain following lactational exposure. An extended 1-generation reproductive toxicity study on 2,4-D was submitted to Agency in February of 2010, in response to the data requirements identified in the 2005 RED, which included a developmental neurotoxicity assessment. The young rat was exposed during gestation, lactation via the milk, and directly via the diet following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (FOB), landing foot splay, grip strength, motor activity, habituation, and acoustic startle response (ASR). The young rats were perfuse d for central nervous system (CNS) and peripheral nerve neuropathology evaluation and brain morphometry, and a special stain was used to evaluate brain myelination. Three dose levels were tested. At the highest dose (calculated intake: 82 mg/kg bw/day in males/59 mg/kg bw/day in females), there were no treatment-related adverse effects on any of the numerous parameters evaluated to assess the developmental neurotoxicity potential of 2, 4-D. The parental rats were assessed for clinical signs of toxicity and maternal behavior, among other parameters. Maternal behavior towards the offspring was comparable among the groups, and there were no clinical signs such as circling to the right observed in the dams or offspring. Although neurotransmitter levels and brain enzymes were not monitored in the extended 1-generation reproduction study, the dose levels were adequate (approaching saturation of renal clearance), and the no observed adverse effect level (NOAEL) for developmental neurotoxicity is greater than the PODs utilized for the human health risk assessment supporting the 2,4-D RED. The PODs used in the RED risk assessm ent are protective of chronic effects identified in the extended 1-generation study and of those identified in the cited study.

The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. Additionally, the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. An exposure pathway identified in the NRDC petition is lactational exposure to 2, 4-D. The Agency currently is addressing the issue of a lack of information regarding the levels of 2, 4-D that can occur in human breast milk. Preliminary studies performed to determine the doses to be used in the extended 1-generation reproductive toxicity study provide data on the levels of 2, 4-D in the milk of the dams and plasma levels of both the dams and offspring. Currently, it cannot be determine what the actual dose to each pup was due to the lack of information on the amount of milk ingested by the pups.

CITED BY NRDC (63) – cited as showing potential effects on the brain of neonatal rats exposed lactationally; alterations in brain enzymes levels and neurotransmitters in pups exposed to 2,4-D through maternal milk and as support for the need to consid er lactational exposure to 2, 4-D in aggregate RA.

CITED BY TF – study conducted at high doses (well above pharmacokinetic nonlinearity) and included use of an unrepresentative route and mode of administration (intraperitoneal), which would further amplify nonlinear pharmacokinetic performance. The Task Force commented that EPA was aware that 2, 4-D may be present in breast milk (discussed in the 2, 4-D RE D). Compared to the high doses used in the animal studies, the Task Force noted that EPA estimated of the maximum dietary exposure from food to human females ages 13-50 to be 0.01018 mg/kg/day and the average exposure to be 0.000642 mg/kg/day (USEPA memorandum, 2002). These values range from 4900 to 1 million times lower than the values in the cited rat study. Additionally, EPA’s manner of doing risk assess ment for infants is protective of any pesticide exposure to infants from human breast milk because the exposure values for pesticides in cow’s milk greatly exceed the values that could be present in breast milk (US Federal Register 2005 at 46706, 46735).

(Study 37) Citation : Giasson, BI, Lee, VM. (2000). A New Li nk Between Pesticides and Parkinson’s Disease. Environ Hlth Perspect. 103: 113. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Neurotoxicity] Methods Summary: not applicable; paper discusses another paper (Betarbet et al., 2000; rats exposed chronically (i.v.) to rotenone, a mitochrondrial inhi bitor), which reports that chronic administration of rotenone can induce the major featur es of Parkinson’s disease in rats ; states that this “remarkable observation not only provides a new animal model for the study of PD, but will also reinvigorate interest in the possible role of exposure to pesticides and other toxins as a cause of human neurodegenerative disease.”

### Page 105 of 186 Results Summary: in the Betarbet paper discussed, rats develop neuropathological and behavioral symptoms of Parkinsonism following iv exposure;
CONCLUSION: Giasson concludes that this (Betarbet) study will revitalize the search for environmental toxins, including other pesticides that may contribute to the etiology of this disease.

COMMENT/ HED : paper is not 2, 4-D specific and i.v. exposure is not a normal route of human exposure; the extended 1-generation reproductive toxicity study (discussed previously) addresses developmental neurotoxicity endpoints, including assessment of moto r activity, FOB, myelination. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. Additionally, the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D.

CITED BY: 2,4-D TF: cited in response to NRCD’s use of the Ton, et al., 2006 zebrafish paper to support their claim that 2,4-D is associated with developmental neurotoxicity in mammals, including decreased motor activity and Parkinson’s-like tremors. TF states that the support for the Ton claim is a “news and view commentary” by Giasson and Lee, 2000, who discuss using the pyrethroid insecticide and pesticide

rotenone as a model for Parkinson’s disease.

(Study 59) Citation : Konjuh, C.; Garcia, G.; Lopez, L.; Evangelis ta de Duffard A.; Brusco, A.; and Duffard,

R. (2008) Neonatal hypomyelination by the herbicide 2,4-dichlorophenoxy acetic acid. Chemical and ultrastructural studies in rats. Toxicol. Sciences 104 (2), 332-340. [ Human Studies Considerations Apply: No ]

Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Pregnant Wistar rats administered 70 mg 2,4-D/kg/day i.p. (DMSO) to dams on PND 9- 22; litters of 14 pups (UN - undernourished) vs litters of 8 pups (WN – well nourished) per litter ; pups

exposure via maternal milk (oral) Results Summary: myelin proteins (analyzed by Western blot and/or immunohistochemical study) showed a significant decrease in WN70, UN, and UN70 with respect to control; UN hypomyelination expressed by a diminution of the number of sheets and the MDLt, whereas 2,4-D hypomyelination had a normal number of sheets but presented altered compacti on of myelin sheaths, increased ID and the presence of a higher number

of blebs.

Table 1. Myelin Chemical Composition

Treatment Total proteins (mg/g brain) Total lipids (mg/g brain) Cholesterol (mg/g brain) Galactolipids (mg/g brain) Phospholipids P (nm P/g brain)

WN 6.0±0.7 683.6±117.2 3.2±0.2 1.00.3 9.1±1.6

WN70 4.3±0.3** 478.6±71.2* 2.0±0.3* 0.6±0.1* 4.9±1.4**

UN 4.5±0.6** 526.2±94.6 1.9±0.2* 0.5±0.2** 6.8±2.1

UN70 3.4±1.0***# 218.3±121.1***# 1.7±0.2* 0.3±0.2** 4.4±1.9*** N=5; * p<0.05; ** p<0.01; *** p<0.001 (WN); #0.001 (UN)

Table 2. Morphometric Study at ultrastructural Level of Myelin around Corpus Callosum Axons Treatment # of sheets ID (nm) MDL t (nm)

WN 7.90±1.90 9.12±0.39 2.70±0.12

WN70 6.00±1.50 8.01±0.68 2.18±0.30**

UN 5.60±2.00** 8.82±1.00 2.30±0.17**

UN70 5.90±1.50** 10.83±0.96*** 1.78±0.34*** ** p<0.01 (WN); *** p<0.001 (UN); MDL t thickness of major dense line; ID interperiod distance

CONCLUSION: 2,4-D and undernourishment are two independent hypomyelinating factors.

COMMENT/ HED: The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Study does not provide any dose-response

information ( do not know dose to pups). The only dose used (70 mg/kg/day) exceeds rat renal clearance levels following oral exposure and does not provide information regarding potential effects of 2, 4-D on myelination

at dose levels below the saturation level for renal cl earance. An extended 1-generation reproductive toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and

### Page 106 of 186 directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity and neuropathology (including an evaluation of myelin in the PND 22 and PND 60 offspring following exposure during gestation and lactation) at dose levels that do not saturate metabolic processes

. There were no treatment-related microscopic changes in the central or pe ripheral nervous systems in the perfused offspring, no exposure-related changes on myelin deposition in the offspring, or changes in microscopic measurements of structures in the cerebral cortex, cerebellum, thalamus, or hippocampus. T he no observed adverse effect level (NOAEL) for developmental neurotoxicity is greater than the PODs utilized for the human health risk assessment supporting the 2,4-D RED. The PODs used in the RED risk assessment are protective of chronic effects identified in the extended 1-generation study and of those identified in the cited study. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D.

As noted by the Task Force, EPA was aware that 2, 4-D may be present in milk. There was a discussion of this issue in the 2, 4-D RED. Co mpared to the high doses used in the animal studies, the EPA estimates of the maximum dietary exposure from food to human females ages 13-50 is 0.01018 mg/kg/day and the average exposure is 0.000642 mg/kg/day (USEPA memorandum , 2002). These values range from 4900 to 1 million times lower than the values in the cited rat study. Additionally, EPA’s manner of doing risk assessment for infants is protective of any pesticide exposure to infants from human breast milk because the exposure values for pesticides in cow’s milk greatly exceed the values th at could be present in breast milk (US Federal Register 2005 at 46706, 46735). The Agency currently is addressing the issue of a lack of information regarding the levels of 2, 4-D that can occur in human breast milk. Preliminary studies performed to determine the doses to be used in the extended 1-generation reproductive toxicity study provide data on the levels of 2, 4-D in the milk of the dams and plasma levels of both the dams and offspring. Currently, it cannot be determine what the actual dose to each pup was due to the lack of inform ation on the amount of milk ingested by the pups.

CITED BY NRDC (38)– study referenced as one that shows that 2,4-D appears to impair normal deposition of myelin in developing brain; effects of 2,4-D make it likely that fetuses, infants, and children will be more susceptible to long-term adverse health effects from exposure to 2. 4-D

CITED BY TASK FORCE: excessive doses (ip); only one dose (70 mg/kg/day); addressed undernutrition (14 pups/litter vs 8/litter); small sample size

CITED BY NCASI: petition fails to mention that dose levels likely exceed renal cleara nce levels and route of exposure is not appropriate

(Study 94) Citation : Rosso, SB; Garcia, GB; Madariaga, MJ; Ev angelista de Duffard A.; and Duffard, R. (2000). 2, 4-Dichlorophenoxyacetic Acid (2,4-D) in Developing Rats Alters Behaviour, Myelination and Regions Brain Gangliosides Pattern. NeuroToxicology 21 (1-2): 155-164. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Wistar rats: pups administered 2,4-D (0, 70, or 100 mg/kg/day) intermittently (sc )

, every 48 hours, 8 pups/dam; exposure from PND 7-25 (100 mg/kg) or PND 12-25 (70 mg/kg); control at PND 25 injected with vehicle (water :ethanol). Neurobehavioral test pe rformed each 48 hrs, 4 hrs before dose (surface righting reflex, negative geotaxis, forelimb support, open fi eld). Histological studies: myelin staining (histochemical and immunohistochemical); gangliosides analyses (GM 1 GD 1a GD 1b GT 1b ) Results Summary: at 100 mg/kg, decreased body and brain we ight at PND 25; surface righting reflex (took more time to return to normal position from PND 11-21); negative geotaxy (showed major latency from PND 11-21; decreased motor activity (both groups) and rearing frequency (100 mg/kg); alterations in forelimb support (both group); decreased myelin staining; changes in ganglioside levels (Table 2) in various brain areas (both groups).

Table 1. Brain Weight of PND 25 Pups Treated with 100 mg/kg 2,4-D (g) Control 70 mg/kg 100 mg/kg 1.41±0.03 1.34±0.04 1.19±0.03 ** ( ↓ 16%)
** p<0.01; mean±SE

### Page 107 of 186 Table 2. Gangliosides Levels in Different CNS Regions

Ganglioside Control 100 mg/kg 70 mg/kg

GT 1b Olfactory bulbs 222.4±9.7 174.6±10.3** ( ↓ 21%) 23.3±6.6 Prefrontal cortex 249.5±11. 257.4±97 220.0±20.0 ( ↓ 12%)

Striatum 254.4±7.77 269.8±12.2 237.0±13.2 Hippocampus 260.1±10.4 282.7±16.8 297.2±21.2

Cerebellum 341.7±13.8 280.8±9.43** ( ↓ 18%) 238.0±4.6** ( ↓ 30%)

GD 1b Olfactory bulbs 161.8±9.5 157.±7.1 196.9±6.7** ( ↑ 22%)

Prefrontalortex 26.13.9 237.2±19.1 284.7±9.4 Striatum 297.9±15.2 268.6±15.1 369.3±14.5* ( ↑ 24%) Hippocampus 234.5±7.2 207.9±17.2 256.9±1.9

Cerebellum 120.7±10.4 132.9±14.7 23.4±8.5

GD 1ab Olfactory bulbs 394.4±28.8 321.6±13.5* ( ↓ 18%) 342.0±8.1

Prefrontal crtex 51.04.1 527.3±21.2 542.9±12.4

Striatum 759.6±28.6 740.8±29.1 865.3±25.1 Hippocampus 370.1±12.1 459.1±19.6** ( ↑ 24%) 398.66.8

Cerebellum 236.7±9.0 210.4±15.5 40.7±9.4

GM 1a Olfactory bulbs 191.4±12.1 145.2±10.5* ( ↓ 24%) 130.9±17.8* ( ↓ 32%) Prefontal cortex 363.6±10.4 263.7±7.6** ( ↓ 28%) 329.2±10.8* ( ↓ 10%) Striatum 338.5±7.1 286.0±11.1** ( ↓ 16%) 305.2±10.7** ( ↓ 10%) Hippocampus 243.7±12.6 226.3±17.5 210.2±7.3

Cerebellum 76.8±6.9 49.8±7.4* ( ↓ 35%) 53.3±2.8* ( ↓ 31%) nmol ganglioside/g tissue; * p<0.05;* *p<0.01; mean±SE

CONCLUSION: early exposure of pups to high dose of 2, 4-D resulted in a deficit in myelin deposition and behavioral alterations together with GM1 reduc tion, which could be reflecting a 2,4-D CNS injury, although a weak undernutrition could be involved also; when 2,4-D dose was lower, body and brain weight effects were not observed, but changes in GM1 contents and in myelin deposition, and alterations in muscular force and motor activity were still observed.

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. The dose levels are above level for saturation of renal clearance mechanism (oral route) and the route of exposure (sc) of pups is not relevant to human pesticide exposure. However, the reported deficit on myelin deposition was also reported following pup

exposure via milk (Duffard, 1996). The extended 1-generation reproductive toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity and neuropathology (including an evaluation of myelin in the PND 22 and PND 60 offspring following exposure during gestation and lactation) at dose levels that do not saturate metabolic processes (described previously)

. There were no treatment-related adverse effects on myelinati on at dose levels approaching renal saturation levels. Additionally, there were no treatment-related microscopic changes in the central or peripheral nervous systems in the perfused offspring, or changes in microscopic measurements of structures in the cerebral cortex, cerebellum, thalamus, or hippocampus. The no observed adverse effect level (NOAEL) for developmental neurotoxicity is greater than the PODs utilized for the human health risk assessment supporting the 2,4-D RED. The PODs used in the RE D risk assessment are prot ective of chronic effects identified in the extended 1-generation study and of those identified in the cited study. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D

CITED BY NRDC (36) – study referenced as one that shows that 2,4-D appears to impair normal deposition of myelin in developing brain; effects of 2, 4-D make it likely that fetuses, infants, and children

### Page 108 of 186 will be more susceptible to long-term advers e health effects from exposure to 2, 4-D.

CITED BY TASK FORCE – excessive doses (s c); litter not stat unit; effect may be due to undernutrition

CITED BY NCASI: petition fails to mention that dose leve ls likely exceed renal clearance levels and route of exposure is not appropriate

(Study 92) CITATION : Rosso, SB; Caceres, AO.; Evangelista de Duffard AM.; Duffard , R. and Quiroga, S. (2000). 2, 4-Dichlorophenoxyacetic acid disrupts the cytoskeleton and disorganizes the Golgi apparatus of cultured neurons. Toxicological Sciences 56, 133-140. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Neurotoxicity] Methods Summary: cultured cerebellar granule cells shown to be excellent model system for studying neurite outgrowth and the expression of cytoskeletal proteins involv ed in nerve cell morphogenesis; Results Summary: An in vitro study (cultured neurons) cell cultures; examined effects of 2,4-D in primary cultures of cerebellar granule cells; 24-hr exposure: dose-dependent inhibition of neurite extension; paralleled by a significant reduction in cellular content of both dynamic and stable microtubules, a disorganization of Golgi apparatus, and inhibition in synthesis of complex gangliosides.

Table 1. Gangliosides Biosysthesis in Cultured Cerebellar Granule Cells Treated with 2,4-D (24 hours)_ Ganglisides Control 0.2 mM 0.5 mM 1.0 mM 2.0 mM

GT1b

GD1b

GD1a

GD3

GM1

GM3 1.00

1.00

1.00

1.00

1.00

1.00 0.96

1.16

0.96

1.05

1.07

0.83 1.10

1.10

0.87

2.29***

1.26

0.91 0.79*

0.57**

0.79*

2.87***

1.11

1.49** 0.13***

0.10***

0.24***

4.56***

0.15***

13.27*** * p<0.05; ** p<0.01; *** p<0.001

CONCLUSION: primary toxic effects of 2,4-D on neurons are most probably due to inhibition of microtubule polymerization; observations raise possibility that at least one basic mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly.

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity

. The mechanistic information ( in vitro ) is not directly applicable to human risk assessment. Agency identified the need for additional data on neurotoxicity. An extended 1-generation reproductive toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity and neuropathology (including an evaluation of myelin in the PND 22 and PND 60 offspring following exposure during gestation and lactation) at dose levels that do not saturate metabolic processes (des cribed previously). There were no treatment-related microscopic changes in the central or peripheral nervous systems in the perfused offspring or changes in myelin, or changes in microscopic measurements of st ructures in the cerebral cortex, cerebellum, thalamus, or hippocampus. Do not know how mM dose relates to levels attained in the brain in vivo. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D

CITED BY: NYS DOH: identified as additional studies that should be considered in any new assessment along with those identified in the NRDC petition.

(Study 93) Citation : Rosso, SB; DiPaolo, OA; Evangelista de Duffard A.; and Duffard, R. (1997). Effects of 2, 4- Dichloro phenoxyacetic Acid on Central Nervous Syst em of Developmental Rats. Associated Changes in Ganglioside Pattern. Brain Research 769: 163-167. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Wistar rats: pups administered 2,4-D (0, 70, or 100 mg/kg/day) intermittently (sc ) , every 48 hours, 10 pups/dam; exposure from PND 7 to PND 17 or PND 25; control at PND 17 or PND 25 injected with vehicle (water:ethanol). Body and brain weights, brain DNA and protein levels, and gangliosides (GM 1 GD 1a GD 1b

### Page 109 of 186 GT 1b ) were assessed. Results Summary: no changes in body weight and DNA/protein content of brain at PND 17; decreased body and brain weights, protein and DNA levels were observed at PND 25; decreased brain weight was observed on PND 17 at both dose levels when exposure started on PND 7 and at 100 mg/kg/day when initiated at PND 12.

Table 1. Effects of 2, 4-D on Body/B rain Weights; Protein/DNA Content; Tota l/Individual Brain Gangliosides Levels

Dosed from PND 7 Dosed from PND 12 Parameter Control 70 mg/kg/day 100 mg /kg/day 70 mg/kg/day 100 mg/kg/day 17 days old Body weight (g) Brain weight (g) Protein (mg/brain) DNA (mg/brain) 30.11 ± 0.65

1.29 ± 0.02

150.46 ± 6.03

1.84 ± 0.12 30.31 ± 0.89

1.16 ± 0.03**(10)

155.29 ± 14.89

1.95 ± 0.07 28.31 ± 2.40

1.08 ± 0.02**(16)

134.43 ± 5.67

1.97 ± 0.14 29.59 ± 0.63

1.22 ± 0.04

133.72 ± 12.46

2.17 ± 0.02 28.21 ± 0.28

1.15 ± 0.01**(11)

148.65 ± 35.42

2.13 ± 0.00 25 days old Body weight (g) Brain weight (g) Protein (mg/brain) DNA (mg/brain) 57.50 ± 2.45

1.41 ± 0.03

143.21 ± 8.33

2.30 ± 0.07 49.81 ± 1.65*(14)

1.24 ± 0.02**(12)

125.17 ± 12.21

2.34 ± 0.11 44.9 ± 2.34**(22)

1.19 ± 0.03**(16)

111.1 ± 2.9*(22)

1.84 ± 0.08*(20) 52.71 ± 2.10

1.34 ± 0.04

137.56 ± 4.35

2.14 ± 0.05 48.77 ± 0.85*(16)

1.33 ± 0.01

150.06 ± 10.55

2.55 ± 0.06**(11) 17 days old

GT 1b GD 1b GD 1a GM 1 201.97 ± 7.2

302.68 ± 11.9

427.36 ± 13.7

222.9 ± 11.4 206.1 ± 17.94

321.3 ± 16.38

484.6 ± 20.6*(13)

197.7 ± 10.1 211.78 ± 8.99

276.45 ± 15.30

410.97 ± 17.98

187.69 ± 9.09 206.73 ± 13.07

318.17 ± 15.58

498.43 ± 24.56

227.53 ± 3.75 219.43 ± 14.81

185.43 ± 12.37**(39)

446.79 ± 15.43

215.35 ± 5.48 25 days old

GT 1b GD 1b GD 1a GM 1 428.14 ± 17.6

362.69 ± 14.2

559.09 ± 15.

298.39 ± 11.8 363.25 ± 17.4*(15)

364.56 ± 17.73

505.4 ± 13.15*

217.54 ± 20.9**(27) 338.2 ± 1.2*(21)

275.7 ± 29.79**

422.0 ± 27.03**

197.5 ± 8.63 298.71 ± 8.02**(30)

501.56 ± 24.75**

633.95 ± 40.72*

283.73 ± 20.56 354.42 ± 17.49*(17)

346.48 ± 48.73

513.53 ± 16.98

261.23 ± 8.9 Data from Tables 1 and 2 (pages 165-166); * p<0.05; ** p<0.01; (% ↑↓ ); gangliosides levels : nmol/brain; n=10

CONCLUSION: results suggest a delay in CNS development when pups exposed to 100 mg/kg/day (sc); at lower dose, brain capable of triggering biochemical mechanisms producing a plasticity response, which is expressed as changes in ganglioside content and composition.

COMMENT/ HED : The cited study is one of several research stud ies from the same group of investigators looking into possible mechanism of 2, 4-D neur otoxicity. The dose levels would be ex pected to exceed the threshold level for saturation of renal clearance mechanism; route of exposure (sc) to pups is not relevant to human health pesticide risk assessment. An extended 1-generation toxicity study on 2, 4-D, in which offspring were exposed to 2, 4-D during gestation, lactation via the milk, and directly via the diet through PND 22, 70, 139, includes an assessment of developmental neurotoxicity and neuropathology (includi ng an evaluation of myelin in the PND 22 offspring following exposure during gestation and lactation) at dose le vels that do not saturate metabolic processes (described previously). There were no treatment-related delays in CNS development at dose levels approaching renal clearance saturation. No treatment-related microscopic changes in the central or peripheral nervous systems in the perfused offspring or changes in myelin, or changes in microscopi c measurements of structures in the cerebral cortex, cerebellum, thalamus, or hippocampus were observed. The no observed adverse effect level (NOAEL) for developmental neurotoxicity was established at 59 mg/kg/day. This NOAEL is greater than the Points of Departure (PODs) utilized for the human health risk assessment supp orting the 2,4-D RED. The PODs used in the RED risk assessment are protective of chronic eff ects identified in the extended 1-generation study and of those identified in the cited study. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. The extended 1-generation reproduction study on 2, 4-D confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NYS DOH: identified as additional studies that should be considered in any new assessment along with those identified in the NRDC petition.

(Study 102) Citation : Sturtz N, Bongiovanni B, Rassetto, M.; Fe rri, A.; Evangelista de Duffard A.; and

### Page 110 of 186 Duffard, R. (2006). Detection of 2, 4-dichlorophenoxy acetic acid in rat milk of dams exposed during lactation and milk analysis of their major components. Food & Chem Toxicology 44, 8-16. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Wistar rats: 0, 15, 25, 50, or 70 mg/kg/day in diet to dams during PND 1-16; milk collected on PND 16; serum samples from dams and pups on PND 16; effect on milk components assessed

RESULTS: 2,4-D can affect the composition of milk; dose-dependent reduction of PUFAs; these significant changes in milk fatty acid composition indicate a disturbance in the supply of PUFAs to the neonate, which could alter normal development of the brain.

Dietary exposure of dams at 15, 25, 50, or 70 mg 2,4-D/kg/day resulted in comparable reductions in total lipids in PND 16 maternal milk at the 3 highest dose levels ( ↓ 29.6%, 27.8%, and 31.4% at 25, 50, and 70 mg/kg/day, respectively); ↓ pup body weight gain at all dose levels ( ↓ 11.4%, 11.6%, 17.8%, 16.7%, respectively, on PND 16); decrease in BWG evident from PND 6-16; residues of 2,4-D in dam and pup

serum and milk at each dose level were provided and show that milk content is greater than in maternal serum; pup serum levels are ≈ 30% (24-32.9%) of the dams’ levels

amount to dam amount in milk A amount in dam serum A amount in pup serum A serum ratio d/p
15 mg/kg/day 28.92±3.04 26.09±4.24 6.34±1.68 4.12 (4.56) B
25 mg/kg/day 59.56 ± 12.03 38.18 ± 10.59 11.52 ± 3.01 3.31 (5.17)
50 mg/kg/day 86.69±22.04 51.30±10.70 16.90±3.24 3.04 (5.13) 70 mg/kg/day 115.08±21.26 74.23±11.54 18.96±4.50 3.92 (6.07)

A μg /mL of sample; B ( ratio of amount in milk to amount in pup serum)

CONCLUSIONS: Maternal dietary exposure during lactation results in damage to pups’ nutritional status; 2,4-D produced a dose-related reduction of polyunsaturated fatty acids (PUFAs); significant changes in milk fatty acid composition indicating a disturbance in the supply of PUFAs to the neonate, which can alter normal developmental of the brain.

COMMENT/ HED : The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Provides data following oral doses to dam that are below, at, and above renal clearance capacity; do not know dose to pup, but provides information regarding oral dose to dam and amount in milk (dose to nursing pups) and resultant amount of 2,4-D in pup serum. Although a decrease in pup body weight gain was observed at all dose levels, the Agency notes that the cited paper only provides pup body weight gain data (in the form of a graph), and data on pup body weights were not provided. Additionally, the lack of body weight gain is not necessarily considered an

adverse effect. It is a calculated value and can be misl eading. For example, if the control and treated groups both show a body weight of 50.9 grams on day X and the control group gains 1.1 grams while the treated group gains 0.8 gram, this would show a 24% decrease in body weight gain for the treated group compared to the control. However, 52 grams vs 51.7 grams is a 1% difference in body weight. Preliminary studies performed in conjunction with the extended 1-generaion reproductive toxicity study also provide data with respect to the 2, 4-D levels in the plasma of pups and dams and levels in the milk. In the extended 1- generation reproductive toxicity study of 2, 4-D (described previously), there were no treatment-related delays in CNS development at dose levels appro aching renal clearance capacity. No treatment-related microscopic changes in the central or peripheral nervous systems in the perfused offspring or changes in myelin, or changes in microscopic measurements of st ructures in the cerebral cortex, cerebellum, thalamus, or hippocampus were observed. The no adverse effect level ( ≈ 30 mg/kg/day) for decreased pup body weight identified in the extended 1-generation study is greater than in the cited study. An exposure pathway identified in the NRDC petition is lactational exposure to 2, 4-D. The Agency cu rrently is addressing the issue of a lack of information regarding the levels of 2, 4-D that can occur in human breast milk. Preliminary studies performed to determine the doses to be used in the extended 1-generation reproductive toxicity study provide data on the levels of 2, 4-D in the milk of the dams and plasma levels of both the dams and offspring. Currently, it cannot be determine what the actual dose to each pup was due to the lackof information on the amount of milk ingested by the pups. However, the doses in the extended onegeneration reproduction study were adjusted during th e lactational period to prevent excessive dosing both to the maternal rat and to the pups in early lactati on and due to a “double expo sure” when pups are both nursing and starting to consume diet. Adjustments to the diet were also performed in the Sturtz study, although the procedures used were different and may, to some extent, explain the apparent differences in

### Page 111 of 186 the Sturtz study compared to the extended one-generation reproduction study. The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency. Additionally, the results in the cited study do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D

CITED BY NRDC (60) – NRDC says study confirms lactational exposure; decreased offspring body weights where dam exposed to 15 mg/kg/day (LOAEL) for 16 post natal days; 2,4-D accumulated by about 1.6 fold in maternal milk; nutritional content of milk altered (fat content dropped significantly; altered some proteins in maternal milk); suggests a lower NOAEL than that being used for short-term (25 mg/kg/day).

CITED BY Task Force – results not duplicated in recent rat dietary life-stage study (Marty, 2009);

demonstrated ↓ pup BW only when dams treated with 2,4-D ≥ 800 ppm in diet (premating through PND 21 (52, 61, 71-117 mg/kg/day); no effects at 400 ppm (25, 28, 37-57 mg/kg/day); at 800 ppm, 2,4-D PK behavior in lactating dams was distinctly non-linear (Saghir, 2009)

(Study 103) Citation : Sturtz, N.; Evangelista de Duffard A.; and Duffard, R. (2000). Detection of 2, 4- Dichlorophenoxy Acetic Acid (2,4-D) Residues in Neonates Breast-Fed by 2,4-D Exposed Dams. NeuroToxicology 21 (1-2): 147-154. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Wistar rats: dams administered 2,4-D (0, 50, 70, or 100 mg/kg/day) intermittently (i.p.), every two days, to obtain a maintained 2,4- D neonates’ exposure; litters culled to 8 pups/dam; exposure from PND 1-15 or PND 1-9 (then dams tr eated only with DMSO until pups were 15 days old). Measured levels of 2,4-D in kidney, blood, stomach, and brain; pups exposed via milk Results Summary: Decreased pup body weight was observed at all dose levels by PND 7, and the 100 mg/kg/day pups failed to gain weight from PND 7-13 when body weight loss was evident. On PND 16, pup body weights were 70%, 65%, and 40% of control at 50, 70, and 100 mg/kg/day, respectively. Brain weights were decreased significantly at the 70 and 100 mg/kg/day dose levels on PND 16. The quantity of 2, 4-D in the blood of the dams on PND 16 (24 hours after last dose) was 4.21, 7.02, and 15.46 μg/mL at the 50, 70, and 100 mg/kg/day. A comparison of the magnitude of the residues of 2, 4-D in the pup brain between the 50 and 100 mg/kg/day dose levels on PNDs 4 and 8 shows a 5-fold difference, whereas the same comparison at PND 16 sh ows a 2.6-fold difference.

Table 1. Organ Weights of Neonates Breast Fed by 2,4-D Treated Dams

Tissue Dose Age of neonates (days)

4 8 16

Brain 50

70

100 -

-

- ↓ 13.5% ↓ 16% ↓ 24% - ↓ 13.9% ↓ 323% Kidney 50

70

100 -

-

- ↓ 33.4% ↓ 39.5% ↓ 28.6% -

-

≈ 50% N=16pups/group, data from Figure 1 (page 149)

Table 2. 2,4-D Residues (μg/g) in Neonat es Breast Fed by 2,4-D Treated Dams

Tissue Dose Ag of neonates (days)

4 8 6

Brain 50

70

100 0.58 ± 0.16

0.98 ± 0.19

2.94 ± 0.81 1.27 ± 0.41

2.84 ± 0.94

6.45 ± 1.33 2.28 ± 0.88

3.82 ± 1.65

5.95 ± 3.24

Blood 50

70

100 2.56 ± 0.64

4.98 ± 0.38

6.77 ± 2.3 2.01 ± 0.33

4.18 ± 0.67

25.33 ± 7.86 8.06 ± 5.92

20.54 ± 13.87

30.65 ± 10.30 Kidney 50

70

100 6.82 ± 1.29

18.54 ± 1.41

12.39 ± 2.92 6.87 ± 1.31

15.84 ± 2.78

79.01 ± 13.92 9.85 ± 2.68

18.62 ± 6.76

91.18 ± 20.53

Stomach content 50

70

100 8.83 ± 2.26

19.17 ± 6.15

42.16 ± 11.53 17.98 ± 1.49

57.73 ± 1.19

100.97 ± 19.44 27.57 ± 12.90

77.03 ± 37.91

114.09 ± 32.45

### Page 112 of 186 N=16 pups/group, data from Table 1 (page 150)

Table 3. 2,4-D Residues (μg/g) in Neonates Breast Fed by 2,4-D Treated Dams (100 mg/kg/day)

Age of neonate Tissue Brain Blood Kidney Stomach content

8 11 (2) 13 (4) 14 (5) 15 (6) 16 (7) 6.45 ± 1.33 [100]

0.64 ± 0.52 [9.92]

0.50 ± 0.14 [7.64]

0.29 ± 0.08 [4.49]

0.28 ± 0.02 [4.34]

0.27 ± 0.01 [4.18] 25.33 ± 7.86 [100]

11.7 ± 5.94 [46.2]

9.92 ± 3.66 [39.2]

8.76 ± 2.73 [34.58]

4.33 ± 0.54 [17.1]

2.52 ± 2.08 [9.95] 79.01 ± 13.92 [100]

6.19 ± 0.18 [7.83]

3.57 ± 0.89 [4.52]

1.91 ± 0.23 [2.41]

0.89 ± 0.09 [1.12]

0.58 ± 0.39 [0.73] 100.97 ± 19.44 [100]

10.52 ± 4.36 [10.4]

10.55 ± 4.48 [10.44]

0.55 ± 0.21 [6.49]

6.24 ± 2.76 [6.18]

2.92 ± 2.12 [2.89] N=16 pups/group; (days after withdrawal); [% of 2,4-D content]; data from Table 2 (page 151)

CONCLUSION: Residues of 2,4-D in stomach content, blood, brain, kidneys of PND 4 pups and older; residues were dose- and exposure-time dependent; at 100 mg/kg/day, decreased pup BW and tissue weight and diminished stomach content; demonstrates that 2,4-D is transferred to neonates and decreased body and organ weights could be due to a diminished milk intake or/and to the direct 2,4-D toxic effect; when 2,4-D withdrawn from dams, 2,4-D residues remained in stomach content for at least a week

COMMENT/ HED : Study provides dose-response information at dose levels at or above level for saturation of renal clearance mechanism. Although the dams were dosed i.p., pups were exposed via the milk, although the dose to the pups is not known; however, this study, in conjunction with another Sturtz study, may provide information for determining pup do se. Additionally, preliminary results submitted to the Agency to support the dose levels selected fo r the extended 1-generation reproduction study provide similar exposure values to the pups. At this time, it is not possible to calculate the dose to the pup from exposure to 2, 4-D via the milk during lactation, since the amount of milk each pup receives is not known. The extended 1-generation reproductive toxicity study provided numerous detailed measures on the offspring following exposure across all life stages, including via the milk during lactation. The LOAEL for PND 22 offspring is based on decreased body weight, which was observed at the highest dose tested (600 ppm-800 ppm; currently, the dose on a mg/kg/day basis has not been calculated). The intended mg/kg/day dose to the parental rats at these ppm levels was 30-40 mg/kg/day.

The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NRDC (59) – cited as showing exposure to 2,4-D through maternal milk; when maternal exposure stopped, 2,4-D continued to be excreted in maternal milk for a week; EPA failed to include any lactational exposure in its aggregate risk assessment

CITED BY TASK FORCE – 2,4-D treatment rates to lactating rats were at doses in excess of saturation of renal clearance of 2,4-D; toxicity observed in the resulting non-linear pharmacokinetic range does not inform human risk assessment, beca use nonlinear doses are exceedingly unlikely to occur in humans.

CITED BY NCASI: petition fails to mention that dose leve ls likely exceed renal clearance levels and route of exposure is not appropriate

(Study 104) Citation : Sturtz, N.; Deis, RP; Jahn, GA; Duffard, R; and Evangelista de Duffard A (2008). Effect of 2, 4-Dichlorophenoxyacetic acid (2,4-D) on rat maternal behavior. Toxicology 247: 73-79. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: Wistar rats: dams administered 2,4-D via diet (0, 15, 25, 50 mg/kg/day); litters 8 pups/dam; exposure from PND 1-7; on PNDs 5 and 7, dams were tested for maternal behavior in home cage; maternal behavior parameters: nursing behavior with retrieval, crouching, licking (of whole body of pup), anogenital licking, time spent out of nest and eatin g; also measured latency and length of retrieval, defined as the time elapsed between the beginning of experiment and the retrieval of the first and last pups, respectively; latency of crouching (time elapsed between the beginning of experiment and the moment when mother assumed a crouching position with pups under her). Serum prolactin levels; endogenous monoamines (noradrenaline (NA), dopamine (D A), 3,4-dihyrdoxyphenyl acetic acid (DOPAC), homovanillic acid (HVA), serotonin (5 -HT), and 5-hydroxyindolacetic aci d (5-HIAA) and their metabolites

### Page 113 of 186 determined. Results Summary: maternal behavior impaired; pup care parameters, such as licking and grouping of pups

were decreased and latencies of retr ieval and crouching were increased, as was time spent out of nesting area; however, pups did not show body-weight loss during testing period, probably because milk supply was normal. Serum prolactin levels were diminished by 61%, 67%, and 71% at 15, 25, and 50 mg/kg/day, respectively; drastic decrease in in dolamine levels (5-HT, 5-HIAA) in arcuate nucleus (AcN) of treated dams; increased catecholamine levels.

Table 1. Maternal Be havior Parameters Parameter 0 mg/kg/day 15 mg/kg/day 25 mg/kg/day 50 mg/kg/day

PND 5 # pups retrieved 8 8 6.7 ± 0.3 8

Latencies of retrieval 17.3 37.9 ± 4.4* 40.2 ± 4.4** 38.7 ± 3.7* Length of retrieval of 8 pups 54.0 ± 2.9 80.8 ± 4.2*** 84.7 ± 6.4*** 88.0 ± 2.9*** # pups in nest 6.4 ± 0.9 5.1 ± 1.0 4.9 ± 1.0 4.2 ± 1.1 Latencies of crouching (seconds) 216 ± 30 348 ± 32** 328 ± 33* 528 ± 29*** % dams licking pups 100 50*** 42*** 16*** % dams licking ano-genital region of

pups 83 0*** 0*** 0***

% dams leaving nest 25 100*** 100*** 100*** Time spent out of nest 13 ± 8 132 ± 10*** 148 ± 8*** 138 ± 11*** % dams going out to eat 0 17 25 33

PND 7 # pups retrieved 8 8 7.6 ± 0.2 8

Latencies of retrieval 26.2 ± 4.8 59.3 ± 6.1*** 66.5 ± 7.8*** 78.7 ± 7.3*** Length of retrieval of 8 pups 48.9 ± 3.1 87.5 ± 5.7** 95.3 ± 5.2*** 93.5 ± 5.6*** # pups in nest 8 2.8 ± 1.1* 1.7 ± 0.9* 1.1 ± 0.7** Latencies of crouching (seconds) 222 ± 26 332 ± 40* 428 ± 37*** 600 ± 0*** % dams licking pups 100 0*** 0*** 0*** % dams licking ano-genital region of

pups 75 0*** 0*** 0***

% dams leaving nest 42 67* 83*** 100*** Time spent out of nest 26 ± 12 136 ± 23*** 141 ± 24*** 120 ± 10*** % dams going out to eat 0 17 25 17 Data from Table 1 (page 76)

Table 2. AcN Catecholamines and Indoleamines Leve ls in Control and 2,4-D-Treated Dams on PND 7 0 15 mg/kg/day 25 mg/kg/day 50 mg/kg/day

NA

DA

DOPAC

HVA

5-HT

5-HIAA 72.0 ± 6.5

35.5 ± 1.5

10.4 ± 0.7

14.5 ± 1.7

252.1 ± 3.0

85.8 ± 3.5 49.9 ± 4.5

41.3 ± 2.0

9.4 ± 2.1

25.8 ± 3.2* ( ↑ 78)

71.1 ± 1.9***( ↓ 72)

59.7 ± 2.5*** ( ↓ 30) 50.6 ± 4.5

49.9 ± 1.5***( ↑ 40)

17.2 ± 1.8*( ↑ 65)

25.5 ± 2.1*( ↑ 76)

40.4 ± 1.7***( ↓ 84%)

40.6 ± 1.5***( ↓ 53) 51.1 ± 8.4

51.7 ± 1.9***( ↑ 46)

18.6 ± 0.9**( ↑ 79)

28.1 ± 2.4 ( ↑ 94)

30.9 ± 0.9***( ↓ 88)

38.7 ± 2.7*** ( ↓ 55) * p<0.05; ** p<0.01; *** p<0.001; ↓↑ (%); fmol/mg wet weight; data from Table 2 (page 77)

CONCLUSIONS: differences observed at all dose levels (latencies of retrieval; latencies of crouching; increased time for retrieval of 8 pups; decreased % dams licking pups; none of 2,4-D dams licked anogenital area of pups; incr eased time spent out of nest); decreas ed 5-HT and 5-HIAA (all doses, DR); increased DA, DOPAC (25 and 50 mg/kg/day); decreased HVA (all doses).

COMMENT/ HED: The cited study is one of several research studies from the same group of investigators looking into possible mechanism of 2, 4-D neurotoxicity. Doses of 15, 25, 50 mg/kg/day are all at or below level of renal clearance saturation. Duration of expo sure to the dams was 7 days (PND 1-7), with an assessment of behavior and monoamines, etc. following 5 and 7 days of exposure via the diet. Prolactin

levels have been related to the onset and maintenance of maternal behavior in rats. Serum levels of prolactin were measured on PND 7 and were found to be significantly reduced by 2,4-D treatment at all dose levels (61%, 67%, and 71% at 15, 25, and 50 mg/kg/day, respectively). Maternal behavioral

### Page 114 of 186 assessment is one parameter assessed in the extended 1-generation reproductive toxicity study (described previously) in which the dams were exposed pre-mating, throughout gestation and lactation via the diet at dose levels of 5, 15, and 40 mg/kg/day. No treatment-related adverse effects were observed on parental behavior or on any of the numerous parameters evaluated to assess reproductive toxicity potential following oral exposure to 2, 4-D. Monoamines were not monitored in the extended 1-generation reproductive toxicity study. The cited study identifies an effect of 2, 4-D on maternal behavior towards her offspring that were associated w ith decreased 5-HT and increased DA levels in the AcN and decreased circulating prolactin following exposure of the dams to 2, 4-D for 5 and 7 days after the birth of their litters. Maternal neglect was not observed in the extended 1-generation reproductive toxicity study at dose levels at or approaching renal clearance saturation. The cited study provides an endpoint for consideration in the overall weight of evidence, although it does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY Beyond Pesticides –Changes to maternal behavior in rats, along with increased catecholamine levels and decrease in indolamine levels are among effects that need to be considered along with the scientific literature, which abounds with evidence that 2,4-D is a dangerous chemical

CITED BY Supplement to NRDC petition indicates that study shows that even short-term exposure to 2, 4- D at dose levels below toxic levels severely inhibits normal maternal hormones in brain and thereby adversely affects maternal behavior.

CITED BY NYS DOH – one of studies cited as additional studies that have accumulated since the RED, in addition to those in NRDC petition, which should be considered in any new 2, 4-D assessment.

(Study 106) Citation : Ton, C.; Lin, Y.; and Willett, C. (2006). Zebrafish as a Model for Developmental Neurotoxicity Testing. Birth Defects Research (Part A) 76: 533-567. [ Human Studies Considerations Apply: No ] Principal Endpoint : [Bin: Neurotoxicity] Methods Summary: NA - screening tool assessment; assay model; Results Summary: sublethal exposure to 2,4-D in fish tank water showed specific neurotoxicity; caused significant increase in brain cell death (apoptosis), disrupted motor neuron growth at high concentrations, decreased motility at moderate concentrations; slightly teratogenic in zebrafish;

CONCLUSION: 2,4-D showed specific neurotoxicity (induction of apoptosis or necrosis is an indicator of neurotoxicity) in zebrafish; zebrafish is a predictive animal model for neurotoxicity screening

COMMENT/ HED : 2,4-D was used as a one of several well-characterized compounds to examine several parameters of neurotoxicity during development (screening tool to identify potential neurotoxic compounds). Study is a screening assay and not directly applicable to human hea lth risk assessment. These data suggest that zebrafish may be an appropriate a lternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pesticides and aid in identification of compounds that should be further tested in mammalian systems. Neurotoxicity is already identified as a potential endpoint for 2, 4-D and more appropriate studies have been performed to assess 2, 4-D’s developmental neurotoxicity potential. In the extended 1-generation reproduction study on 2,4-D (MRID 47972101), young adult rats were administered 2, 4-D via the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly (via the diet) following weaning. Offspring were subjected to a developmental neurotoxicity (DNT) assessment, which included functional observational battery (FOB), motor activity and acoustic startle response (ASR). An evaluation of myelin in the PND 22 offspring following exposure during gestation and lactation was performed. On PND 60, the young rats were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation, myelination assessment, and brain morphometry. The extended 1-generation reproductive toxicity study on 2, 4-D was performed at three dose levels up to the saturation level for renal clearance. At the highest dose tested, there was no evidence of deve lopmental neurotoxicity and no adverse effect on myelination.

### Page 115 of 186 The cited study does not identify a more sensitive endpoint for human health than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D. Additionally, the extended one-generation reproduction study confirms the protectiveness of the existing Points of Departure as to neurotoxic effects.

CITED BY NRDC (32): evidence of neurotoxicity; sublethal exposure to 2,4-D in fish tank water significantly increased brain cell death, disrupted motor neuron growth, and decreased motility; in mammals, 2,4-D has been shown to exert related developmental neurotoxicity, including decreased motor activity and Parkinson’s like tremors.

CITED BY Task Force: a method development exercise; lowest conc affecting motor neuron growth

exceeded LC50 for zebraf ish; not appropriate

### Page 116 of 186 BIN: ENDOCRINE

(Study 5) Citation : Blair, R.M., Fang, H., Branham, W.S., Hass,, B.S., Dial, S.L., Moland, C.L., Tong, W., Shi, L., Perkins, R., Sheehan, D.M. (2000). The Estrogenic Recep tor Relative Binding Affinities of 188 Natural and Xenochemicals: Structural Diversity of Li gands. Toxicological Sciences 54: 138-154. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: a validated (standardized) estrogen recepto r (ER) competitive-binding assay was used to determine the ER affinity; uteri from ovariectomi zed Sprague-Dawley rats were the ER source for the competitive binding assay; chemicals were screened at high concentrations to determine whether chemical competed with [ 3 H]–estradiol for the ER. Those with affinity were assayed using a wide range of

concentrations to characterize the binding curve and to determine each chemical’s IC and relative binding affinity (RBA) values. Results Summary: 2,4-D (99%) was reported as negative in this investigation

CONCLUSION: 2,4-D in this test system did not exhibit any estrogenic activity at the concentrations

tested.

COMMENT/ HED : Study examined the effects of 2, 4-D in a validated (standardized) estrogen receptor (ER) competitive-binding assay, which was used to determine the ER affinity. 2, 4-D was one of many chemicals that were screened at high concentrations to determine whether the chem ical competed with [3H]–estradiol for the ER. 2, 4-D did not exhibit any estrogenic activit y at the concentrations tested. The Agency used an exposure based approach to select

the first list of chemicals to receive Test Orders for the Endocrine Disrupt or Screening Program (EDSP) Tier 1 screening battery; 2,4-D was included in the list of chemicals. Additionally it should be noted that this study was cited by the Test Order recipient as Other Scientifically Rele vant Information (OSRI) in response to the Agency’s EDSP Test Order and was evaluated by th e Endocrine Disruptor Review Team (TXR No. 0055461). The EDRT concluded that while 2, 4-D as reported was negative in this investigat ion, no data were supplied to substantiate this conclusion, and this study did not meet Test Orde r requirements (TXR No. 0055461, page 17). EPA waived the Tier 1 tests most relevant to human endocrine effects for 2,4-D due to the availability of the extended one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacetic Acid (2,4-D) - Report of the Endocrine Disruptor Review Team - Test Order #: EDSP–031001-120” (December 20, 2010). ) EPA has received all required final study reports and data from the Ti er 1 battery of tests for 2,4- D, which include an in vitro Estrogen Receptor Binding Assay (890 .1250). The submitted EDSP Tier 1 tests will be considered with regard to potential en vironmental effects and the need for Tier 2 in vivo

studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., wh ether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the qu antitative endocrine risks posed by 2,4-D for humans given the availability of the extended one-generation reproduction study (an in

vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with
endocrine system endpoints



### . CITED BY Task Force: This is one of several studies referenced by the Task Force that they identified as demonstrating that 2,4-D does not bind to or activate estrogen re ceptors, contrary to NRDC’s assertions that 2, 4-D is a “potent” endocrine disruptor. 2 , 4-D did not exhibit any estrogenic activity at the concentrations tested.

(Study 42) Citation : Haddow JE, Palomaki GE, Allan WC, Williams JR, Knight GJ, Gagnon J, O'Heir CD, Mitchell ML, Hermos RJ, Waisbren SE, Faix JD, Klein RZ. (1999) . Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. The New England Journal of Medicine 341 (8): 549-555. [ Human Studies Considerations Apply: Yes Principal Endpoint : [ Bin: Endocrine] Methods Summary: thyrotropin was measured in stored serum samples collected from 25216 pregnant women between January 1987 and March 1990; 47 women located with serum thyrotropin concentrations

at or above the 99.7 th percentile of values for all pregnant women, 15 women with values between the 98 th

and 99.6 th percentiles, inclusive, in combination with low thyroxine levels, and 124 matched women with normal values; their 7-9 year old children were tested (15 tests) for intelligence, attention, language, reading ability, school performance, and visual-motor performance. Results Summary: children of the 62 women with high serum thyrotropin concentrations performed

### Page 117 of 186 slightly less well on all 15 tests; their full-scale IQ scores on Wachsler Intelligence Scale for Children averaged 4 points lower than those of the children of the 124 matched control women; 15% ha scores of 85 or less, and compared with 5% of the matched control children; of the 62 women with thyroid deficiency, 48 were not treated during pregnancy, and the full-scale IQ scores of their children averaged 7 points lower than the 124 matched control children; 19% had scores of 85 or less. Eleven years after the pregnancy, 64% of the untreated women and 4% of matched control women had confirmed hypothyroidism.

CONCLUSION: undiagnosed hypothyroidism in pregnant women may adversely affect their fetuses; therefore, screening for thyroid deficien cy during pregnancy may be warranted.

COMMENT/ HED : This is a general paper (not 2,4-D specific); describes children of women who had thyroid deficiency during pregnancy; assessed for vari ous abilities (15 tests of intelligence, attention, etc.); contrary to statement in NRDC petition, concern for pote ntial thyroid effects were identified in the 2005 2, 4-D RED; the Data Call-In issued for a 2-generation reproduction study on 2, 4-D specifically required examination of the thyroid.

The extended 1-generation reproductive toxicity study on 2,4-D (described previously) provides an assessment of the thyroid (thyroid weight, thyroid hormone levels, histopathology) of the adult and offspring. In the 2, 4-D extended 1-generation reproductive toxicity study in rats, the expected hormone response consistent with perturbation in thyroid function was seen in the GD 17 satellite dams. These findings correlated with the thyroid alterations histologically. There was a comparable increase ( ↑ 9%) in thyroid weights at the low and high dose levels but no change in thyroid weight at the mid dose. In the F1 PND 4 pups, there were decreases in T3 levels at the low ( ↓ 8%) and mid ( ↓ 13%) dose groups, but not at the high dose. Decreased T4 levels were seen at the mid ( ↓ 15) and high ( ↓ 12%) dose groups, but there was no dose response. TSH level was increased ( ↑ 19%) at the high dose. Thyroid weight and histopathology data were not collected for this group. In the F1 PND 22 offspring, there was a decrease in T4 ( ↓ 20%) only at the high dose. Thyroid weight and histopathology data were not collected for this age group. In the F1 PND 62-64 offspring, T3 level was increased ( ↑ 11%) only at the high dose. T4 levels were increased ( ↑ 19%) both at the mid and high dose levels. There was a dose-dependent increase in TSH levels at the mid ( ↑ 11%) and high ( ↑ 24%) dose levels. Female offspring (PND 70) displayed decreased absolute thyroid weights at the mid ( ↓ 9%) and high ( ↓ 5%) dose levels. No treatment-related histopathological lesions were seen in the thyroid glands of P1 rats or F1 offspring. The thyroid effects noted in the extended 1-generation reproductive toxicity study were considered to be ad aptive and not adverse. Additionally, no robust effects on auditory startle or statistically significant brain morphometric change s were seen in the DNT component of the extended one-generation toxicity study; findin gs otherwise expected as sequelae to any marked thyroid hormone deficiency. The no observed adverse effect level (NOAEL) for thyroid toxicity was

established at ≈ 40 mg/kg/day. This NOAEL is greater than the PODs utilized for the human health risk assessment supporting the 2,4-D RED. It is to be noted that 2, 4-D was tested in rats, which are much more sensitive to thyroid perturbations than are humans because the serum half-life of the thyroid hormone, thyroxine, is much shorter in rats (less than 1 day) than in humans (5-9 days). The 10X interspecies uncertainty factor applied to 2, 4-D to account for the possibility that humans are more sensitive than the test animals is therefore more than adequate to pr otect humans. The 10X intras pecies factor accounts for variability in sensitivity among species and gives protection for women with iodine deficiency.. The extended one-generation reproduction study confirms th e protectiveness of the existing Points of Departure as to thyroid effects.

CITED BY NRDC (19): cited in support of their conclusion that EPA failed to incorporate information on the endocrine disrupting effects of 2, 4-D; NRDC states that thyroid hormone plays a critical role in development of brain; slight thyroid suppression advers ely affects neurological de velopment in fetus, with lasting effects on child learning and behavior

(Study 49) Citation : Hurst MR, Sheahan DA. (2003). The potential for oestrogenic effects of pesticides in headwater streams in the UK. The Science of the Total Environment 301: 87-96. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: Yeast Estrogen Screen (YES) or yeast two-hybrid screen Results Summary: negative

### Page 118 of 186 CONCLUSION: human YES negative

COMMENT/ HED : This is an in vitro study, which cannot be directly extrapolated to the complex in vivo situation. The referenced study [yeast two-hybrid screen; Yeast Es trogen Screen (YES)] is a screening study in which 2, 4-D was reported as negative. The Task Force referenced this study to dispute NRDC’s claim that 2, 4-D is an endocrine disruptor. Concern for endocrine disruption was identified in the RED. The Agency used an exposure-based approach to select the first list of chemicals to receive Test Orders for the EDSP Tier 1 screening battery; 2,

4-D was included in this list of chemicals. This c itation was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (see TXR No. 0055461). EDRT did not accept yeast-based assays as other scientifically relevant information (OSRI) primarily because some test chemicals are unable to penetrate the cell wall of the yeast cells and this may result in a false negative. Another issue with these assays is cytotoxicity. Tier 1 screening assays are limited to ev aluating whether a substance is capable of interacting with the endocrine system and are not sufficient to determine whether a chemical may have an effect in humans that is similar to an effect produced by naturally occurring hormones. The fact that a substance may interact with a hormone system does not mean that when the substance is used, it will cause adverse effects in humans or ecological systems. EPA waived the Tier 1 tests most relevant to human endocrine effects for 2,4-D due to the availability of the extended one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacetic Acid (2,4-D) - Report of the Endocrine Disruptor Review Team - Test Order #: EDSP–031001-120” (December 20, 2010). ) EPA has received all required final study re ports and data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 te sts will be considered with regard to potential

environmental effects and the need fo r Tier 2 in vivo studies for effect s in wildlife. Although the submitted

Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in ma mmals), the studies will not affect EPA’s conclusionson the quantitative endocrine risks posed by 2,4-D for humans given the availability of the extended onegeneration reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints

**.** CITED BY TF: one of several studies referenced by TF to dispute NRDC’s clai m that 2,4-D is an endocrine disruptor; states that Agency has on several occasions rejected the position that data gathered under Endocrine Disruptor Screening Program is a prerequisite to a safety determination.

(Study 54) Citation : Jung J, Ishida K, Nishihara T. (2004). Anti-estrogenic activity of fifty chemicals evaluated by in vitro assays. Life Sciences 74: 3065-3074. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: Yeast two-hybrid assay system with rat estrogen receptor (ER) α and the coactivator, T1F2; introduced 2 expression plasmids, pGBT9-ERLBD and –GAD424-T1F2, into yeast cells ( Saccharomyces cerevisiae Y190). The yeast two-hybrid system transfected with a rat ER receptor, a transcriptional activation assay using MCF-7 ce lls, and an ER binding assay using fluorescence polarization. Results Summary: 2,4-D was negative

CONCLUSION: 2,4-D was not found to inhibit estrogen activity

COMMENT/ HED : 2,4-D is among the group of 58 pesticide active ingredient s on the initial list to be screened under EDSP (test orders issued). Concern for endocrine disruption was identified in the RED. The Agency used an exposure-based approach to select the first list of chemicals to receive Test Orders for the EDSP Tier 1 screening battery; 2, 4-D was included in this list of chemicals. This citation was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (see TXR No. 0055461). EDRT did not accept yeast-based assays as other scientifically rele vant information (OSRI) prim arily because some test chemicals are unable to penetrate the cell wall of the yeast cells and this may result in a false negative. Another issue with these assays is cytotoxicity. Th is citation was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (see TXR No. 00 55461, page 17). EDRT did not accept yeast-based assays as OSRI primarily because so me test chemicals are unable to penetrate the cell wall of the yeast cells and this may result in a false negative. Another issue with these assays is cytotoxicity. The test order recipient did not cite this study as OSRI. EPA waived the Tier 1 tests most relevant to human endocrine

### Page 119 of 186 effects for 2,4-D due to the availability of the extend ed one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacetic Ac id (2,4-D) - Report of the Endocrine Disruptor Review

Team - Test Order #: EDSP–0 31001-120” (December 20, 2010). ) EPA has received all required final study reports and data from the Tier 1 battery of te sts for 2,4-D. The submitted EDSP Tier 1 tests will be considered with regard to potential e nvironmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the quan titative endocrine risks posed by 2,4-D for humans given the availability of the extended one-generation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints.

CITED BY TF: one of several in vitro studies referenced by TF to dispute NRDC’s assessment of Xie, et al (2005) report that 2,4-D has “relatively potent estr ogenic effects in fish”; st ates that Agency has on several occasions rejected the position that data gath ered under Endocrine Disruptor Screening Program is a prerequisite to a safety determination.

(Study 55) Citation : Jungbauer A, Beck V. (2002). Yeast reporter system for rapid determination of estrogenic activity. J. Chromatography B 777: 167-178. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: an in vitro test system for the determination of estrogens, xeno- and phytoestrogns, based on the activation of human estrogen receptor- α , was examined for ability in monitoring environmental estrogens; system consists of an expression plasmid for the human estrogen receptor- α and a reporter plasmid containing the lacZ under the contro l of the vitellogenin hormone response element. These plasmids have been transformed into S. cerevisiae. Cultivation of yeast in the presence of estrogenic substances leads to activation of the estrogen recept or and induces the expression of the reporter lacZ. Β

Galactosidase activity of the translated gene lacZ is a measure of the estrogenic activity of a compound. Results Summary: 2,4-D – no activity (reference cited is Soto et al., 1995);

CONCLUSION: 2,4-D no activity (E-Screen);

COMMENT/ HED : paper cited data from Soto, et al., 1995. As noted above, 2,4-D was included on the list

of chemicals to receive test orders for the Endocrin e Disruption Screening Program (EDSP) Tier 1 battery. This citation and the paper by Soto, et.al., 1995 were included in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461, page 17). Th e concentrations were not identified nor were data provided in this citation. This study was not cited as OSRI by the test order recipient. EDRT did not accept yeast-based assays as OSRI primarily because some test chemicals are unable to penetrate the cell wall of the yeast cells and this may result in a false negative. Another issue with these assays is cytotoxicity. EPA waived the Tier 1 tests most relevant to human endocrine effects for 2,4-D due to the availability of the extended one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacetic Acid (2,4-D) - Report of the Endocrine Disruptor Review Team - Test Order #: EDSP–031001-120” (December 20, 2010). ) EPA has received all required final study re ports and data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 te sts will be considered with regard to potential

environmental effects and the need fo r Tier 2 in vivo studies for effect s in wildlife. Although the submitted

Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in ma mmals), the studies will not affect EPA’s conclusionson the quantitative endocrine risks posed by 2,4-D for humans given the availability of the extended onegeneration reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints.

CITED BY TF: listed in table of 11 assays that indicate that 2,4-D is negative for estrogenicity; one of

several in vitro studies referenced by TF to dispute NRDC’s assessment of Xie, et al (2005) report that 2,4- D has “relatively potent estrogenic effects in fish”.

(Study 56) Citation : Kim HJ, Kim WD, et al. (2002). Mechanis m of phenoxy compounds as an endocrine

### Page 120 of 186 disruptor. J Toxicology Public Health 18:331-339. (in Korean ). [ Human Studies Considerations Apply:

No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: Dose of 50 mg 2,4-D/kg po gavage for 10 days; dose of 100 mg DCP/kg po gavage for 10 days; an androgenicity assay system (Hershberg er assay), to assess potential endocrine disruption; 2,4-D/DCP may act as androgenic endocrine disruptors by binding to androgen receptor, as well as by inhibiting metabolism of testosterone. Results Summary: administration of both compounds to rats caused an increase in tissue weight of ventral prostate, Cowper’s gland, and glands penis; these increases were additively potentiated when rats were simultaneously treated with low dose of testosterone (1 g/kg s.c.)

CONCLUSION: results collectively suggest that 2,4-D a nd DCP may act as androgenic endocrine disruptors by binding to the androgen receptor as we ll as by inhibiting the metabolism of testosterone.

COMMENT/ HED : concern for endocrine disruption was identifie d in 2005 RED. This article was written in Korean and was not available to the Agency in English. The Agency identified concern for potential endocrine effects in the 2005 RED. In response to this and other concerns raised, the registrant conducted the extended one-generation reproductive toxicity study. This study measures several key endpoints. These endpoints include an evaluation of potential effects on parental male and female reproductive function offspring, survival, growth including endocrine and systemic toxicity parameters such as estrous cyclicity (female adult rats and offspring), evalua tion of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and balano-preputial separation), and thyroid gland weight, hormone and histopathology assessment. In the extended 1-generation reproductive toxicity study on 2,4-D (MRID 47972101) submitted in response to the data ca ll-in, young adult rats were administered 2, 4-D via

the diet for ≈ four weeks prior to mating and continuing through mating, gestation, and lactation at dose levels up to those that saturate metabolic processes. The young rat was exposed during gestation, lactation ( via the milk), and directly ( via the diet) following weaning. Paramete rs assessed include sperm parameters,sex gland weights and histopathology; AGD, nipple retention; puberty timing. There were no treatmentrelated, adverse effects on any of the parameters assessed, including prostate and other sex organ weights



### . The citation was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461). EDRT concluded that this citation cited as OSRI did not satisfy the requirement for the Androgen Receptor Binding Assay (G uideline 890.1150). The requiremen t for the Hershberger Assay is satisfied based on the extended 1-generation reproductive toxicity study (MRID 47972101) of 2, 4-D (TXR No. 0055461, page 37).

CITED BY NRDC (24) – paper cited as evidence of endocrine disrupting effects of 2,4-D; exposure to 2,4- D caused increases in prostate size in laboratory rats

CITED BY TF: Effect on prostate occurred at dose known to be above threshold for saturation of renal clearance; in vitro study of questionable value; effects of dietary 2,4-D treatment on prostate weight and pathology is being examined in the data call-in studies requested by EPA

(Study 57) Citation : Kim H-J, Park YI, Dong M-S. (2005). Effects of 2,4-D and DCP on the DHT-induced androgenic action in human prostate cancer cells. Toxicological Sciences 88 (1): 52-59. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: To elucidate the mechanism of the synerg istic effects of 2,4-D and DCP on the androgenicity of testosterone, the androgenic action of 2,4-D and DCP was characterized using a mammalian detection system in prostate cancer cell lines. 2,4-D (>98%); 2 human prostate cancer cell lines were used: AR expressed 22Rv1 and androgen-independent PC3; The 22Rv1 cells are derived from a relapsed tumor and have been characterized as a valuab le tool for the study of prostate cancer progression; a representative androgen-insensitive cell line PC3 was used for validation; Results Summary: 2,4-D treatment alone did not increase the proliferation rate of 22Rv1 cells; the combined effects of 2,4-D with DHT on prostate cell proliferation showed that whereas stimulation with

DHT alone showed a 1.5-fold increas e in cell growth rate, co treatment with 2,4-D exhibited a 2.5-fold increase in cell growth rate, which suggests to authors that 2,4-D acted either additively or synergistically with DHT on the proliferation of prostate cancer cells.

CONCLUSION: provides clear evidence for androgenic actions of phenoxy compounds using mammalian

### Page 121 of 186 system; enhance DHT-induced AR transcriptional activity; promotes proliferation of androgen-sensitive

cells

COMMENT/ HED : Kim et al., (2005) studied the ability of 2,4-D and its metabolite to increase the proliferation of 22Rv1 human pros tate cancer cells. Negative results were reported. Trancriptional activation studies were conducted with various cell lines. All were reported negative, except 2,4-D was reported to increase the response when coadministered with testos terone. NRDC cited the paper as evidence of endocrine disrupting effects of 2, 4-D, stating that the combination of 2, 4-D and testosterone resulted in synergistic increases in the proliferati on of prostate cancer cells. The Task Force commented

that this in vitro mechanistic study cited by NRDC is based on the earlier report that 2, 4-D increased prostate weight in rats following oral (gavage) exposure at a high dose above the threshold for saturation of renal clearance, which they state renders th e usefulness and interpretation of this in vitro study of questionable value to potential human risk

The Agency used an exposure-based approach to select the first list of chemicals to receive Test Orders for the EDSP Tier 1 screening battery; 2, 4-D was included in this list of chemicals. This study was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461); it should be noted that due to the limitations of these studies, the submitter did not claim them as OSRI.

Effects of dietary 2, 4-D treatment on prostate weight and pathology were examined in extended 1- generation reproductive toxicity study on 2, 4-D, and there were no treatment-related effects. The NOAEL (45 mg/kg/day) is greater than the PODs utilized for the human health risk assessment supporting the 2, 4- D RED. The objective of the EDSP Tier 1 battery assays is to screen for the potential to interact with the estrogen, androgen and thyroid hormone pathways. EPA waived the Tier 1 tests most relevant to human endocrine effects for 2,4-D due to the availability of the extended one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacet ic Acid (2,4-D) - Report of the Endocrine Disruptor Review Team - Test Order #: EDSP–031001-120” (December 20, 2010). ) EPA has received all required final study reports and data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 tests will be considered with regard to potential environmental effects and the need for Tier 2 in vivo

studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., wh ether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the qu antitative endocrine risks posed by 2,4-D for humans given the availability of the extended one-generation reproduction study (an in

vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints. The purpose of Tier 2 testing is to identify and establish a dose response relationship for any adverse effects that might results from interactions identifie d through Tier 1 screening. The Tier 2 mammalian study is currently the 2-Generation Reproduction and Fertility Effects Study (Note: the OECD Guideline for the Extended F1 One Generation Reproduction Test Guideline is also under consideration). The existing 2, 4-D hazard database includes an Extend ed F1 One Generation Reproduction Test Guideline.

CITED BY NRDC (23): paper cited as evidence of endocrine disrupting effects of 2,4-D; shows that the combination of 2,4-D and testosterone resulted in syne rgistic increases in the pr oliferation of prostate cancer cells, including up to a 32-fold incr ease in activation of these cancer cells.

CITED BY TF: this in vitro mechanistic study is based on their earlier report that 2,4-D increased prostate weight in rats at 50 mg/kg/day (oral gavage); use of high dose (above threshold for saturation of renal clearance) renders usefulness and interpretation of this in vitro study of questionable value to potential risk; states that effects of dietary 2,4-D treatment on prostate weight and pathology is currently being examined in extended 1-generation reproduction study

(Study 58) Citation : Kojima H, Katsura E, Takeuchi S, Niiyama k, Kobayashi K. (2004). Screening for estrogen and androgen receptor activities in 200 pesticid es by in vitro reporter gene assays using Chinese hamster ovary cells. Environm Hlth Perspect 112 (5): 524-531. [ Human Studies Considerations Apply:

No ] Principal Endpoint : [ Bin: Endocrine]

### Page 122 of 186 Methods Summary: CHO-K1 cells; reporter gene assays for human ER α (hER α ) and hER β and AR (hAR) expression Results Summary: 2,4-D not identified as showing estrogen or androgen receptor activities

CONCLUSION: 2,4-D did not show estrogenic effects or antiestrogenic properties in the hER α transactivation assay; did not show androgenic transcrip tional activity; did not show an inhibitory effect on the androgenic activity induced by DHT

**COMMENT/** HED **:** In an in vitro assay (hER α transactivation assay; reporter gene assays for human ER α (hER α ) and hER β and AR (hAR) expression), 2,4-D did not show estrogenic eff ects or antiestrogenic properties in the hER α transactivation assay; did not show andr ogenic transcriptional activity; and did not show an inhibitory effect on the androgenic activity induced by DHT. Concern for endocrine disruption potential was identified in the RED. The Agency used an exposure-based approach to select the first list of

chemicals to receive Test Orders fo r the EDSP Tier 1 screening battery; 2,4-D was included in this list of

chemicals. This is one of several studies cited as Other Scientifically Relevant Information (OSRI) in response to the EDSP Tier 1 Test Orders and by th e Task Force to refute the NRDC evidence of the endocrine disrupting effects of 2,4-D. This citation was evaluated as OSRI in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461, Table 7, page 35). The citation does not provide any data to substantiate the reported negative results. T ranscriptional assays do not measure receptor binding and involve post-binding events; therefore, they are not an adequate substitute for receptor binding assays. The F1-extended one generation reproductiv e toxicity study of 2,4-D evaluated several key endpoints. These endpoints include an evaluation of potential effects on parental male and female reproductive function, offspring survival, growth including endocrine and systemic toxicity parameters such as estrous cyclicity (female adult rats and of fspring), evaluation of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and balano-preputial separation), and thyroid gland weight, hormone and histopathology assessment. In addition the test guideline includes options toevaluate development of the immune and nervous systems. The findings for 2, 4-D for the extended onegeneration reproductive toxicity study determined ther e were no adverse effects observed on any of the numerous parameters evaluated, and the NOAEL was established at 45 mg/kg/day in males and 40 mg/kg/day in females. Both NOAELs are greater than the Points of Departure utilized for the human health risk assessment supporting the 2, 4-D RED. The extended 1-generation reproduction study confirms the protectiveness of the existing Points of Departure.

CITED BY TF: one of several studies cited that indicate that 2,4-D is negative for estrogenicity; human estrogen receptor-alpha, estrogen r eceptor-beta transactivation: negative

(Study 66) Citation : Lin, N.; Garry, VF (2000). In vitro studies of cellular and molecular developmental toxicity of adjuvants, herbicides, fungicides, Minnesota. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: YES: Yeast Estrogen Screen or yeast two-hybrid screen Results Summary: commercial-grade 2,4-D LV4 & 2,4-D amine induced MCF-7 cells; adjuvants induced proliferation of MCF-cells but at lower concentra tion; reagent-grade 2,4-D inactive; did not induce proliferation of MCF-7 cells

CONCLUSION: 2,4-D negative in this assay

COMMENT/ HED : The Agency used an exposure-based approach to select the first list of chemicals to receive Test Orders for the EDSP Tier 1 screening batte ry; 2,4-D was included in this list of chemicals.

This is one of several studies cited as Other Scientif ically Relevant Information (OSRI) in response to the EDSP Tier 1 Test Orders and by the Task Force to refute the NRDC evidence of the endocrine disrupting effects of 2,4-D. This citation was evaluated as OSRI in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461; Table 4, page 18). EDRT noted that a cell proliferation assay does not measure receptor binding and may be affected by factors other than binding with the receptor. Study may be useful supplementary information. EPA waived the Tier 1 tests most relevant to human endocrine effects for 2,4-D due to the availability of the extend ed one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacetic Ac id (2,4-D) - Report of the Endocrine Disruptor Review

Team - Test Order #: EDSP –031001-120” (Decemb er 20, 2010). ) EPA has received all required final study reports and data from the Tier 1 battery of te sts for 2,4-D. The submitted EDSP Tier 1 tests will be

### Page 123 of 186 considered with regard to potential e nvironmental effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on the quan titative endocrine risks posed by 2,4-D for humans given the availability of the extended one-generation reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints.

CITED BY TF: one of several studies cited that indicate th at 2,4-D is negative for estrogenicity; YES for 2,4-D ai was negative; YES formulated material, which may contain estrogenic surfactants was positive.

(Study 67) Citation : Liu, RCM; Hahn, C.; and Hurtt, ME (1996). The Direct Effect of Hepatic Peroxisome Proliferators on Rat Leydig Cell Function in vitro . Fundamental & Applied Toxicol. 30: 102-108. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] (cancer MOA) Methods Summary: Leydig cells, isolated from adult Crl:CDBR ra ts (12-16 weeks old), were treated with 2,4-D for 21 hours and medium was assayed for estradiol; function of treated Leydig cells was evaluated by measuring the release of testosterone in response to human chorionic gonadotropin (hCG); addressing MOA for Leydig cell adenomas and PP Results Summary: 2,4-D did not significantly reduce the hCG-stimulated release of testosterone (70% of C); concentration that decreased response to 50% of control response (IC50) testosterone was 1147 μM; basal estradiol production: estimated concentration that increased control response to half-maximum EC50 (μM) estradiol was 221

CONCLUSION: results suggest that peroxisome proliferators, as a class of compounds, directly modify the steroidogenic function of Leydig cells in vitro. Further investigations necessary to address the

mechanism for the in vitro effects on Leydig cells and to clarify the apparent relationship between peroxisome proliferator-induced changes in Leydig cell function and the development of Leydig cell

tumors.

COMMENT/ HED : The referenced study is an in vitro study designed to address the mode of action for Leydig cell adenomas and peroxisome proliferation. The results suggest that peroxisome proliferators, as a class of compounds, directly modify the steroidogenic function of Leydig cells in vitro . Investigators stated that further investigations were neces sary to address the mechanism for the in vitro effects on Leydig cells and to clarify the apparent relationship between peroxisome proliferator-induced changes in Leydig cell function and the development of Leydig cell tumors. This study is one of several studies cited by NRDC as the basis for their concern for potentia l endocrine disrupting effects of 2, 4-D. The Task Force stated that no significant effects in hCG-stimul ated release of testosterone were seen in rat Leydig cell cultures. 2,4-D is a known weak peroxisome proliferator and effects on reproductive organs/hormones were identified in RED; 2, 4-D is not associated with Leydig cell tumors. The citation is of ongoing research into the possible relationship between peroxisome proliferators and changes in Leydig cell function; 2, 4-D, as a known peroxisome proliferators , was used in this MOA assessment of peroxisome proliferators. Effects on reproductive hormones were identified in the RED and were addressed in extended 1-generation reproductive toxicity study (MRID 47942101). The F1-extended one generation reproductive toxicity study of 2,4-D evaluated several key endpoints. These endpoints included an evaluation of potential effects on parental male and female reproductive function, offspring survival, growth including endocrine and systemic toxicity parameters such as estrous cyclicit y (female adult rats and offspring), evaluation of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and balano-preputial separation), and thyroid gland weight, hormone and hi stopathology assessment. There were no adverse effects observed on any of the numerous parameters evaluated in the extended 1-generation reproductive study (TXR No. 0055341), and the NOAEL was established at 45 mg/kg/day in males and 40 mg/kg/day in females. Both NOAELs are greater than the Points of Departure utilized for the human health risk assessment supporting the 2, 4-D RED.

CITED BY NRDC (22): one of several studies cited that NRDC believes establishes the dangerous endocrine disrupting effects of 2,4-D that requires that 2,4-D be banned; 2,4-D causes slight decrease in testosterone release, significant increase in estrogen release from testicular cells;

### Page 124 of 186 CITED BY TF: states that n o significant effects in hCG-stimulated re lease of testosterone in rat Leydig cell cultures; indicates that NRDC fails to mention mini mal effect concentration fo r effect on estrogen was 500 μM, which translates to ≈ 110 μg/mL; is > max 2,4-D plasma conc reported in rats treated w/ 100 mg/kg/day (above saturation of renal clearance) (Saghir, et al, 2006); given 100 mg/kg is well above saturation of renal clearance, these in vitro findings ar e not considered indicative of an endocrine risk by

TF.

(Study 70) Citation : in Russian – Malysheva LN, and Zhavoronkov AA. (1997). Morphological and histochemical changes in the thyroid gland after a single exposure to 2,4-DA herbicide. Biull Eksd Biol Med 124 (12),676-677. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: not available for review Results Summary: as cited in BP: decreases in thyroid gl and transport and production functions and impairment of hormone iodination in thyroid were observed after acute exposure to rats

CONCLUSION: don’t have translation (in Russian).

COMMENT/ HED : The study is in Russian and no translation is available. There are no details regarding test methods or dose levels used, and no data are available to substantiate the reported effects. As cited by Beyond Pesticides, decreases in thyroid gland transport and production functions and impairment of hormone iodination in thyroid were observed after acute exposure to 2, 4-D. Thyroid effects were identified in the 2, 4-D RED and a Data Call-In was issued for a 2-generation reproduction study that specifically required examination of the thyroid. The F1-extended one-generation reproductive toxicity study of 2, 4-D provides an assessment of the thyroid following repeat exposure at adequate dose levels; i.e., an evaluation of thyroid hormones (T3, T4 and TSH), thyroid weight and histopathology of the thyroid across life stages, and an evaluation of auditory startle and brain morphometry data (reported to be sensitive to thyroid disruption). The thyroid toxicity NOAEL was established at ≈ 40 mg/kg/day, the highest dose tested. This NOAEL is greater than the Points of Departure utilized for the human health risk assessment supporting the
2, 4-D RED.

The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D

CITED BY BP - additional evidence that 2,4-D poses unreasonable risks to human health and environment; its registration must be revoked

(Study 73) Citation : McKinlay, R.; Plant, JA; Bell, JNB; et al. (2008). Endocrine Disrupting Pesticides: Implications for Risk Assessment. Environment International 34, 168-183. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: NA – review article Results Summary: cited paper by Kim, et al. (2005); Synergistic androgenic effects when combined with testosterone (Kim 2005) promotes pro liferation of androgen-sensitive cells

CONCLUSION: more data and research ne eded to quan tify exposure

COMMENT/ HED : The extended 1-generation reproductive toxicity study on 2,4-D includes a detailed assessment of endocrine endpoints [thyroid, estrus cyclicity, sexual maturation (delays in vaginal opening and preputial separation), andrology, ovarian staging]. 2,4-D was included on the list of chemicals to receive test orders for the Endocrine Disrupti on Screening Program (EDSP) Tier 1 battery. Antiandrogenicity or androgenicity sensitive parameters were evaluated in the F1-extended 1-generation reproductive toxicity study of 2, 4-D, which included: balano-preputial separation, anogenital distance, quantitative nipple retention, evaluation of sperm parameters, male reproductive organ weights and detailed histopathological evaluation of male reproductive organs

. There were no treatment-related adverse effects on any of the numerous parameters assessed across life stages. T he no observed adverse effect level (NOAEL) for androgenic effects was established at 45 mg/kg/ day. This NOAEL is greater than the Points of Departure utilized for the human health risk assessment supporting the 2,4-D RED.

### Page 125 of 186 The cited study does not identify a more sensitive endpoint for human health risk assessment than already identified by the Agency, and the results do not yield information that would cause the Agency to modify its conclusions from the 2005 RED for 2, 4-D

CITED BY BP: cited study as showing that 2,4-D promotes the proliferation of androgen-sensitive cells by acting synergistically with its main metabolite, DCP, also known for its endocrine disrupting effects

(Study 79) Citation : Nishihara T, J Nishikawa, T. Kanayama, F. Dakeyama, K. Saito, M. Imagawa, S.Takatori, Y. Kitagawa, S. Hori, h. Utsumi. (2000). Estrogenic Activities of 517 Chemicals by Yeast TwoHybrid Assay. Journal of Health Science 46 (4): 282-298. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: yeast two-hybrid assay system with estrogen receptor (Er α and the coactivator, T1F2; introduced 2 expression plasmids, pGBT9-ERLBD and –GAD424-T1F2, into yeast cells ( Saccharomyces

cerevisiae Y190), which carry a β -galactosidase reporter gene and require tryptophan and leucine for growth; results evaluated by relative activity, expre ssed as REC10 (10% relative effective concentration), that is the concentration of test compound showing 10% of agonist activity of 10 -7 M E2, which is the optimum concentration for E2. when the activity of test compound was higher thanREC10 within the concentration tested, chemical judged as positive. Results Summary: 2, 4-D negative (REC10 2,4-D >1 x 10 -3 )

CONCLUSION: positive chemicals demonstrate a common stru cture: phenol with a hydrophobic moiety at the para- position without bulky groups at the ortho- position. 2, 4-D was reported as negative (page 296)


### COMMENT/ HED : The referenced study is a yeast two-hybr id assay system, in which 2, 4-D was reported to be negative. When the Agency used an e xposure-based approach to select the first list of

chemicals to receive Test Orders for the EDSP Tier 1 screening battery, 2,4-D wa s included in the list of chemicals. This study was referenced in a document submitted to the Agency by the 2,4-D Consortium in response to the EDSP Tier 1 Test Order, however it was not cited as Other Scientifically Relevant Information (OSRI). This citation was included in th e Endocrine Disruptor Review Team assessment for 2, 4-D (TXR No. 0055461, page 18). In yeast-based assays some test chemicals are unable to penetrate the cell wall and this failure may result in a false negative. Another issue with these assays is cytotoxicity. EPA waived the Tier 1 tests most relevant to human endocrine effects for 2,4-D due to the availability of the extended one generation reproduction study with 2,4-D. (Ref. Office of Chemical Safety and Pollution Prevention, EPA, Memorandum from Greg Ackerman to Katie Weyrauch, “ 2, 4-Dichlorophenoxyacetic Acid (2,4-D) - Report of the Endocrine Disruptor Review Team - Test Order #: EDSP–031001-120” (December 20, 2010). ) EPA has received all required final study re ports and data from the Tier 1 battery of tests for 2,4-D. The submitted EDSP Tier 1 tests will be considered with regard to potential environmental

effects and the need for Tier 2 in vivo studies for effects in wildlife. Although the submitted Tier 1 in vitro studies may inform EPA on mechanistic issues in mammalian systems (e.g., whether 2,4-D can bind to the estrogen or androgen receptor in mammals), the studies will not affect EPA’s conclusions on thequantitative endocrine risks posed by 2,4-D for humans given the availability of the extended onegeneration reproduction study (an in vivo study in rats) that comprehensively examined the risks to human health from 2,4-D’s interaction with endocrine system endpoints.

CITED BY: 2,4-D Task Force: one of several in vitro studies referenced by TF to dispute NRDC’s assessment of Xie, et al (2005) report that 2,4-D has “relatively potent estrogenic effects in fish”; states that Agency has on several occasions rejected the position that data gathered under Endocrine Disruptor Screening Program is a prerequisite to a safety determ ination; yeast two-hybrid screen: negative (states that Xie, et al . , 2005, incorrectly indicated 2,4-D “estrogenic at a concentration of ≈ 0.2 g/L”).

(Study 83) Citation : Oakes, et al. (2002) A Study of the Potential for a Herbicide Formulation Containing 2,4-D and Picloram to Cause Male-Mediated Developmental Toxicity in Rats. Toxicological Sciences 68: 200-206. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] (Reproductive/Developmental) Methods Summary: male Sprague-Dawley rats gavaged for 5 days/week for 9 weeks with mixture (0.125,

### Page 126 of 186 0.25, or 0.5 mL/kg/day 2,4-D and Picloram); 3 dose leve ls (150, 75, 37.5 mg/kg/day 2,4-D; distilled water); + control: cyclophosphamide; each male mated with 2 untreated females during weeks 2 and 4, 4 and 5, and 8 and 9 of treatment, and with 4 untreated females after an 11-week recovery period; females sacrificed on GD 20 and fetuses were weighed and examined fo r either structural malf ormations or skeletal development. Results Summary: mean # corpora lutea, preimplantation lo ss (except 2-3 week exposure HD 15.2% vs 6.1% control), postimplantation loss, litter size, fetal weight, and malformation rate (except 2-3 week treatment 5.8%* HD vs 1.2% control) were unaffected.

CONCLUSION: results did not show any evidence that 2,4-D re peat exposure of male rats is likely to cause male-mediated birth defects in offspring or other adverse reproductive outcomes.

COMMENT/ HED : Study examines the effects of 2, 4-D on male rats following 9 weeks of exposure with a mixture of 2,4-D and picloram. The treated males were mated with untreated female rats, and the females were sacrificed on GD 20, and fetuses were weighed and examined for either st ructural malformations or skeletal development. The results did not show any evid ence that repeated exposure of male rats was likely to cause male-mediated birth defects in offspring or other adverse reproductive outcomes. Information provided in the report includes 2,4-D serum levels a ttained at 150 mg/kg/day dose level [daily dose Cmax values of 249-284 μg 2,4-D/mL serum]; serum half-life of ~6 hours; commercial applicators and forestry workers (no protection) estimated exposures ranging from 0.9 to 160 μg/kg/day, which represents exposure to 0.1-11 mg for 70 kg man (referenced Draper and Street, 1982; Kolmodin-Hedman and Erne, 1980; Lavy, et al, 1987; Nash, et al, 1982); in terms of estimated serum levels, men given an oral dose of 5 mg 2,4-D/kg (=350 mg for 70-kg man) had a Cmax of 40 μg/mL (Kohli, et al., 1974); an 11-mg intake would give an estimated plasma level of 1 μg/mL; negative study at doses up to 150 mg/kg/day 2,4-D, which exceeds

renal excretion mechanism. This citation was in cluded in the Endocrine Disruptor Review Team assessment of 2, 4-D (TXR No. 0055461, page 94), but was rejected in the EDRT’s weight of evidence.

Endocrine/reproductive concerns were identified in the RED and are addressed in the extended 1- generation reproductive toxicity study. The F1-extended one generation reproductive toxicity study of 2, 4- D evaluated several key endpoints. These endpoints included an evaluation of potential effects on parental male and female reproductive function, offspring survival, growth including endocrine and systemic toxicity parameters such as estrous cyclicity (fema le adult rats and offspring), evaluation of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and balano-preputial separation), and thyroid gland weight, hormone and histopathology assessment. In addition the test guideline includes options to evaluate development of the immune and nervous systems. There were no adverse effects on any of the numerous parameters monitored in the extended 1-generation reproductive toxicity study of 2, 4-D (reproductive performance, sperm parameters assessed, sex gland weights and histopathology; AGD, nipple retention; puberty ti ming). The NOAEL was established at 45 mg/kg/day, which is greater than the Points of Departure utilized for the human health risk assessment supporting the 2, 4-D RED. As noted above, EPA has received all require d study reports and data from the Tier 1 battery of test for 2, 4-D.

CITED BY: 2, 4-D Task Force: study cited as providing evidence that exposure to a herbicide formulation containing 2,4-D and picloram is not likely to cause male-mediated birth defects or other adverse reproductive outcomes; no effects character istic of endocrine-mediated response.

(Study 100) Citation : Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO. (1995). The E-SCREEN assay as a tool to id entify estrogens: An update on estrogenic environmental pollutants. Environ Hlth Perspect. 103: 113. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Endocrine] Methods Summary: E-screen is a quantitative assay that compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrog ens (negative control) and in the presence of 17 β -estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. Results Summary: negative for 2,4-D

CONCLUSION: 2, 4-D was not identified as estrogenic

COMMENT/ HED : This citation was included in the Endocrine Disruptor Review Team assessment of 2, 4-D (see TXR No. 0055461). The results of the MCF-7 cell proliferation a ssay were described as negative,

### Page 127 of 186 but no data were provided to substantiate this result. An extended 1-generation reproductive toxicity study on 2,4-D, in which offspring were exposed to 2, 4-D during gestation, lactation, and directly through PND 22, 70, 139, includes an assessment of 2, 4-D on several key endpoints. These endpoints include an evaluation of potential effects on parental male and female reproductive function offspring survival, growth including endocrine and systemic toxicity parameters such as estrous cyclicity (female adult rats and offspring), evaluation of sperm parameters, anogenital distance, nipple retention, puberty onset (vaginal opening and balano-preputial separation), and thyroid gland weight, hormone and histopathology assessment. In addition the test guideline includes options to evaluate development of the immune andnervous systems. In the extended one generation reproductive toxicity study, there were no treatmentrelated adverse effects on any of the measured parame ters evaluated to assess the estrogen, androgen or thyroid endocrine systems in intact rats following 2, 4-D exposure at dose levels approaching saturation of renal clearance. As noted above, EPA has received a ll required study reports and data from the Tier 1 battery of test for 2, 4-D.

CITED BY: 2,4-D Task Force: cited to dispute NRCD’s claim that 2,4-D has extensive hormone disrupting activity that may occur at low doses; 2,4-D not found to exhibit estrogenic properties; MCF7 cell proliferation: negative

BIN: DERMAL PENETRATION (Study 11) CITATION : Brand, RM and Jendrzejewski, JL (2008). The Effect of Chronic Ethanol Ingestion on Skin Penetration of 2,4-D – title cited (could not find paper with this title). Chronic Ethanol Ingestion Alters Xenobiotic Absorp tion Through the Skin: Potential Role of Oxidative Stress. Food & Chemical Toxicol 46: 1940-1948. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: rats pair-fed a diet containing 36% etha nol for 12 weeks; then switched to a nonalcohol diet and monitored for 4 weeks; non-invasive measurements for changes in dermal blood flow using laser Doppler velocimetry (LDV), damage to skin barrier via transepidermal water loss (TEWL), and changes in skin moisture content were performed; at 0, 1 day, or 1, 2, 3, 4 weeks after alcohol removal, rats sacrificed and their skin analyzed for alcohol and aldehyde dehydrogenase, and lipid peroxidation; transdermal penetration of DEET an d 2,4-D was determined also. Results Summary: Transdermal absorption, LDV, TEWL, sk in alcohol and aldehyde dehydrogenase (enhancement ratio 1.6), and lipid peroxidation ( ↑ 2.3 fold) were significantly increased after continuous ethanol exposure; factors remained el evated for up to 4 weeks after alcohol consumption stopped; reflect fundamental changes in the skin

CONCLUSIONS: alcohol ingestion is correlated with several skin disorders; link between ethanol ingestion and skin disorders associated with alcohol use

COMMENT/ HED : Alcohol is known to alter the skin, which can alter dermal function (barrier) and lead to increased dermal absorption (not unique to 2, 4-D). Alcohol shown to trigger increases in levels of enzymes associated with ethanol me tabolism, induce physical changes in the skin leading to increased uptake of xenobiotics (e.g., 2,4-D); higher generation of reactive oxygen species; ethanol induces changes in the skin that mimic those seen in the liver, including increases in enzymes related to alcohol metabolism, oxidative stress and blood flow. The exposure conditions are questionable, especially with regard to the regimens used for ethanol administration. Dose level appears very high (36% of diet) compared to what would be expected for those drinking alcohol and then attempting to treat their lawns or work in their treated lawns. Many factors can alte r dermal function (barrier) and lead to increased dermal absorption. Several human dermal absorption studies were considered in the Agency assessment as part of the weight of evidence for determining the dermal absorption factor. A mean-percent absorbed value of 5.7% of the applied dose was derived from four different human dermal absorption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the variability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for po tential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertainty factor for extrapolation fr om animal to human (UF A ; intraspecies). Generally when an endpoint is deri ved from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X. In this case, in spite of using a dermal absorption value from a human study, the full 10X UF H was retained. It should also be noted that the UF H factor is partially intended to

account for uncertainties associated w ith the relative sensitivity to a pe sticide within a population, which includes factors such as differences in dermal absorption that could possibly be attributed to the use of

### Page 128 of 186 sunscreen, clothing, or occlusion. The UF A is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has

been eliminated. Therefore, the Agency is conf ident that the dermal absorption value used does not under estimate dermal risk and is adequately protective of any potential enhancement to dermal absorption from

sunscreen/alcohol use.

CITED BY : NRDC (supplement; provided only authors names and year of publication). One of several studies cited by NRDC by the same investigators to support concern for enhanced dermal penetration of 2, 4-D following alcohol consumption; states that study s howed an average of a doub ling of absorption of 2,4- D through skin with acute and chronic ethanol intake; suggested that homeowner applying a “weed and feed” type product on a Saturday afternoon after drinking a beer would likely have a skin penetration of 2,4-D at double the rate of EPA’s estimate and using DEET would enhance dermal absorption even more; concludes that EPA should use a dermal absorp tion factor of at least 30% to protect public.

(Study 12) CITATION : Brand RM, Charron AR, Dutton L, Gavlic TL, et al. (2004). Effects of Chronic Alcohol Consumption on Dermal Penetration of Pesticides in Rats. J. Toxicol Environ Health A 67, 153- 161. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: male Wistar rats fed Lieber & DeCarli liquid diets (35% fat, 18% total protein,, 36% ethanol, remainder as carbohydrate) for 6-8 weeks; skin shaved, rem oved, frozen; skin placed in an in vitro flow-through diffusion cell system; surface area 0.79 cm 2 (circular chamber 1 cm diameter); epidermal side exposed to 100 μL of [ 14 C]-2,4-dichlorophenoxyacetic acid (specific activity 92.6 μCi/mg) diluted 1:40 with water and 50000 dpm [ 14 C]-2,4-D; commercial formulation (Agriliance) used instead of neat herbicides because previous studies have demonstrated that the inert ingredients lead to enhanced transdermal penetration and are probably more realistic predictor of human exposure; 2,4-D consists of 47.3% 2,4-D DMA salt (473 g/L, 2.1 M) and 52.7% inert ingredients Results Summary: feeding rats with ethanol (36% of calorie s) for 6-8 weeks leads to enhanced (by a factor of 2.2) transdermal penetration

CONCLUSIONS: if ethanol consumption has same effect on human skin, it could potentially have adverse health effects on people exposed to 2,4-D.

COMMENT/ HED : this is a generic issue; EPA agrees that in vitro studies indicate that ethanol consumption could potentially enhance the dermal absorption of 2, 4-D under the test conditions. Alcohol consumption has been shown to adversely affect the sk in itself (Brand & Jendrzejewski, 2008, Fd & Chem Tox 46: 1940-1948), which can alter dermal function (barrier) and lead to increased dermal absorption. The exposure conditions in the study cited are questionable, especially with regard to the regimen used for ethanol administration (36% of the diet). The Agency is already aware of the kinds of variables that impact the magnitude of the dermal absorption factor (e.g. anatomical site of ap plication, presence or absence of penetration enhancers in the pesticide formation, whether liquid formulations are aqueous or organic, species differences, magnitude of dose, and duration of exposure). The human volunteer dermal absorption studies that were consider ed in the 2005 risk assessment addressed several of these variables (skin site, dose level, organic or aqueous dose vehicles) and a conservative, upper-bound estimate of 10%

was selected that accounts for these variables. As such, the Agency considers the 10% DAF adequately protective for a wide range of the variables known to impact the magnitude of the dermal absorption factor. It is to be noted that there is a 10X uncertainty f actor applied for potential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertaint y factor for extrapolation from animal to human (UF A ; intraspecies). Generally when an endpoin t is derived from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X . In this case, in spite of using a dermal absorption value from a human study, the full 10X UF H was retained. The UF A is typically used to account for the uncertainty associated with the ex trapolation of animal testing results to humans for risk assessment, including dermal absorption. As such, the risk asse ssment results are inherently more protective in this case because this uncertainty has been eliminated. Therefore, the Agency is confident that the dermal absorption value used does not under estimate dermal ri sk and is adequately protective of any potential

enhancement to dermal ab sorption from alcohol use.

CITED BY NRDC : (66) one of several research papers from the same investigators cited by NRDC to show that the 2007 Brand study confirms prior research showing alcohol consumption enhances 2,4-D

### Page 129 of 186 penetration.

(Study 13) CITATION : Brand, RM; McMahon, L; Jendrzejew ski, JL; et al. (2007). Transdermal absorption of the herbicide 2, 4-dichlorophenoxyacetic acid is enhanced by both ethanol consumption and sunscreen application. Food and Chemical Toxicology 45: 93-97. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: Skin from rats ingesting 1.5 g/kg, 4.3 g/kg, or 6 g/kg of ethanol (control saline) was treated with a commercially availa ble sunscreen containing titanium di oxide and octyl methoxycinnimate and transdermal absorption of 2,4-D amine [ 14 C] was monitored by excising the skin and placing it into a

diffusion chamber. Results Summary: Ethanol ↑ penetration by a factor of 1.9, 2.0, and 2.5, respectively, demonstrating an ethanol-induced dose response; sunscreen applicatio n to skin from ethanol-gavaged rats caused 2,4-D absorption above that in duced by ethanol alone by an additional factor of 1.3, 2.1, and 2.9, respectively;

CONCLUSION: Results emphasize the importance of limiting excessive alcohol consumption in individuals with potential herbicide exposure rather than discouraging the use of sunscreens; states that studies demonstrate that personal be havior must be accounted for when estimating potential exposure in both occupational and recreational settings.

COMMENT/ HED : study indicates that the use of sunscreens and ethanol can enhance dermal absorption of 2, 4-D. This is a generic issue; alcohol consumption has been shown to adversely affect the skin itself; exposure of skin to the sun has its own adverse eff ects and the use of sunscreens is important (Brand & Jendrzejewski, 2008, Fd & Chem Tox 46: 1940-1948). Many factors can alter dermal function (barrier) and lead to increased dermal absorptio n. Several human dermal absorption studies were considered in the Agency assessment as part of the weight of eviden ce for determining the dermal absorption factor. A meanpercent absorbed value of 5.7% of the applied dose was derived from four different human dermal absorption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the variability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X. In this case, inspite of using a dermal absorption value from a human study, the full 10X UF A was retained. Therefore, the Agency is confident that the dermal absorption value used does not underestimate dermal risk and is adequately protective of any potential enhancement to dermal absorption from sunscreen/alcohol use. 

### T he exposure conditions are questionable, especially with regard to the regimens used for ethanol administration. Dose levels appear very high compared to what would be expected for those drinking alcohol and then attempting to treat their lawns or work in their treated lawns (dosed with ethanol at 1.5 g/kg to 6 g/kg BW). [Note: For a 60 kg woman, these dose rates equate to the consumption of 6 to 24 one ounce shots of 100- proof vodka.] Also, any dermal absorption enhancements associated with ethanol consumption is moot when considering children’s risk since alcohol consumption by minors is illegal.

CITED BY NRDC : (64) states that EPA ignored data showin g that dermal absorption is enhanced by alcohol consumption, sunscreen and DEET (didn’t incorporate into risk assessment or adopt risk mitigation measures); alcohol consumption ↑ skin penetration by 1.9-2.5 fold; sunscreen further enhanced skin penetration of 2,4-D; one of several papers by same investigators cited to support concern.

(Study 14) CITATION : Brand, RM; Spalding, M; Mueller, C. (2002). Sunscreens can increase dermal penetration of 2, 4-dichlorophenoxyacetic acid. Journal of Toxicology Clinical Toxicology 40 (7): 827-832. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: Hairless mouse (CRL:SK1) skin (dorsal) pretreated with co mmercially-available sunscreens; 2,4-D placed on top of epidermis in an in vitro diffusion chamber for 24 hours
Results Summary: total penetrating through skin in 24 hours ranged from 39.1% ± 1.7% for no sunscreen

control to 81.0% ± 2.8% for Neutrogena Oil Free Sunscreen

### Page 130 of 186 Table 1. Effect of Sunscreen Pretreatme nt on Transdermal Penetration of 2,4-D

Peak (% dose/1.5 hr) Time to Peak (hr) Cumulative (% dose/24 hr) Lag time (hr) Mean

enhancement ratio

C to Go C Sport

C Oil Free

NoAd Neutrogena S Neutrogena OF

Banana Boat Off Skin (DEET) Off Deep (DEET)

Control 12.9 ± 1.1

8.8 ± 2.3

13.0 ± 1.4

12.5 ± 2.2

7.7 ± 0.5

14.1 ± 2.7

7.3 ± 2.3

14.4 ± 1.2

17.9 ± 2.5***

8.4 ± 0.3 7.5 ± 0.9

7.8 ± 1.2

7.5 ± 0.9

8.0 ± 1.5

9.5 ± 2.2

9.8 ± 2.3

9.5 ± 2.0

6.0 ± 0

6.5 ± 0.5

8.8 ± 0.4 56.8 ± 6.6**

48.1 ± 6.3

68.0 ± 1.5**

57.6 ± 1.3**

59.2 ± 4.9**

81.0 ± 2.8**

39.6 ± 3.4

46.8 ± 2.6

62.4 ± 5.1**

39.1 ± 1.7 4.0 ± 0.5

3.9 ± 0.6*

3.1 ± 0.1**

3.3 ± 0.6**

3.6 ± 0.5

4.0 ± 0.2

4.1 ± 0.2

3.1 ± 0.0**

2.8 ± 0.2**

4.6 ± 0.1 1.5

1.2

1.7

1.5

1.5

2.1

1.0

1.2

1.6

1.0

mean ± SEM; * p<0.05; ** p<0.01; *** p<0.001; lag time = x-inte rcept of line extending from the linear portion of cumulative penetration curve; enhancement ratio = 2,4-D cumulative transport after sunscreen treatment/mean 2,4-D cumulative transport for control

CONCLUSION: Commercially-available sunscreens can enhance dermal penetration of the moderately hydrophilic herbicide 2,4-D. Careful selection of sunscreens during pesticide application could reduce potential exposure.

COMMENT/ HED : previous EPA response on this issue suggested that one means of dealing with this particular issue would be to add a statement to the label of such products informing users that use may enhance dermal absorption of various substances en countered in everyday life, including pesticides. Dermal absorption factor being used is already cons ervative: human average % dermal absorption (5.8%) vs 10% dermal absorption factor being used. Many factors can alter dermal function (barrier) and lead to increased dermal absorption. Several human dermal absorption studies were considered in the Agency assessment as part of the weight of evidence for de termining the dermal absorption factor. A mean-percent absorbed value of 5.7% of the applied dose was derived from four different human dermal absorption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the variability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspecies factor can either be re duced to 1 or a 3X. In this case, in spite of using a dermal absorption value from a human study, the full 10X UF H was retained. It should also be noted that

the UF H factor is partially intended to account for uncerta inties associated with the relative sensitivity to a pesticide within a population, which includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscreen, clothing, or occlusion. The UF A is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorption. As su ch, the risk assessment results are i nherently more protective in this

case because this uncertain ty has been eliminated. Therefore, the Agency is confident that the dermal absorption value used does not under estimate dermal risk and is adequately protective of any potential enhancement to dermal absorptio n from sunscreen/alcohol use.

CITED BY NRDC : (67) one of several research papers cited to show that 2007 Brand study confirms prior research showing alcohol consumption and sunscreens enhance 2,4-D penetration

(Study 27) CITATION : Feldmann R.G., Mailbach, H. I.. (1974). Percutaneous penetration of some pesticides and herbicides in man. [ Human Studies Considerations Apply: Yes ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: [ 14 C] 2,4-D applied to forearm skin of 6 ma le humans (vehicle acetone); treated area

washed after 24 hours.

RESULTS: 2,4-D absorption of 5.8%±2.4% (range 3.4-8.2%)

CONCLUSION: 2,4-D absorption of 5.8%±2.4% (range 3.4-8.2%)

COMMENT/ HED: Several human dermal absorption studies, which included this study, were considered in the Agency assessment as part of the weight of ev idence for determining the dermal absorption factor. A mean-percent absorbed value of 5.7% of the applied dose was derived from four different human dermal absorption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the

### Page 131 of 186 variability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity among memb ers of the human population (UF H interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X. In this case, in spite of using a dermal absorption value from a human study, the full 10X UF H was retained. It should also be

noted that the UF H factor is partially intended to account for uncertainties associated with the relative sensitivity to a pesticide within a population, whic h includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscreen, clothing, or occlusion. The UF A is typically used to account for the unce rtainty associated with the extrapol ation of animal testing results to humans for risk assessment, including dermal absorp tion. As such, the risk assessment results are inherently more protective in this case becau se this uncertainty has been eliminated. Therefore, the Agency is confident that the dermal absorption valu e used does not under estimate dermal risk and is adequately protective.

CITED BY: NRDC : petition stated that EPA used a dermal absorption factor of 10% based on a 1974 study of 6 adult men, which they indicate is inappropri ate, “especially considering the synergistic effects of other exposures.” states that the de rmal absorption factor of 10% in ag gregate risk assessment is woefully

insufficient.

CITED BY Task Force – regarding NRDC’s allegation of enhanced dermal absorption with alcohol/sunscreen use, cite s this study as one considered by EPA (5.8%±2.4; range: 3.4%-8.2%); indicates that study was an extreme test usi ng 2,4-D applied with acetone vehicle, which tends to denature the skin, a process in which proteins or nucleic acids of the skin lose their 3-D structure, thus allowing for increased potential dermal absorption; skin not protected and area not washed for 24 hours for maximum absorption. Task Force states that EPA has prev iously considered and rejected NR DC’s claim that dermal absorption may be enhanced through other factor s (sunscreen/DEET/alcohol consumption)

(Study 43) CITATION : Harris SA, Solomon KR. (1992). Percutaneous penetration of 2, 4- dichlorophenoxyacetic acid and 2, 4-d dimethylamine salt in human volunteers. J Toxicol Environ Health 36 (3): 233-240. [ Human Studies Considerations Apply: Yes ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: 2, 4-D and 2, 4-D dimethylamine salt (DMA) ; five male volunteers (each dosed on separate occasions with both chemicals); urine sa mples, collected for 144 hours following dermal application of 10 mg to dorsum of the hand (9 cm 2 ), were analyzed for 2,4-D.

RESULTS: an average of 4.46 ± 0.85% was recovered in urine following 2,4 –D exposure, and 1.76 ± 0.57% following DMA; greater amounts of DMA (7.68 ± 0.49%) were washed off at 6 hours compared to 2,4-D (5.35 ± 0.38); average ≈ half-lives for excretion were 39.5 ± 8.1 hours (2,4 –D) and 58.5 ± 13.2 hours (DMA).

CONCLUSION: 2,4-D was applied as a solution in acetone, whic h can be expected to increase penetration into corneum where it is resistant to removal by washing; thus overestimating dermal penetration under

field conditions

COMMENT/ HED: These data were evaluated previously by HED (TXR# 010044, 2/23/1993; several publications including bystander exposure, professional applicators); results were considered in previous

assessments.

CITED BY Task Force – one of studies cited; stated that EPA’s decision regarding glove use consistent with robust peer-reviewed literature, which repeatedly demonstrates that use of a protective rubber gloves alone dramatically reduces to tal absorbed dose of 2,4-D

(Study 74) CITATION : Moody RP, Franklin CA, and Ritter L, (1990). Dermal absorption of the phenoxy herbicides 2,4-D, 2,4-D isooctyl, and 2,4,5-T in rabbits, rats, rhesus monkeys, and hum ans: a cross-species comparison. J. Toxicology and Environmental Health 29: 237-245. [ Human Studies Considerations Apply: Yes ] Principal Endpoint : [ Bin: Dermal Penetration]

### Page 132 of 186 Methods Summary: M ale New Zealand rabbits and male Sprague-Dawl ey rats (shaved middorsal region), male rhesus monkeys (shaved middorsal forearm or forehead regions); constant dose rate of μg/ cm 2 ; nonocclusive gauze patches used to protect dosed areas for 24 hours (except forehead); male human volunteers (0.7μCi of 14 C radiolabel

dissolved in either acetone or Es teron LV96 was applied to a 45-cm 2 area of the forehead); 14 C ring-labeled 2,4-D (>98%), dimethylamine, and isooctyl ester: 14 C-2,4-D amine in acetone; 2,4-D isoo ctyl in acetone or Esteron LV96. Urine samples (animals) collected at 4 and 8 hrs on first day, 24-hr intervals for 14 days; human urine collected at 4, 8, and 12 hrs and daily until post-treatment day 7; dose site washed at 24 hrs post dose (all); % dermal absorption and

excretion half-lives were calculated

RESULTS: Table 1. Total Cumulative Dermal Absorption

Pesticide Rabbit (back) Rat (back) Rat (tail) M onkey (FA) Monkey (FH) Human (FA) Human (FH)

2,4-D

acid

% rec ± sd

T 1/2 N 36 ± 10.9

2.14

3 - - 15 ± 10.0

1.94

8 29 ± 7.6

1.47

8 6 ± 2.4 ♪

nr 6 -

2,4-D

amine

% rec ± sd

T 1/2 N 12 ± 3.7

1.65

8 20 ± 3.0

2.55

8 - - - - 58 ± 22.6

nr 6
2,4-D

amine

% rec ± sd

T 1/2 N - - 14 ± 6.4

1.35

4 6 ± 3.4

1.83

8 31 ± 13.1

2.13

8 - -

2,4-D isooctyl

% rec ± sd

T 1/2 N 50 ± 12.9

nr 6 - - 40 ± 24.2

2.07

8 56 ± 15.2

2.04

8 - 3 ± 3.0

1.33

4
2,4-D isooctyl

% rec ± sd

T 1/2 N 34 ± 7.6

0.74

8 - - - - - 6 ± 2.6

1.63

4 Data from Table 1, page 241 of pa per; FA forearm; FH forehead; ♪ data from Feldmann and Maibach, 1974; nr not reported

CONCLUSION: paper stated that anatomical site-related differences in sk in permeability are one of a large number of variables known to affect permeability to chemicals; in Discussion section it states: it is noteworthy that the i.m.

correction factor used for the animal da ta permits interspecies comparison of dermal absorption data and circumvents a potential problem that differential interspecies excretion of the pesticide radiolabel between urinary and fecal routes precludes direct extrapolation. They note that their human data were not correct ed for non-urinary excretion since ivpercent recovery was 100% for 2,4-D acid in humans (Feldmann, 1974). Investigators indicated that assuming a nonurinary excretion of the amine salt and is ooctyl ester similar to that obtained in monkeys would tend to increase the human dermal absorption at most by 26-28%, a value consiste nt with the 20-25% estimated by Grover, et al. (1986) for non-urinary excretion and body storage of 2, 4-D acid ingested by humans (Grover, R., Cessna, AJ, Muir, NI, et al. 1986. Factors Affecting the Exposure of Ground-Rig Applicators to 2, 4-D Dimethylamine Salt. Arch Environ Contam Toxicol. 15: 677-686).

COMMENT/ HED: The Agency is aware of the kinds of vari ables that impact the magnitude of the dermal absorption factor (e.g. anatomical site of application, presence or absence of penetration enhancers in the pesticide formation, whether liquid formulations are aqueous or organic, species differences, magnitude of dose, and duration of exposure). The human volunteer dermal absorption studi es that were considered in the 2005 risk assessment addressed several of these variables (skin site, dose level, organic or aqueous dose vehicles) and a conservative, upper-bound estimate of 10% was selected that accounts for these variables. As such, the Agency considers the 10% DAF adequately protective for a wide range of the variables known to impact the magnitude of the dermal absorption

factor.

CITED BY: Task Force

– findings of study not quoted (not specifically addressed; just listed as a reference)

### Page 133 of 186 (Study 75) CITATION : Moody RP, Nadeau B. (1992). Effect ofthe mosquito repellent DEET and longwave ultraviolet radiation on permeation of the herbicid e 2, 4-D and the insecticide DDT in natural rubber gloves. Am Industrial Hygiene Assoc journal 53: 436-441. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: examined total 14 C-ring labeled 2,4-D permeability across a 1.5 cm 2 cut-out of natural rubber latex gloves incubated in laboratory apparatu s at 37°C for 48 continuous hours; mean thickness

0.51 ± 0.008 mm (n=4)

RESULTS: Deet itself rapidly permeated the glove, no significant effect of DEET was observed on glove permeability to 2,4-D (2.4 ±1.8% with DEET vs 3.2± 3.46% without DEET); phot oenhanced (long-wave ultraviolet (UVA) radiation) permeation of 2,4-D was observed in a natural rubber glove (6.2% ± 0.73%

+UVA vs 0.3% ± 0.14% -UVA).

CONCLUSION: No significant effect of DEET on glove perm eability to 2,4-D; glov e permeability to 2,4- D enhanced when glove exposed to long-wave ultraviolet radiation

COMMENT/ HED: As discussed elsewhere, Agency is conf ident that the dermal absorption value used does not under estimate dermal risk and is adequately protective.

CITED BY: NRDC (71) – infers that glove use will not afford adequate dermal protection; study cited as focusing on rubber gloves commonly worn by farmers; NRDC cited study as showing that these gloves were highly permeable to 2,4-D penetration when th ere was simultaneous exposu re to DEET and sunlight.

CITED BY: Task Force – states that study primarily intended as hypothesis-generating experiment; does not provide definitive conclusions for enhanced risk for following reasons: 1) authors concluded DEET did not significantly impact 2,4-D pe netration through rubber (NRDC cl aims DEET alone enhanced 2,4-D permeability); 2) n=2; permeability of 2,4-D was ↑ to 6.2±0.73% when rubber was simultaneously exposed to UV lamp emitting UVA (not “sunlight”; non-UVA control 0.3±0.14% 2,4-D permeation); 3) neither DEET alone nor UVA alone rendered the rubber “highl y permeable to 2,4-D despite the relatively severe experimental conditions; 4) only permeation of total [ 14 C] was measured and thus authors could not exclude that the postulated enhanced permeation with UVA_cotreatment was represented by 2,4-D photolysis product(s); and 5) authors stated the field relevance of observations remained to be established.

(Study 76) CITATION : Moody RP, Wester RC, Melendres JL, Mailbach HI. (1992). Dermal Absorption of the Phenoxy herbicide 2,4-D Dimethylamine in Humans: Effect of DEET and Anatomic Site. J. Toxicol Environ Health 36 (3): 241-250. [ Human Studies Considerations Apply: Yes ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: 14 C-ring labeled 2,4-D-dimethylamine (>99%) (1) in water applied to left palmar region; (2) with DEET in water to left palmar region; (3) in water to left ventral forearm; (4) in acetone to left ventral forearm; 0.5μCi of 14 C radiolabeled 2,4-D amin e (7 μg) dissolved in 10 μL water or acetone was applied topically to a 4.2 cm 2 area on palm or forearm (dose rate: 1.7 μg/ cm 2 ) ; for (2) 10 μL DEET was applied to treatment region on palm just prio r to application of the 2,4-D amine; nonocclusive polypropylene chambers were affixed over treatment sites; after 24 hours, site washed vigorously with 5 successive gauze swabs wet, respectiv ely with Radiac soap solution, water, Radiac soap solution, water, water; site protected after wash; urine samples collect ed for 5 days at 24-hr intervals; on day 5 treatment site tape stripped 10 times with cellophane tape, which was analyzed for residues

RESULTS:
Table 1. Average % Dose Recoveries of Soap and Water Washes
Wash 2,4-D-DMA -DEET (aq)

on palm 2,4-D-DMA +DEET (aq)

on palm 2,4-D-DMA -DEET (aq)

on forearm 2,4-D-DMA -DEET (ac)

on forearm Soap

Water Soap

Water

Water

Total 20±15.3

7±4.9

3±1.6

2±1.5

2±1.0

34±23.0 11±7.0

5±2.3

3±2.0

1±0.4

1±0.5

21±11.8 19±9.9

7±4.6

4±3.4

2±1.6

2±1.7

34±20.3 18±11.3

10±8.1

3±2.7

2±1.4

2±1.4

34±23.4 Tape strips 2±1.6 1±0.6 1±0.9 2±1.4

### Page 134 of 186 Chambers 22±3.4 37±7.6 18±3.4 19±4.9

Urine excretion 10±11.5 14±4.5 7±6.2 13±5.0 Data from Tables 1&2, pages 244-245 of paper; average % dose recoveries ± sd; aq (aqueous); ac (acetone)

Most of residue present in initial soap wash; plastic chambers covering treatment site showed 18%-37% recovery 24 hr post treatment; no significant diff erence between average % recoveries for any of the sample groups involving soap wash es, tape strips, and urine samples between each of their respective treatment groups; significan tly greater % recovery was obtained for the plastic chambers in sample group where DEET was employed; urinary data demonstrated that there was no significant difference between palmar and forearm skin permeability to 2,4-D-amin e, that DEET had no significant effect on total cumulative palmar permeability, and there was no sign ificant difference between % recoveries obtained with acetone or aqueous vehicles.

CONCLUSION: investigators indicated that, from regulatory viewpoint, these data should raise concern since 2, 4-D-amine has been shown to permeate the pa lm, and hands are the anatomical sites most often contaminated by pesticide applicators. Although this exposure may be somewhat reduced by wearing gloves, study shows significant dermal absorption even following rigorous soap and water washing at 24 hr

post treatment.

COMMENT/HED: Several human dermal absorption studies were considered in the Agency assessment as part of the weight of evidence for determining the dermal absorption factor. A value of 10% was used by the Agency based on these data and given the variability noted in the results. Additionally, a 10X uncertainty factor applied for potential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A ; intraspecies) were applied. It is to be noted that although a dermal absorption value from human data was used, the full

10X UF H was retained to account for unce rtainties associated with the re lative sensitivity to a pesticide within a population. The UF A is typically used to account for th e uncertainty associated with the extrapolation of animal testing results to humans fo r risk assessment, includin g dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has

been eliminated. Therefore, the Agency is confident that th e dermal absorption value used (10%) does not under estimate dermal risk and is adequately protective. It is also pointed out that if the results from the trial combining 2, 4-D and DEET (14%) are included in the overall average of dermal absorption values from the human studies (Ross, paper), the average dermal absorption only increases from 5.7% to 6.4%. Both numbers round to 6%.

CITED BY: NRDC (68) – cited as research demonstrating presence of DEET on skin significantly enhances absorption of 2,4-D (14% palmar absorption of 2,4-D after applying DEET to skin).

CITED BY: Task Force – states that EPA previously responded to this allegation (the conclusion that the data demonstrate a difference between exposure w ith (14±4.5%) and without (10±11.5%) DEET is not supported given the magnitude of the standard deviation); also points out that in the 2005 RED, the PPE changes implemented mitigated concerns for potential enhancement by DEET, etc.

(Study 84) Citation : Pelletier, O; Ritter, L; Caron, J; and Somers, D (1989). Disposition of 2,4- Dichlorophenoxyacetic Acid Dimethylamine Salt by Fischer 344 Rats Dosed Orally and Dermally. J. Toxicology and Environmental Health 28: 221-234. [Human Studies Considerations Apply: No] Principal Endpoint : [Bin: Dermal Penetration (comparison of oral and dermal route) ] Methods Summary: dimethylamine salt of 14 C-ring-labeled 2,4-D administered to Fischer 344 rats orally (1 and 0.4 mg/kg) and dermally (10 mg/kg); designed to compare the pharmacokinetics of 2, 4-D administered orally and dermally to better understand the relationship between oral toxicity studies and typical dermal field exposure ; stated that 2, 4-D is rarely commercially available in the acid form due to its poor water solubility and that commercial formulations are usually composed of alkali or amine salts or

esters. Results Summary: dermal dose (10 mg/kg) as within upper range that might be expected from typical agricultural use; oral dose (1 and 0.4 mg/kg) from preliminary work, which indicated a 4-10% absorption factor for 2,4-D amine thro ugh skin, as compared to almost comple te absorption from oral route; 94-96% systemically available within 6 hours of oral dose/ 10% over 72 hours of dermal dose; peak concentrations in blood and kidneys achieved within 30 minutes (both routes); by 1.5 hrs after dosing, 2,4-D conc. in blood, muscle, liver, kidneys had decreased (both routes); plateau between 2-8 hrs (dermal) in these tissues

### Page 135 of 186 while urinary excretion increased; concentrations in orally-dose animals continued to decrease; at 7 hrs, skin wash removed 63% of applied dose; oral dose almost completely excreted within 24 hours

CONCLUSION: oral dose almost completely excreted w ithin 24 hours; dermal exposure resulted in slower decline due to continued absorption from site of application (72-hour study)

COMMENT/ HED : Based on urinary excretion values, systemic availability ≈ 10% of dermal dose, while that of the oral dose was virtually 100 % within 72 hours after exposure. In terms of 2, 4-D concentration, about 1 mg of 2, 4-D was excreted following either a dermal dose of 10 mg/kg or an oral dose of 1 mg/kg. Peak concentrations in blood and kidneys were achieved within 30 minutes (both routes). Oral dose almost completely excreted within 24 hour s, whereas about 13% of the dermal dose was systemically available at

72 hours.

CITED BY: Task Force : in reference list, but did not find where it was discussed/cited

(Study 86) CITATION : Pont AR, Charron AR, Brand RM. (2004). Active ingredients in sunscreens act as topical penetration enhancers for the herbicide, 4-dichlorophenoxyacetic acid. Toxicology and Applied Pharmacology 195: 348-354. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: full-thickness dorsal skin of female hair less mice (CRL:SKH1); Ke l-F Bronaugh-style flow through diffusion chambe r (exposed surface area of 785 cm 2 ) with epidermal side facing donor chamber; 2,4-D amine in ethanol; 9 sunscreen formulations Results Summary:

Table 1. Sunscreen ingredient Cumulativ e (% dose/24 hr) Lag time (hr)

Hairless mouse skin in vitro Octyl methoxycinnamate Octocrylene Oxybenzone

Homosalate Octyl salicylate

DEET 9.5%

DEET 19.0%

Padimate-o

Sulisobenzone

Control 84.2 ± 1.8**

65.6 ± 4.3

74.9 ± 4.8

72.5 ± 3.5*

74.1 ± 3.1**

83.3 ± 2.4**

82.8 ± 3.4

86.9 ± 2.5**

81.2 ± 3.1**

54.9 ± 4.7 1.7 ± 0.2**

3.7 ± 0.3

3.7 ± 0.4

2.6 ± 0.4

1.1 ± 0.2**

2.6 ± 0.2**

2.4 ± 0.1**

2.5 ± 0.4**

1.5 ± 0.2**

4.4 ± 0.2

Human skin in vitro

Padimate-o Octyl salicylate

Octocrylene

Control 41.3 ± 3.3**

35.7 ± 11.5*

14.5 ± 1.9

13.0 ± 1.0 1.9 ± 0.4**

0.9 ± 0.1**

2.7 ± 0.4*

4.2 ± 0.4

mean ± SEM; * p<0.05; ** p<0.01

CONCLUSION: Most of the sunscreens increased the total amount of 2, 4-D flux through the skin vs control and decreased lag time vs control (both hairless mouse and human skin).

COMMENT/ HED The existing dermal absorption value of 10% is based on the high end of the range of results in an extensive set of high quality human research results. The Agency considers this the best available data because these data eliminate the interpretative cons iderations necessary to ex trapolate from a different species and an in vitro test methodology. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertainty factor for extrapolation from animal to human (UF A ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X. In this case, inspite of using a dermal absorption value from a human study, the full 10X UF H was retained. Additionally,

the UF A is typically used to account for th e uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorption. As such, the risk assessment results are inherently more protective in this case becau se this uncertainty has been eliminated. Therefore, the Agency is confident that the dermal absorptio n value used does not under estimate dermal risk and is adequately protective of any potential enhancement to dermal absorption from sunscreen use.

### Page 136 of 186 Current OPP policy precludes using in vitro dermal absorption data as a stand-alone for quantitative use in risk
assessment. Corroborating in vivo data is also required. This is because OPP does not consider in vitro
methods to have been sufficiently validated for stand-alone regulatory use 1 Additionally, there are important

limitations to in vitro models for dermal absorption, one of which is lack of an intact vasculature.

NAFTA Dermal Absorption Group Position Paper on Use of In Vitro Dermal Absorption Data in Risk

Assessment (2009).

CITED BY: NRDC– (65) one of several research papers by the same investigators cited to show that 2007 Brand study confirms prior research showing alcohol consumption and sunscreens enhance 2,4-D penetration; NRDC considers EPA’s previous response to public comments (12/16/2004) urging EPA to consider these effects in the RA a cursory and highly deficient response

(Study 89) CITATION : Riviere JE, Baynes RE, Brooks JD, Yeatts JL, Monteiro-Riviere NA. (2003). Percutaneous absorption of topical N, N-diethyl-m-to luamide (DEET): effects of exposure variables and coadministered toxicants. J. Toxicol Environ Health A, 66(2): 133-151. [ Human Studies Considerations Apply: No ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: isolated perfused porcine sk in flaps (IPPSFs were prepared and perfused in a nonrecirculating perfusion chamber; flow-through diffusion cells Results Summary: an important finding is that vehicle and co ncentration seemed to have minimal effects on absorption, while exposure to systemic drugs or a topical toxicant such as subvesicating doses of sulfur mustard did increase absorption, ther eby accentuating any role that DEET might play in a syndrome such as the Gulf War Illness; most obvious finding, and one easily addressable in an occupational environment, was that covering DEET dose areas with an occlusive dressing, or even fabric, enhances absorption. Care should be taken to avoid this scenario after DEET has been applied.

CONCLUSION: results suggest that co-exposure to a number of chemicals that potentially could be encountered in a military environment may modulate the percutaneous absorption of topically applied DEET beyond that seen for normal vehicl es at typically applied concentrations.

COMMENT/ HED : study is on DEET and cofactors that could modulate transdermal flux of topically applied DEET as it relates to contributory factor to th e Gulf War Illness; a generic issue; Generally when an endpoint is derived from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X. In this case, inspite of using a dermal absorption value from a human study, the full 10X UF H was retained. Therefore, the Agency is confident that the dermal absorption value used does not under estimate dermal

risk. UF H factor is partially intended to account for uncerta inties with dermal absorp tion such as the use of sunscreen, clothing, or occlusion. Additionally, the UF A is typically used to account for the uncertainty associated with the extrapolation of animal testing results to humans for risk assessment, including dermal absorption. As such, the risk asse ssment results are inherently more pr otective in this case because this uncertainty has been eliminated. The Agency is awar e that occlusive coverings increase skin hydrationstatus by preventing water loss, and that hydrated skin is generally more permeable to pesticides than unhydrated skin Therefore, occlusive coverings woul d be expected to increase exposure via the dermal route, resulting in increased risk. However, in EPA’s evaluation of this study, it found that the Agency’s risk assessment and margins of safety established through the application of uncertainty factors, already account for any uncertainty raised by NRDC relative to the findings in this study

CITED BY: NRDC – (70) states that occlusion can significantly enhance skin absorption of dermallyapplied materials

(Study 91) CITATION : Ross RH, Driver JH, Harris SA, Maibach HI. (2005). Derm al absorption of 2, 4-D: a review of species differences. Regulatory Toxicology and Pharmacology 41: 82-91. [ Human Studies Considerations Apply: Yes ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: comparison of dermal absorption studies in animals (rat, mouse, rabbit, Rhesus monkey) and

humans Results Summary: dermal penetration: rat 17.3%; rabbit 18.5%, monkey 10.5%; mouse 21%; references 5 human studies where either 2,4-D or DMA were applied to forearm or hand, which show consistent results

### Page 137 of 186 Table 1. Dermal Absorp tion Studies in Humans Reference (acid/salt) Methods (site , n, dose, vehicle, time of wash, sample collection time % absorbed (mean ± sd) Feldmann (acid) Maibach (DMA) Maibach (DMA) Moody (DMA) Moody (DMA) Moody (DMA) Harris (acid) Harris (DMA) Wester (DMA) Forearm; n=6; 4 μg/cm 2 ; acetone; 24 hours, at 120 hours Forearm; n=6; 4 μg/cm 2 ; na; 4 hours, at 120 hours Forearm; n=6; 4 μg/cm 2 ; na; 4 hours, at 120 hours Palm; n=4; 1.7 μg/cm 2 ; water; 24 hours; at 168 hours Forearm; n=4; 1.7 μg/cm 2 ; water; 24 hours, at 168 hours Forearm; n=4; 1.7 μg/cm 2 ; acetone; 24 hours, at 168 hours Backhand; n=5; 1100 μg/cm 2 ; acetone; 6 hours, at 144 hours Backhand; n=5; 1100 μg/cm 2 ; water; 6 hours, at 144 hours Forearm; n=6; 39.7 μg/cm 2 ; ethanol; 24 hours, at 144 hours 5.8 ± 2.4

3.7 ± na
2.8 ± na
10 ± 11.57 ± 6.2

13 ± 5.0

4.5 ± 0.9

1.8 ± 0.7

1.1 ± 0.3

Weighted Average ♪ 5.7 ± 3.4

♪ sum of % absorption from each data set multiplied by the # of replic ates in that data set divided by the total # of replicates (34) that excluded Maibach and Feldmann, 1974

CONCLUSION: For purposes of estimating potential human health risks associated with sy stemic absorbed doses, there is far less uncertainty in using carefully collected human data than in using dermal absorption estimates from small numbers of inbred laboratory animals. For purposes of developing simplistic, deterministic estimates of potential human (e.g., consumer or professional worker) daily absorbed doses of 2,4-D following skin exposures to liquid formulations across a range of applied dose densities (1-1000 μg of 2,4-D per cm 2 of skin), and for contact durations of 24 hours or less, a mean percent absorbed value of 5.7% of the applied dose (derived from 4 different human dermal absorption studies) is appropriate.

COMMENT/ HED : Several human dermal absorption studies were cons idered in the Agency assessment as part of the weight of evidence for determining the dermal absorptio n factor. A mean-percent abso rbed value of 5.7% of the applied dose was derived from four different human dermal absorption studies. Ultimately, a value of 10% was used by the Agency based on these data and given the variability noted in the results. It is to be noted that there is a 10X uncertainty factor applied for potential variation in sensitivity among members of the human population (UF H interspecies) and a 10X uncertainty factor fo r extrapolation from animal to human (UF A ; intraspecies). Generally when an endpoint is derived from a human study, the 10X intraspecies factor can either be reduced to 1 or a 3X. In this case, in spite of using a dermal absorp tion value from a human study, the full 10X UF H was retained. It should

also be noted that the UF H factor is partially intended to account for uncertainties associated with the relative sensitivity to a pesticide within a population, which includes factors such as differences in dermal absorption that could possibly be attributed to the use of sunscreen, clothing, or occlusion. The UF A is typically used to account for the uncertainty associated with the extr apolation of animal testing results to humans for risk assessment, including dermal absorption. As such, the risk assessment results are inherently more protective in this case because this uncertainty has been eliminated. Therefore, the Agency is co nfident that the dermal absorption value used does not under estimate dermal risk and is adequately protective.

CITED BY: Task Force – cited in support of EPA selection of 10% dermal absorp tion factor as a protective value given study variation; review and summary of numerous dermal absorption studies; determined that human percutaneous absorption of 2,4-D has been well characterized; 5 published studies using human subjects showed remarkable reproducibility across a span of 3 decades and multiple laboratories, formulations, and methods; 5.7% dermal absorption from these studies in close agreement with fi ndings from Feldmann and Maibach, 1974 (5.8%).

(Study 134) CITATION : Wester RC, J Melendres, L Sedic, H Maibac h, and JE Riviere. (1998). PercutaneousAbsorption of Salicylic Acid, Theophylline, 2,4-D Dimethylamine, Diethyl Hexyl Phthalic Acid, and pAminobenzoic Acid in the Isolated Perfused Procine Skin Flap Compared to Man In Vivo . Toxicology and Applied Pharmacology 151: 159-165. [ Human Studies Considerations Apply: Yes ] Principal Endpoint : [ Bin: Dermal Penetration] Methods Summary: study compares percutaneous absorption in the isolated perfused porc ine skin flap (IPPSF) system12 with that in man in vivo

. Human : Six subjects (males, ages 18-85 years; postmenopausal women 50-65 years) topically dosed at 8.7 μg/cm 2 with [ 14 C] 2, 4-dichlorophenoxyacetic acid dimethylamine (99.9%; 0.022 mCi/mg; solubilized in 50 μL ethanol) on the ventral forearm (spread over a 10- cm 2 skin surface area. Ethanol vehicle was allowed to dry; site not occluded. Urine collected for day of dosing and subsequent 6 days; at 24 hr, site washed with 50% Liquid Ivory soap (1:1, v/v) and water; wash sequence: soap (50%), water rinse, soap, followed by 2 water rinses using a cotton swab. At 7 days after appli cation, skin dosing site was cellophane tape-stripped 10, times for residual [ 14 C]-chemical. Percutaneous absorption determined from 14 C–urinary excretion. IPPSF : 5 IPPSF were surgically prepared, harvested and perfused in a nonrecirculating chamber and then dosed with [ 14 C] 2,4-D –

### Page 138 of 186 dimethylamine one hour after equilibrium; a 10- cm 2 area on the IPPSF was dosed in a manner identical to the in vivo human study. Skin flap viability was assessed by monitoring of glucose utilization; venous effluent was collected at 30-minute increments for 8 hours after which the perfusions were terminated; after termination, the skin flaps were weighed and the dosing area was swabbed 2 times with gauze dipped in a mild soap solution; dosed skin was they stripped 12 times with cellophane tape; treated area was then excised and removed from the subcutaneous tissue and weighed; the remainder of the skin flap was collected for mass balance purposes; total penetration after 8 hours was estimated as the radiolabel detected in perfusate plus treated skin samples (iv dosing of Rhesus monkey (Moody et al., 1990) to account for that part of dose excret ed by other routes (feces) or retained in body).

RESULTS: Human: recovery of dose in urinary excretion was 0.77±0.21%; only 2.0±0.7% was recovered in soap and water wash; only 0.06±0.06% was recovered in tape stripping. IPPSF: dose in perfusate was 1.5±0.24% and skin content after tape stripping was 2.4±0.5%, giving a predicted absorption of 2, 4-dimethylamine of 3.8±0.6%; only

21.4% dose was recovered in the surface wa sh and 4.9±2.6% was recovered in tape strips, giving a material recovery

of 31.2±5.6%.

CONCLUSIONS: human in vivo percutaneous absorption (% dose absorbed 1.1±0.3) compared to penetrated dose in IPPSF (% dose absorbed 3.8±2.4)

COMMENT/ HED : IPPSF system tested as a model for predicting pe rcutaneous absorption relative to man; appears to support a 10% dermal absorption factor

CITED BY: Task Force – in reference list but did not find where Task Force cited

BIN: METABOLISM

(Study 97) Citation : Saghir, SA; Mendrala, AL; Bartels, MJ; Da y, SJ; Hansen, SC; Sushynski, JM;, and Bus, JS (2006). Strategies to Assess Systemic Expo sure of Chemicals in Su bchronic/Chronic Diet and Drinking Water Studies. Toxicology and Applied Pharmacology 211: 245-260. [Human Studies Considerations Apply: No] Principal Endpoint : [Bin: Metabolism] Methods Summary: dietary administration 2,4-D (97.3%); Fischer 344 rats (9 weeks old); 5 and 100 mg/kg/day Results Summary: 2,4-D concentrations in plasma and AUC values increased in a distinctly nonlinear fashion between 5 and 100 mg/kg/day (4-week exposure); nonlinear increase was apparent from the 89-fold

increase in C

max , and a 69-fold higher plasmaAUC-24 hr, far exceeding the expected 20-fold difference between doses; higher than expected plasma AUC-24 hr was also supported by the saturation of urinary excretion at high dose; only 28-fold higher 2,4-D was recovered during 18 hr of collection instead of 69- fold as expected from difference in circulating levels;

Table 1. Data From Literature

Diurnal Blood/Plasma Levels after Steady-State Achieved (diet) Time 5 mg/kg/day 100 mg/kg/day Ratio (100/5)

12 am

3 am

6 am

9 am 12 pm 3 pm 6 pm 9 pm 0.59±0.17

0.62±0.06

0.72±0.15

0.48±0.03

0.41±0.08

0.42±0.34

0.44±0.08

0.64±0.04 29.54±4.90

36.08±4.76

63.95±6.02

50.27±9.76

35.65±8.18

38.64±14.12

19.28±8.51

23.54±8.86 50

58

89

105

87

92

44

37 AUC (μg h mL -1 ) # time points ♪ 12.95

13.08

13.92

11.54 890.88

1207.51

998.79

1206.49 68

92

72

105 n=3/time point (from literature)/2-7 animals ♪

amount in diet amount in blood/plasma 5 mg/kg/day 0.41-0.72 μg /mL 100 mg/kg/day 19-64 μg /mL

### Page 139 of 186 CONCLUSION: general - development of strategies to assess systemic exposure levels of chemicals to better design long-term studies

COMMENT/ HED : demonstrates that a dose of 2, 4-D of 100 mg/kg/day can be considered an excessive dose; HED aware of saturation kinetics of 2,4-D.

CITED BY: Task Force : cited with respect to Sturtz (2000) citation by NRDC regarding lactational exposure at 50, 70, 100 mg/kg/day; TF states that toxicity observed in the resulting non-linear pharmacokinetics range does not info rm human risk assessment because nonlinear doses are unlikely to

occur in humans

(Study 96) Citation : Saghir, SA; Marty, MS; Clark, AJ; Zabl otny, CL; Bus, JS; Perala, AW; Yano, BL; and Neal, BH (2009). A Dietary Dose Range-Finding and Toxicokinetic (TK) Study of 2,4- Dichlorophenoxyacetic Acid (2,4-D) in Adult Crl:CD( SD) Rats and Their Offspring. 1. Toxicokinetics.

Abstract. [Human Studies Considerations Apply: No] Principal Endpoint : [Bin: Metabolism – Toxicokinetics] Methods Summary: dietary administration of 2,4-D (0, 100, 200, 400, 600, 800, 1200, or 1600 ppm) from pre-breeding through weaning; F1 offspring exposed through PND 35; 2,4-D AUC 24 h was calculated from 3 plasma samples collected at 6 am, 9 am, 5 pm on designated sample days; 2,4-D in milk determined on lactation days 4 and 14 and in plasma of nursing pups by collection of single 9 am sample on PND 4, 14, 21, and 28; due to saturation of renal clearance, nonlinear TK was observed in parental males at 1600 ppm (test day 28) and 1200 ppm (test day 71); onset of nonlinear TK in parental females was observed at <400 ppm (test day 29 and 95; AUC 100 ppm = 25; AUC 400 ppm = 201 μg h mL -1 ); during gestation and lactation, female 2,4-D levels were higher due to higher diet consumption; nonlinear TK occurred at <400 ppm (GD 17 and LD 14) and > 400 ppm and <800 ppm (LD 4); a shift in nonlinear TK on LD 4 may have

been due to excretio n into milk (levels ≈ 50% of female plasma); plasma 2,4-D in pups 30-70% lower than dam on LD 4 (M/F TK nonlinear <800 ppm) but similar to or higher than dam on LD 14 (M/M TK nonlinear <400 ppm); maximal 2,4-D in pups was r eached on PND 21 when pups were consuming 2,4-D diet in addition to nursing, but dropped to levels = to dams on test day 29 and 95 levels on PND 35 Results Summary: kinetically-derived maximum dose was 400 ppm ( ≈ 25 mg/kg/day) for females and 1200 ppm ( ≈ 65 mg/kg/day) for males; increased dam and pup AUCs during lactation supported dietary adjustment during lactation

CONCLUSION: TK data support a definitive study high dose for females and pups lower than that derived by traditional toxicity testing

COMMENT/ HED : These dose selection data were discussed with Agency prior to extended 1-generation reproductive toxicity study initiation (MRID 47414902) a nd were included in the review of the extended 1- generation reproductive toxicity study of 2, 4-D (MRID 47972101). Study was evaluated by Agency and classified acceptable/non-guideline. Th e results do not yield information that would cause the Agency to modify its conclusions from the 2005 Reregistration Elig ibility Document for 2, 4-D that pertain to hazard.

CITED BY: Task Force: cited with respect to Sturtz (2000) citation by NRDC regarding lactational exposure at 50, 70, 100 mg/kg/day; states that toxicity observed in the resulting non-linear pharmacokinetics range does not info rm human risk assessment because nonlinear doses are unlikely to

occur in humans

(Study 130) Citation : Van Ravenzwaay, B; Harwick, TD; Needham, D; Pethen, S. and Lappin, GJ (2003). Comparative Metabolism of 2,4-Dichlorophenoxyacetic Acid in Rat and Dog. Xenobiotica 33 (8): 805-821. [Human Studies Considerations Apply: No] Principal Endpoint : [Bin: Metabolism] Methods Summary: rats and dogs of both sexes dosed orally with either 5 or 50 mg/kg 14 C-2,4-D; rates and routes of excretion and plasma toxicokinetics and biotransformation were assessed Results Summary: elimination from rat plasma significantly faster than in dog; t 1/2 were 1.3-3.4 hrs for rat and 99-134 hr for dog following 5 or 50 mg/kg, respectively; AUC for rat 21-57 μ eq.hg -1 and 4889-5298 μ eq.hg -1 for dog at 5 mg/kg; 122-2358 μ eq.hg -1 for rat and 34100-44296 μ eq.hg -1 for dog at 50 mg/kg; major route of excretion for rat was in urine; essentially complete after 24 hrs (low)/48 hrs (high) dose; elimination incomplete in dog over sampling period (5 0% of dose recovered); ur ine principal route at low

### Page 140 of 186 dose, but equal amounts excreted in urine and feces at high dose over 120 hrs; body burden of 2,4-D in dog significantly higher than in rat for an equivalent dose, which is consistent with the increased sensitivity of dog to 2,4-D toxicity.

CONCLUSION: In the case of certain organic acids (2, 4-D) , the rat is a better model for assessing the relevance to man of the toxicity of these compounds than the dog.

COMMENT/ HED : these data were considered previously; the toxicological endpoints for use in human risk assessment for 2,4-D were selected from the rat da tabase, based on the determination that the rat is a better predictor than the dog of the potential toxicity of 2,4-D to man; the data at 50 mg/kg in the rat suggested the clearance m echanism was saturated.

CITED BY: Task Force: used this reference in discussion of lactational exposure (Sturtz 2006 study)

where ↓ pup BW observed at 15 mg/kg/day; TF contends that this effect level was not replicated in 2009 Marty dietary life-stage study, wh ere BW decreases were not observed at 400 ppm (25, 28, 37-57 mg/kg/day); were observed at 800 ppm, which occurred under conditions in which 2,4-D pharmacokinetic behavior in lactating dams was distinctly non-linear, thus rendering these responses of limited relevance to human risk assessment; also, cited w ith respect to Sturtz (2000) citation by NRDC regarding lactational exposure at 50, 70, 100 mg/kg/day; toxicity observed in the resulting non-linear pharmacokinetics range

does not inform human risk assessment because n onlinear doses are unlikely to occur in humans.



### BIN: MUTAGENICITY

REEVALUATION of the GENETIC TOXICOLOGY PROFILE on 2, 4-D

Eighteen recent Genetic Toxicology publicatio ns were referenced by NRDC in the document submitted to the Agency. These articl es have been retrieved and reviewed in conjunction with a revisit of a ll of the mutagenicity data presented in the fourth Cancer Peer Review of 2, 4-D. The new information as well as the revisit are discussed below:

A. NEW GENETIC TOXICOLOGY STUDIES on 2, 4-D

1. Genetic Toxicology assays of th e Industry Task Force II on 2, 4- Dichlorophenoxyacetic acid (2, 4-D )

A series of Genetic Toxicology assays, wh ich was supported by the Industry Task Force II on 2,4-Dichlorophenoxyacetic acid (2,4-D, 96.1%), were performed on 2,4-D acid and

seven salts and/or esters listed as:

2, 4-D DEA = 2, 4-D diethanolamine salt (73.8%) 2, 4-D BEE = 2, 4-D 2-butoxyethylester (95.6%) 2, 4-D DMA = 2, 4-D dimethyamine salt (66.2%) 2, 4-D EHE = 2, 4- D 2-ethylhexyl ester (98%) 2, 4-D IPA = 2, 4-D isopropylamine (50.15%) 2, 4-D IPE = 2, 4- D isopropyl ester (81.6%) 2, 4-D TIPA = 2, 4-D triisopropanolamine (70.9%)



### The test articles were evaluated in five studies (4 in vitro: gene mutation in bacterial and mammalian cells, chromosome aberrations in mammalian cells, and unscheduled DNA synthesis in primary rat hepatocytes and 1 in vivo : mouse bone marrow micronucleus assay). Only 2, 4-D IPA, 2, 4-D TIPA a nd 2, 4-D BEE were tested in the mammalian cell culture gene mutation and chromosome ab erration assays. Only 2, 4-D was assayed in the second in vivo test system ( in vivo rat hepatocyte UDS). All assays used the protocols in the US EPA pes ticide guidelines (US EPA, 19 91). The summarized results are presented in Table 1 and each study is discussed below.

### Page 141 of 186 Charles et al (1999a)

Charles et al (1999a) reported the results of the independently conducted Ames test plate incorporation and the preinc ubation modification to the plate incorporation assay. Additionally, independe ntly performed the in vitro primary rat hepatocyte unscheduled DNA synthesis (UDS) assay and the in vivo test procedure with he patocyte treated with 2,4-D acid were investigated. For the Ames assay, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 were exposed to 5 to 7 dilutions of the test articles in the presence or absence of Aroclor 1254-induce ra t liver homogenates. Note: TA 1538, which is cons idered a duplicate of TA98, was not used for all test articles. All bacterial assays were performed for each test ag ent with a high concentration in excess of the limit dose for this test system (5000 μg/plate) or up to a cytotoxic concentration; negative (solvent) and the appr opriate positive controls were included. A minimum of triplicate plates per dose per stra in per condition was also included in each assay. Representative results are presented in Table 1. As shown none of the eight test articles induced a mutagenic response in any of the tester strains at any test article concentration. All negative and positive controls responded in the expected manner. Accordingly, all bacterial assays were cons idered negative and fulfill the regulatory requirements for acceptable bacterial gene mutation assays

. In vitro UDS assays were conducted with isolate primary rat hepatocytes exposed to th e five to six test article dilutions using solubility or cytotoxicity as the starting concentra tion. Hepatocytes were

doses for 18-20 hours and scored for net nucl ear grain (NNG) counts and the percentage

of cells with ≥ 5 NNG. Data from these assays can be seen in Table 1 and clearly show that neither 2, 4-D acid nor its salts or esters induced a genotoxic effect. By contrast, the positive control induced marked increases in NNG and the percentage of cells with ≥ 5

NNG. Accordingly, these assays are negative and fulfill the regulatory requirements for acceptable in vitro UDS assays.

Only 2, 4-D acid was tested in the in vivo UDS assay conducted with male Wistar Han rats administered single oral gavage doses of 400 and 1000 mg/kg. Five animals per dose received the selected test material doses, vehicle (1% methylcellulose) or positive

controls and were sacrificed at 2-4 and 12-14 hours after dosing. Hepatocytes were recovered from three animals per group, cultured with [ 3 

### H] thymidine and scored for

NNG. Dose selection was based on the findi ngs of a preliminary range finding study with 0, 600, 800 or 1000 mg/kg. Clinical signs of toxicity in the range finding study were

loss of balance and red stains on the nose and mouth “shortly after dosing” with 600 mg/kg. At 800 mg/kg, lethargy, abnormal br eathing, unsteady gait and rigid body tone were seen. Similar signs were observed at 1000 mg/kg within 5 hours and two rats were killed in extremis after 3 days (Bus, 2009). Accordingly, 1000 mg/kg was selected as the maximum tolerated dose (MTD). Findings from the main test indicated that 2, 4-D did not increase the frequency of NNG at any dose or treatment time.

Gollapudi et al., 1999



### Gollapudi et al.(1999) continued the Indus try Task Force II efforts on 2, 4-D and

### Page 142 of 186 investigated several 2, 4-D derivatives [ 2, 4-D BEE (95.6%), 2, 4-D IPA (50.15%) and 2, 4-D TIPA (70.9%)] in mammalian cells performing the Chinese hamster ovary (CHO) cell forward gene mutation assay at the HGPRT locus and the rat lymphocyte in vitro cytogenetic assay. For the CHO/HGPRT a ssay, concentrations ranges of 150 to 1400μg/mL +/-S9 (2, 4-D BEE), 500 to 3000 μg/mL +/-S9 (2, 4-D IPA), or 800 to 5000 μg/mL +/-S9 (2, 4-D TIPA) were tested. D uplicate cultures per concentration were exposed to the selected test material dos es and the appropriate solvent and positive controls for 4-5 hours, allowed a 7-9 day expression time, sele cted and plated for mutants and survivors. Each experiment was independe ntly repeated at least once. The highest concentration of each test agents was selected to represent either to the limit dose of 5000 μg/mL for the test system (2, 4-D TIPA), the limit of solubility (2, 4-D BEE) or severe cytotoxicity (2, 4-D IPA +/-S9). None of the 2, 4-D deriva tives induced a mutagenic

effect in CHO cells in ei ther the absence or presence of S9 activation at any

concentration tested.Page143of186Table 1. Results of US EPA –Pesticide Assessment Guideline Genetic Toxicology Studies Supportedby the Industry Task Force II on2, 4-Dichlorophenoxyacetic acid (2,4-D) and salts and estersTest Assay2,4-D acid 2,4-DDEAa2,4-DDMAa2,4-D IPAa2,4-D TIPAa2,4-D BEEa2,4-D EHEa2,4-D IPEaReferenceAmes Neg up tocytotoxiclevels(≥6410μg/plate –S9;(≥9610μg/plate+S9Neg up to10,332μg/plate+/–S9Neg up to6620μg/plate+/–S9Neg up tocytotoxiclevels (≥5010μg/plate +/–S9)Neg up to7090 μg/plate+/–S9Neg up to acytotoxic level(4780 μg/plate+/–S9)Neg up to 9800μg/plate +/–S9Neg up tocytotoxic levels(≥2428 μg/plate–S9; (≥4855μg/plate +S9Charles etal., 1999CHO/HGPRTGene mutation (invitro)ND ND ND Neg. up to acytotoxic dose(3000μL/mL+/-S9)Neg. up to thelimit dose(5000μL/mL+/-S9)Neg. up to thelimit ofsolubility(1400 μL/mL+/-S9)ND ND Gollapudi etal., 1999Chromosomeaberrations ratlymphocytes (invitro)ND ND ND Neg. up tocytotoxicdoses (3068 –S9 3000μL/mL+S9)Neg. up to athe limit dose(5000μL/mL+/-S9)Neg. up to thelimit ofsolubility(1400 μL/mL –S9) orcytotoxicity(700 μL/mL+S9)ND ND Gollapudi etal., 1999Mouse BoneMarrowMicronucleusNeg to theMTD (400mg/kg)Neg to theMTD (443mg/kg)Neg to theMTD (397mg/kg)Neg to theMTD (376mg/kg)Neg to theMTD (542mg/kg)Neg to theMTD (375mg/kg)Neg to the MTD(500 mg/kg)Neg to the MTD(400 mg/kg)Charles etal., 1999bUDS 1°rathepatocytes (inNeg up to96.9Neg up to369Neg up to66.2 μL/mLNeg up to250.5μL/mLNeg up to354.5μL/mLNeg up to478μL/mLNeg up to 24.5μL/mLNeg up to194.2μL/mLCharles etal., 1999aPage144of186Test Assay2,4-D acid 2,4-DDEAa2,4-DDMAa2,4-D IPAa2,4-D TIPAa2,4-D BEEa2,4-D EHEa2,4-D IPEaReferencevitro)bμL/Ml μL/mLRat hepatocyteUDS (in vivo)Neg at 400& 1000mg/kg,MTDNDNDNDNDNDNDNDCharles etal., 1999aa2, 4-D DEA = 2, 4-D diethanolamine salt 2, 4-D BEE = 2, 4-D 2-butoxyethylester2, 4-D DMA = 2, 4-D dimethyamine salt 2, 4-D EHE = 2, 4-D 2-ethylhexyl ester2, 4-D IPA = 2, 4-D isopropylamine 2, 4-D IPE = 2, 4-D isopropyl ester2, 4-D TIPA = 2, 4-D triisopanolamineMTD = Maximum tolerated dosebTested to a cytotoxic level or the limit of solubility.

### Page 145 of 186 For the in vitro chromosome aberration assay, duplicate cult ures of rat lymphocytes were treated, 48 hours after culture initiation, fo r 4 hours with comparable levels of the three test articles and the appropriate solvent and positive controls for 4 hours. Cells were harvest 24 or 48 hours after the termination of treatment; slides we re prepared, staine d and coded. Fifty metaphases/duplicate cultures were score for structural chromosome aberrations; the Mitotic Index (MI) was determined based on the count of 1000 cells/replicate culture. Based on the MI, three concentrations of each agent were scored for aberrant chromosome morphology. The highest concentration of each test agents was selected to repres ent either the limit dose of 5000 μg/mL for the test system (2, 4-D TIPA), severe cytotoxicity, indicated by a ≥ 50% decrease in the MI, (2, 4-D IPA +/-S9) or the limit of solubility (2, 4-D BEE -/+ S9). None of the 2, 4-D derivatives induced a clastogeni c effect in rat lymphocytes in either the absence or presence of S9 activation at any concentration tested . Accordingly, these assays are negative and fulfill the regulatory re quirements for acceptable in vitro mammalian cell gene mutation and chromosome aberration assays.

Charles et al (1999b)

The final publication in this se ries of Industry Task Force II Genetic Toxicology investigations on 2, 4-D details the in vivo mouse bone marrow micronucleus assays conducted with 2, 4-D (96.1%), 2, 4-D DEA (73.8%) 2, 4-D DMA ( 66.2%), 2, 4-D IPA (50.15%), 2, 4-D TIPA (70.9%), 2, 4-D BEE (95.6%), 2, 4-D EHE (98%), a nd 2, 4-D IPE (97.1%). Doses selected for the assays were based on the findings of mortalit y in groups of three male and three female administered 3 to 5 oral gavage doses of each ag ent. In the main study, groups of 5 male and 5

female mice/dose/sacrifice interval s received a single administrati on of three selected doses of each test agent via oral gavage. Animals were sacrificed at 24, 48 and 72 hours postdosing and bone marrow cells were harvested, processed, spr ead on to slides, stained and scored; there was

no indication whether slides were coded prior to analysis. A ppropriate vehicle and positive controls were included in each assay. The incidence of micronucleated polychromatic erythrocytes (MPCE) and the PCE to normochromatic (NCE) ratio were determined by analyzing 1000 PCEs/animal.

All test agents were examined up to the maximum tolerated dose (MTD), based on mortality; the generally comparable MTDs estimate for each test agent were:

• 400 mg/kg 2, 4-D 400 mg/kg 2, 4-D IPE

• 443 mg/kg 2, 4-D DEA 376 mg/kg 2, 4-D IPA

• 397 mg/kg 2, 4-D DMA 542 mg/kg 2, 4-D TIPA

• 500 mg/kg 2, 4-D EHE 375 mg/kg 2, 4-D BEE

Results of the individual micr onuclei analysis indi cated that neither 2, 4-D nor its seven derivatives were cytotoxic to the bone marro w since all PCE: NCE ratios were generally comparable to the vehicle control values. Similarl y, none of the test treatments with 2, 4-D or its seven derivatives induced an aneugenic or clast ogenic effect at any dose or sacrifice time in

mouse bone marrow. Accordingly, the test agents were adequately tested and found to be negative in these acceptable assays that fulfill the regulatory requirements for in vivo mammalian erythrocyte micronucleus assays.

### Page 146 of 186 2. Assays in Yeast, Plants and Insects

Three publications were available that reported th e findings of investigat ions on genotoxicity and cytotoxicity of 2, 4-D in yeast and human tran sformed hematopoietic ce lls (Venkov et al., 2000); in transgenic plants (Fil kowski et al., 2003) and in Drosophila (Tripathy et al., 1993). Summarized results from these studies are pres ented in Table 2 and discussed as follows:

Venkov et al. (2000) In this assay, Venkov et al. (2000) exposed Saccharomyces cerevisiae D7 ts1 to 2, 4-D (100%)

test concentrations at 4, 8, or 16 mM (~900, 1800 or 3600 μg/m L, respectively) for 1 to 8 hours; cells were recovered and plated for survival and the reversion and mitotic gene conversion rates were calculated. Additionally, 100% pure phe noxyacetic acid, 4-chlorophenoxyacetic acid and 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) were tested in this yeast assay following a 4-hour treatment. Results show that 2, 4-D at 8 mM induced gene mutations and gene conversions at levels causing ~40% reduction in cell survival. The effect was time and dose dependent with the peak response occurring after 8-hours of treatment with 8 mM. No eviden ce of genetic activity was seen for phenoxyacetic acid or 2,4, 5-T; 4- chlorophenoxyacetic acid at 16 mM induced mitotic gene conversion but did not increas e reversion to isoleu cine protrotrophy.

These investigators also examined the cytoto xic effects of 2, 4-D (16, 160 and 1600 μM) and 2, 4, 5-T (16, 160 and 1600 μM) in human transforme d hematopoietic cells lines (LAMA-84 cells, derived from chronic myelogenous leukemia cells and U-937 cells, derive d from histocytic lymphoma cells). Both test agents were 100% le thal at the high concentration. At 160 μM 2, 4- D, cell survival was 13.4 and 52.5 % for the LAMA-84 and U-937 cell lines, respec tively. For the lowest dose of 2, 4-D, survival for both cell lines was ≥ 89%. The data i ndicate that 2, 4-D was slightly more cytotoxic th an 2, 4, 5-T since cel l survival at 160 μM 2, 4, 5-T was 41 and 53% for the LAMA-84 and U-937 cell lines, respectively. Based on these findings, the investigators concluded that 2, 4-D is cytotoxi c, mutagenic and recombinogenic in yeast and cytotoxic in transformed human cells. Data on the cy togenetic analysis of C57b1 male and

female mice is discussed under the section on chromosome aberrations and micronucleus

induction.

Filkowski et al. (2003)

Filkowski et al. (2003) exposed transgenic seedlings of Arabidopsis thaliana to 0, 3, 10, 30, 100, 200 and 300 μg/L 2, 4- D (>98%) for 3 weeks under standard growth conditions; the number treated was not reported. Plants were coll ected, stained with 5-bromo-4-chloro-3-indolyl glucuronide, incubated for 48 hours and bleached with ethanol. Intrachromosomal homologous recombinations and point mutations were scored as blue sectors on white pl ants. It was indicated

that “1000 ‘mutation’- and 200 ‘recombinatio n’-monitoring plants were scored”. The experiment was repeated three times. Data were only presented in graphs; thus variability in the

data could not be ascertained. The mid-to lo w-doses of 2, 4-D (3, 10 and 30 μg/L) but not the high doses (100 and 300 μg/L) induced significant (p<0.05) increases in A → G point mutations; no explanation was provided for the lack of an eff ect at the high doses. Results also show that significant (p<0.05) increases in the induction of homologous re combinations (suggestive of DNA double strand breaks) occurred at all tested c oncentrations of 2, 4-D; a clear dose response was, however, not evident. The authors stated, “...the in crease in the recombination frequency at low concentrations of 2, 4-D correlated with the increase in the levels of A → G point mutations”.

### Page 147 of 186 Tripathy et al. (1993)

In a Drosophila melanogaster wing spot test and se x-linked recessive lethal assay, Tripathy et al. (1993), exposed second- and third- instar mwh + / + fl 3 

### transheterozygous larvae to 2.5, 5.0, 10.0 mM 2, 4-D (98%) prepared in 3% ethanol + 1% Tween 80 and in corporated into the feeding medium for 48 and 72 hours (i.e., “t he rest of their larval life”); the high dose represents 50% of

the LD 50. 

### Following exposure, 40 wings/treatment or c ontrol group of “eclosed” flies (i.e., flies that have wings that are highly folded and co mpact; within an hour, each wing expands) were examined for the type and number of spots. Duplicate experiments were performed; the data were pooled because there were no statistically significant differences among experiments.

In the sex-linked recessive lethal assay, Oregon- R larvae were fed diets containing comparable doses of 2, 4-D as described for 3 days; male flies were mated to 3 Basc virgin females for 3 days to obtain the F 1 

### Basc/ Y siblings (Brood 1). Brood 1-males and females were mated (1:1) to produce Brood 2 (Brood 2 represents the sampling of germ cells th at were primarily late or middle spermatids). X chromosomes fr om 60-65 males/test group (~1230-1330 X chromosomes/test group) and 150 pooled vehicle controls (3130 X chromosomes) were scored;

the X-linked recessive lethals were calculated. D uplicate experiments were performed; the data were pooled because there were no statistical ly significant differences among experiments.

Results indicated that after 48 hours of expos ure to 5.0 and 10.0 mM 2, 4-D, a significant (p<0.05) and dose-related increase in the number of spots/per wing (small si ngle and large single wing spots) as well as the tota l spots was induced. Following 72 hours of exposure, significant increases were obtained only at the high dose for small single and large single and twin wing spots and total spots. The low dose (2.5 mM) was not active. Based on these data, it was concluded that 2, 4-D induced somatic cell genot oxic damage in the primordial wing cells of Drosophila

.

Significant (p<0.05) and dose-related increases in th e lethal frequencies were calculated at 5.0 and 10.0 mM 2, 4-D (48-hour exposure ) and at all doses (72-hour e xposure). Acco rdingly, the

authors concluded that in addition to somati c cell damage, 2, 4-D is also genotoxic in the male germinal cells of Drosophila

.

3. Cytogenetic and Micronucleus Assays in Human Lymphocytes

Four assays were available on cy togenetic evaluations of structur al chromosome aberrations or micronucleus induction in human ly mphocytes. Findings from thes e studies are summarized in

Table 2 and discussed below:

Figg et al., 2000

Figg et al. (2000) found no evidence of micronuclei (MN) induction in the peripheral lymphocytes of 12 applicators spraying only 2, 4-D during a 6-month pe riod. In this study, 12 applicators were compared to a control group consisting of 9 cont rols (matched for gender, age, alcohol and tobacco uses, general health and geographical location). Blood and urine samples were collected prior to spraying and at every other week after a typical day of 2,4-D application. The concentration of 2, 4-D in the collected urinary samples was determined. Lymphocyte cultures were grown for 44 hours, and Cytoch alasin B was added to prevent complete cytokinesis. At 72 hours, ly mphocytes were separated; slides were prepared, stained with antikinetochore antibody and coated with fluores cent anti-goat human IgG, counterstained with

### Page 148 of 186 the DNA dye, 4’, 6-diamidino-2-phe nylindole, refrigerated for 1 week, coded and scored. At least 1000 binucleated lymphocytes were scored for micronuclei; cell division kinetics was calculated for the count of 400 cells per sample (duplicate cultures). Th e replicative index (RI) was determined by calculating th e percentage of cells with one , two, three or more nuclei per individual. Hematological end points such as hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentra tion, red cell count, white blood cell count and leukocyte differential examinations were also conducted. The 2, 4-D concentration ‘grand mean among all applicators after spraying 2, 4-D was 240 ± 100 (±SE) ppb. The authors stated, “Urinary concentrations of 2, 4-D increased as hours spent spraying the herbicide increased.” Consequently, the 12 individuals were grouped according to hours spent spraying 2, 4-D according to the following urinary concentrations : <20 ppb (4 subjects), 20-40 ppb (3 subjects) or > 40 ppb (5 subjects). Results of the ly mphocyte analysis reveal ed that there was no significant increase in the incide nce of micronuclei in the exposed workers. However, the replicative index (RI) for lymphocytes was signifi cantly (p<0.05) higher in applicators compared to non-applicants and in applicators after spra ying compared to before spraying (p<0.016). It

was stated that the increase in RI suggests “stimulated cell grow th that could contribute to carcinogenesis.” Nevertheless, the authors concluded th at their findings did not support a “genotoxic pathway” for 2, 4-D at me an urinary levels of 12 to 1285 ppb.

Holland et al., 2002

In the in vitro micronucleus assays of Holland et al. (2002), two experiments were conducted with either 2 or 5 volunteers (t otal of 3 females and 2 males, nonsmokers, 26- 45 years). Note: one of the subjects from the first test particip ated in the second test. Whole blood cultures as well as isolated lymphocyte assays were examin ed in the first test; th e second test only used isolated lymphocytes. Collection of blood sample s, culture preparation, treatment conditions and scoring of binucleated lymphocytes was conducted as described above in Figg et al. (2000). The only procedural exceptions were that lymphocytes smears were not stained with antikinetochore antibody or coated with anti-goa t human IgG and MIs were determined from the count of 1000 cells/culture. In the first test, samples of w hole blood and isolated lymphocytes were exposed in

vitro to pure (98%) 2, 4-D at c oncentrations of 0.001-1.0 mM a nd analyzed for micronucleus induction. In the second test, only isolated lymphocytes were exposed to both pure 2, 4-D or commercial 2, 4-D (9.4%2, 4-D but adjusted to 98% pure 2, 4-D) at concentrations of 0.005 and

0.3 mM.

Cultures exposed to concentra tions ranging from 0.001- 1.0 mM of pure 2, 4-D (98%) for 4 hours showed only “a minimal increase in MN...at doses approaching cytot oxic levels” in either whole blood or isolated lymphocytes. Similar fi ndings were obtained for the commercial product at 0.3 mM 2, 4-D in isolated lymphocytes. Howe ver, the study authors conceded that these increases were within the “lower range of normal MN for healthy adults”. RIs were significantly (p<0.001) decreased (~19%) at 0.3 mM of either pure or commerci al 2, 4-D, regardless of the solvent (ethanol or culture medium) used. Thes e data suggest a cytotoxic effect. Although Holland et al. (2002) claimed that 0.005 mM of commercial grade 2, 4-D induced an increase (nonsignificant) in the RI of isol ated lymphocytes, an examination of the data in Table 1 of this paper revealed that the pooled average RI for both commercial 2,-4 –D groups (10 samples) was 1.65 vs. 1.62 for the pooled solven t control groups. This “incr ease” cannot be considered evidence of a proliferat ive response. Similarly, MIs were un affected by treatme nt with 0.005 or 0.3 mM 2, 4-D (commercial or pure). Thus, the l ack of a meaningful incr eased RI at this low dose is supported by data show ing that MIs were unchanged. The overall conclusions are that 2, 4 –D, regardless of the formulation, did not induce micronuclei in human lymphocytes

### Page 149 of 186 up to levels approaching cytotoxicity.

Zeljezic and Garaj-Vrhovac, (2004 )

Zeljezic and Garaj-Vrhovac (2004) exposed hu man lymphocytes, harvested from two young, healthy non-smoking volunteers, to Deherban A®, a commercial formulation of 2, 4-D adjusted to contain 0.4 or 4 μg/mL 2, 4-D in the absence and the presence of S9. The S9 fraction was

derived from the livers of male Sprague-Dawley rats treated with intraperitoneal (ip) injections of Deherban A® adjusted to contain 500 mg/m L 2, 4-D for 3 consecutive days. Lymphocytes were treated for 24 hours, recovered, processe d, stained with Giemsa and scored (1000 metaphases/concentration) for chromosome aberra tions. Similar cultures were treated for 24 hours with 0.4 or 4 μg/mL 2, 4-D +/-S9; cytoch alasin B was added and cells were harvested, processed, and stained as pr eviously described for the in vitro micronucleus assay. Cells were scored (1000 binucleated lymphocytes/conc entration) for micronuc lei and nuclear buds. Appropriate solvent and positive control cultures were included. The arti cle did not indicate

whether slides were coded to elim inate bias prior to scoring.

Both concentrations of 2, 4-D induced statistically significant (p<0.05) increases in the percentage of cells with aberrant chromosome aberrations (primarily chromatid and chromosome breaks) in lymphocytes from both donors. The nu mber of micronuclei and nuclear buds at both

nonactivated and S9-activated test material co ncentrations was also significantly increased (p<0.05) at 0.4 and 4 μg/mL +/-S9 in cells also harvested from two different donors. The cytogenetic, micronuclei and nuclear bud response s were uniformly and significantly (p<0.05) higher in cells treated in the presence of S9 at both test levels. Hence, it can be concluded that S9 activation was not required to demonstrate the clastogenic effect induced by 2, 4-D in this study.

Venkov et al. (2000) As part of the analysis of 2, 4-D and other substituted phenoxyacetic acids in yeast and cytotoxicity assays in human transformed hemato poietic cells (see Secti on, Assays in Yeast, Plants and Insects), Venkov et al. (2000) also in vestigate 100% pure 2, 4-D and 2, 4, 5- trichlorophenoxyacetic acid (2, 4, 5-T) for the in duction of structural chromosome aberrations following single ip administrations of both agents at 3.5 and 4.0 mg/kg, respectively. Accordingly, bone marrow cells were harvested fro m groups of four male and four female mice at 12 and 24 hours postdosing. The appropriate ve hicle (0.9% NaCl) and positive controls were included. At least 100 metaphases per animal we re scored for aberrant structural morphology

and the MIs were determined from the count of 1000 cells per animal. It was not stated whether slides were coded prior to anal ysis. 2, 4-D at 3.5 mg/kg induced significant (p<0.0.5) increases in the frequency of cells with aberrations at both sacrifice times (3.4 and 5.5% vs. 1.5 % for the vehicle control at 12 and 24 hours, respectively); the predominant type of aberration induced by 2,4-D was chromatid breaks, suggesting cytotoxic ity, and exchanges sugges ting clastogenicity. 2, 4-D also reduced the MIs by 69% at 12 hours and 35% at 24 hours. By c ontrast, 2, 4, 5-T was neither cytotoxic nor clastogeni c in bone marrow cells harveste d 12 postexposure to 4.0 mg/kg. Based on the findings from this investigat ion, the authors concluded that 2, 4-D was clastogenic in mouse bone marrow cells when administered via ip injection.

4. DNA Damage Assays

Five studies were found in the open literature investigating the potential of 2,4-D to induce DNA damage; these include two studies on sister chro matid exchange (SCE) assays in chick embryos

### Page 150 of 186 performed by Arias (2003 and 2007), SCE and co met assays in CHO cells by Gonzales et al, (2005), SCE induction in human whole blood and leukocytes (Sol oneski et al., 2007) and SCE induction in mouse bone marrow and spermatogonia (Madrigal-Bujaidar et al., 2001). Findings from these publications are also summar ized in Table 2 and discussed below:

Arias 2003, 2007

In the first study, concentrations of 0, 0.5, 1, 2, and 4 mg/embryo of either the pure grade of 2,4- D (99%) or the commercial formulation, Agroxone containing 37% 2,4-D were injected into the air cell of White Leghorn chicke n eggs (9-12/dose). Treatment lasted for 3 days; eggs were automatically rotated at 37.8oC and BrdU (150 μg/e mbryo) was injected into the air cell. On Day 4, embryos were removed following a 2-hour exposure to colchicine (0.05%). Chromosome preparations were made from harvested embryos and slides were staine d using the fluorescentplus-Giemsa technique; the report did not indicate whether slides were coded. A total of 30-40 second-division metaphases were scored for SCE in the first five pairs of macrochromosomes. The frequency of first (M1), se cond (M2) and third (M3) divi sion metaphases from 200 cells were scored for mitotic delay and MIs were determ ined from the count of 1000 cells. No tabular presentation of the data was available. Results , shown in graphs, indicated a significant (p<0.05) increase in SCEs for the high concentration of the commercial formulation (~1.43 SCEs/ cell vs. ~1.22 SCEs/cell for the solvent control, acetone ) and a nonsignificant in crease (~1.41 SCEs/ cell vs. ~1.22 SCEs/cell for acetone) for pure 2,4-D. Although the dose respons e was concentration related for both test materials, the increase at the high dose was only 1.2-fold higher, compared to the solvent control and 1.2-fold compared to the average spontaneous background of 1.15 SCEs/cell. Additionally, data were presented as the mean and standard error of the mean, which suggests wide variation in the data. The inves tigator also stated th at cell cycle delay was observed at the high concentration (2 and 4 mg /embryo) and that the MI was significantly (p<0.01) reduced by both test material at the hi gh dose (4 and 8% reductions for the commercial and pure 2, 4-D, respectively). Based on these considerations, the values for SCE induction, cell cycling and MIs provide questionable evidence of a positive response.

For the second study, Arias (2007) used the same purity of the test materials at similar concentrations and the same protocol with th e exception of including a 7- and 10-day extended exposure time and increasing the sample size to 35 embryos/concentration. Results were similar to the previous study showing “s ignificant (p<0.05)” increases in SCEs/cell at 4.0 mg/embryo (1.1-fold increase) at 4, 7 and 10 days for th e commercial product an d at 4.0 mg/embryo (1.1 fold-increase) at 7 and 10 days for pure 2,4-D. Similarl y, slight but significant (p<0.01) effects were reported on cell cycle delay and the MI ( ≈ 23.3 for the commercial product and ≈ 23.1 for the pure substance at the high dose vs. 24 for acet one), which was not associated with prolonged

exposure. Although these effects were statistically significant, the biological relevance is

unclear.

Gonzalez et al., 2005

Gonzalez et al. (2005) evaluated 2,4-D (purity not reported) and commercial grade 2,4-D DMA (containing 60.2% 2,4-D as the acti ve ingredient) for the ability to cause DNA damage in CHO cells using the SCE and Comet tests. Concentra tion ranges of 2- 10 μg/mL were used in both assays in the absence of S9 ac tivation; treatment times for th e SCE assay were 24 and 36 hours

and 90 minutes and 36 hours for the comet test. For both the SCE assay and the Comet test,

### Page 151 of 186 duplicate cultures were prepared for each dose; three independent experiments were conducted; and cultures treated with the solvent (acetone) were included. The percentage of cells in M1, M2 and M3 division in 200 metaphases/sample, the pr oliferative rate and the MIs/2000 cells were determined. There were no significant differences for cell-cycle progression or alterations in the rate of cell proliferation. By contrast, 2, 4-D at 24 hours caused 25 and 35% reductions in the MI at 6 and 10 μg/mL; respectively, and 42 and 46% reductions at 6 and 10 μg/mL, respectively (36 hours). For 2, 4-D DMA, reduction of ~38 % (24 hours) and ~28% (36 hours) were recorded for both 6 and 10 μg/mL. SCE data were presented in graphs and showed significant (p<0.01) and

dose-related increases in SCE/cell at all levels (2, 6 and10 μg/mL) for both test materials, regardless of time. The fold increase at the high dose was 1. 5 and 1.8 X for 2,4-D at 24 and 36 hours, respectively and 1.6 and 2.1 X for 2,4-D DMA, at 24 and 36 hours, respectively. Similar evidence of DNA damage was seen in the comet test with significantly (p<0.01) increased and dose-related percentages of cells with tail comets, indicative of DNA damage, at 2.0, 6.0 and 10.0 μg/mL in the absence of severe cytotoxicity ( ≥ 85% viability) at 90 minutes. At 36 hours, regardless of the test material, all cells had tail comets. These data provide clear and reproducible evidence of the DNA-damaging ac tivity of 2, 4-D and the DMA derivative

.

Soloneski et al., 2007

In another study of SCE induction, Soloneski et al. (2007) assayed 2, 4-D (purity not reported) and commercial grade 2, 4-D DMA (purity not listed) at levels of 10 to 100 μg/mL in human whole blood (WBC) and plasma leukocyte cultu res (PLC). Treatment time was 72 hours. Duplicate cultures were prepared for each dose or the solvent control, acetone and three independent experiments were conducted. Smears were prepared and stained as previously described and metaphases were scored blind fo r cell cycle progression, MIs and SCE induction. Results show that both test materials significantly (generally p<0.05) affected cell cycle progression in WBCs at doses ≥ 25 μg/mL (note: the 100-μg/mL group was not scored for 2, 4- D) and only at 100 μg/mL (2, 4-D) for PLCs. Mean SCE freque ncies were also significantly (p ≤ 0.05) increased at 10, 25 and 50 μg/mL (2, 4-D) and at 25, 50 and 100 μg/mL (2, 4-D DMA) when WBCs were tested. No activity was s een at any dose when PLCs were examined. Significant reductions in the pro liferative rates and th e MIs were found in WBCs and PLCs after treatment with 50 or 100 μg/m L 2, 4-D, respectively. For 2, 4-D DMA reduced proliferative rates and MIs were noted at the high dose but onl y in WBC. The discrepancies in the data between WBCs and PLCs were considered by the authors to be related to the detoxificating properties of erythrocytes, whic h are present in the PLC cultur es but not the purified leukocyte

cultures. The authors, citing severa l investigators, further sp eculated that the cellular response caused by both test age nts in this study could be relat ed to oxidative damage since lipid peroxidation has been reported for 2,4-D.

### Page 152 of 186 Madrigal-Bujaidar et al., (2001)

In the study of Madrigal-Bujaidar et al. ( 2001), SCE induction in mouse bone marrow and spermatogonia were investigated. Groups of five male mice receiv ed oral doses of 0, 50, 100 or 200 mg/kg 2,4-D (98.95%) in alkalinized H 2 

### O (pH 10.5) or the vehicle, followed at 2 hours by an intraperitoneal (ip) injection of BrdU/charcoal and at 21 hours by an ip injection of colchicine. Bone marrow cells were harvested, stained by the fluorescent-plus-Giemsa technique, and scored; it was not reported whethe r slides were coded prior to scoring. Thirty second-division metaphases/animal were scored for SCE induc tion; the MIs, cell cycle delay and average generation time were also determined. For th e examination of spermatogonial cells, groups of

five male mice received the test material and BrdU/charcoal as described; cell division was halted by the subcutaneous injec tion of colchicine at 51 hours after BrdU administration and testes were harvested 4 hours later. Prepared slides were stained as described and 25 mitosis /animal were scored for SCEs. The presented data show that there were no adverse effects on the MI, cell cycle progression on the average generati on time in the bone marrow cells. By contrast, significant (p<0.05) and dose-relate d increases in SCE induction (2 -fold or approaching 2-fold increases) at 100 and 200 mg/kg were recorded for both cell types. It is also of note that the authors speculate that the fate of the genotoxic damage on the spermatogonial cells is unclear since “teratogenic and embryotoxi c studies on rodents treated with 2,4-D have given conflicting results and the dominant lethal mutation assay on mouse and rats has produced negative data. Thus cell repair, cell lethality or zygote damage remains as three possible fates of the observed genotoxic alterations in spermatogonia.” Similar sp eculations can be raised for the fate of DNA damage in bone marrow cells since chronic a nd oncogenic studies in rats and mice were negative. Thus, this somatic cell damage may be repaired or result in cell death.

5. Other Mechanisms

Two articles were placed in this category: the malignant cell transformation in Syrian hamster embryo (SHE) cells by Maire at al . (2007) and the two studies on apoptosis (Maire at al., 2007 and Tuschl and Schwab, 2003). The article of Mair e at al. (2007) also cont ains the results of a RT-PCR determination of gene expression analysis in SHE cells as well as a comet assay of DNA damage. These findings are summarized in Table 2 and discussed below:

Maire et al. (2007)

In a series of in vitro investigations, Maire et al. (2007) exposed Syrian hamster embryo (SHE) cells to 2, 4-D (98%) at concen trations of 0, 4.5, 11.5, 23 or 34 μM (equivalent to 0, 1, 2.5, 5 or 7.5 μg/mL, respectively) and evaluated the cultur es for malignant cell transformation, apoptosis, expression of the c-myc gene and gene product, and DNA damage (Comet test). For the cell transformation assay, X-irradiated SHE feeder ce lls were seeded and grow n for 25 hours. Target cells were seeded on top of the feeder cells, allowed a 24-hour s period of quiescence and 10

dishes /concentration were treated with the selected test article levels of 2, 4-D for 7 days. At the end of exposure, dishes were washed, fi xed and stained. Appropriate solvent (H 2 

### O) and positive controls were included in each assay. One th ousand colonies/concentration were counted and examined for morphology; cloning efficiency (CE) and the transformation frequency (TF) were

determined.

For apoptosis, cells were also sampled 48 hours after seeding, tr ypinized, reseeded and treated with of 0, 4.5, 11.5 and 23 μM 2, 4-D for 5 and 24 hour s. After treatment, cells were suspended in culture medium deprived of growth factors fo r 3 hours, harvested and ce ntrifuged. Cell pellets

### Page 153 of 186 were stained with fluorescent acridine orange and ethidium bromide, mixed, placed on slides, and scored for the number of apoptotic cells/1 00 cells/slide. Three slides/concentration were

examined.

For the RT-PCR determination of gene expres sion, cells were again sampled 48 hours after seeding, trypinized, reseeded and treated with of 0, 4.5, 11.5 and 23 μM 2, 4-D for 5 hours. After treatment, total RNA was extracted and complementary DNA (c- DNA) was synthesized from 1μg of total RNA using oligo d (T) as a primer and reverse transcriptase. PCR amplification of the c-DNA was performed with c-myc, bcl-2 and bax primers specific to SHE cells. Preparations were denatured and PCR products were sequenced, cloned and electrophoresed. Signals were analyzed by dens itometry and data were expressed as density

ratios of the transcri pt corresponding to the c-myc gene. Three different experiments were

conducted with different batches of SHE cells. Western blot an alysis was conducted in parallel

with RT-PCR to assess effects on c-myc protein.

For the comet assay, SHE cells were trypsinized 48 hours after seeding, reseeded and treated with 0, 4.5, 11.5, or 23μM 2, 4-D for 5 and 24 hours. At least three experiments were performed and included in each assay were the appropriate solvent and pos itive control. Cells were recovered and electrophoresed; nuclear DNA was stained with et hidium bromide and scored blind. Cells (75/slide/concentra tion) were scored randomly and the percentage of DNA in the tail was determined as a measure of DNA damage.

Results indicate that 2, 4-D i nduced a dose-dependent and significant (p <0.01) increases in morphological cell transformation of SHE ce lls at 11.5 (2.4%) and 23μM (3.8%); relative cloning efficiency at 11.5 and 23μ M was 14%. No significant incr ease in transformed cells was observed at the high dose of 34 μM 2, 4-D. There were also no significant increases in apoptotic

cells 5 or 24 hours after exposure to the selected doses of 2,4-D. These da ta are supported by the lack of an effect on expression of bcl-2 and bax genes, which are associat ed with regulation of the apoptotic pathways. By contrast

, a significant (p<0.05) increas e in the up-regulation of c-myc RNA at 11.5 and 23 μM was reported; the increases represented 2.3-fold at these levels and immunoblotting confirmed the increase in the c-myc protein

. Thus, 2, 4-D induced over expression of c-myc at both the RNA and the protein level; this finding is consistent with the transforming activity of 2, 4-D at comparable doses . Data from the comet test revealed that treatment with 11.5 and 23 μM for 5 or 24 hours also induced a dose-dependent and significant (p<0.01) increase in the number of DNA-damaged cel ls. No dead cells were found at any dose

or harvest time.

Overall, the investigators concl uded that 2, 4-D induces morpho logic cell transformation in SHE cells at doses that that extend into the cytotoxic range and caused overexpression of cmyc (at both the mRNA and the prot ein level) and DNA damage.

Tuschl and Schwab (2003)

By contrast, Tuschl and Schwab (2003) found th at exposure of the human hepatoma cell line, HepG2 to 2, 4-D (purity no stated) in 1% ethanol induces apoptosis but only at high doses (4, 8 or 16 mM; equivalent to 0.9, 1.8 or 3.6 mg/mL) that induced severe effects on cell cycling,

increased necrotic cells and increased breakdown of mitochondrial membranes. The data in this article were only presented in graphic form. It is of note, however, that these doses are ≈ 1000X

### Page 154 of 186 higher than the doses reported by Maire et al. (2007). The relevance of the findings is unclear because of severe cytotoxicity at all levels.

Table 2. Summarized Results of Recent Geneti c Toxicology Studies on 2, 4- Dicholorphenoxyacetic acid (2, 4-D)

ENDPOINT CONCENTRATION

RANGE RESULT REFERENCE

ASSAYS in YEAST, PLANTS and INSECTS Saccharomyces

cerevisiae D7 ts1 gene mutations & gene conversions

Human

transformed hematopoietic

LAMA-84 & U937 cell lines 0, 4, 8, 16 mM 2, 4-D (100%) for 1-8 hrs

0, 16, 160 & 1600 μM 2, 4-D (100%) for 48 hrs Pos. ; Time & dose-dependent increases in gene mutations & gene conversions

100% lethal at 1600 μM; 13.4% survival (LAMA-84) & 52.5 % (U-937) at 160 μM & ≥ 89% survival (both cell lines) at 16 μM Venkov et al.,

2000

Transgenic Arabidopsis

thaliana point

mutations & homologous

recombination 0, 3,10, 30, 100, 200, 300 μg/mL for 3 weeks Equivocal; significant increases (p<0.05) in A → G point mutations at 3, 10, & 30 μg/mL but not at 100-300 μg/mL; no explanation provided.

Pos. ; significant increases (p<0.05) in induction of homologous

recombinations at a ll concentrations but not clearly dose-related. Filkowski et al.,

2003

Drosophila melanogaster wing spot test (somatic cell gene mutations)

Sex-linked

recessive lethal test (germinal cell

chromo-some damage) 0, 2.5, 5.0, 10.0 mM 2, 4- D (98%) larval feeding 48 and 72 hrs (wing spot test) 3 days (X-linked recessive lethal) Pos .; 5.0 & 10.0 mM induced significant & dose-related increases (p<0.05) in the number of small and large single spots & frequency of lethal

X chromosomes. Tripathy et al.,

1993

MICRONUCLEUS (Mn a ) AND STRUCTURAL CHROMOSOME INDUCTION

Mn in lymphocytes of 12 2, 4-D applicators sampled every

other week for 12

wks Urinary range: 12-1285 ppb Neg. No significant increases in Mn of lymphocytes harvested from 2, 4-D workers compared to matched controls but significant increases in RIs for applicators vs. controls. Figg et al., 2000

Mn in whole

blood & isolated lymphocytes from 5 volunteers ( in

vitro ) 2, 4-D (98%): 0.001- 1.0
mM; Commercial 2, 4-D (adjusted to contain pure 2, 4D): 0.005- 0.3 mM Neg. Minimal increases in Mn for whole blood & isolated lymphocytes at 0.1 mM (98% 2,4-D) & 0.3mM commercial 2,4-D but “within the normal range for healthy adults”. Nonsignificant increases RIs at 0.005 but not 0.3 mM;

at 0.3 mM RI were decreased ≈ 19% (both formulations); no effects on MIs at any dose. Holland et al.,

2002

Mn & structural

chromosome

aberrations in

isolated Deherban A®, adjusted to contain 0.4 and 4μg/mL 2,

4-D +/-S9. S9 derived

from the liver of male rats Pos. Significant (p<0.05) increases in chromosome aberrations (chromatid & chromosome breaks), micronuclei & nuclear buds at 0.4 and 4 μg/mL +/-S9. Zeljezic and Garaj-Vrhovac

2004

### Page 155 of 186 ENDPOINT CONCENTRATION

RANGE RESULT REFERENCE

lymphocytes from 2 volunteers ( in

vitro ) induced with 500 mg/kg 2,

4-D as Deherban A® for 3 days The response was ≈ 20% higher in the presence of S9.

Structural

chromosome

aberrations in

mouse bone marrow (M&F) 0, 3.5 mg/kg (single ip administration); 12 & 24

hrs. harvest times Pos Significant (p<0.05) increases in chromosome aberrations (chromatid breaks & exchanges) Venkov et al.,

2000

DNA DAMAGE

SCEs in chick embryos 0, 0.5, 1, 2, 4 mg/embryo 99% 2, 4-D & 0, 0.5, 1, 2, 4 mg/embryo 37% 2, 4-D as Agroxone; sampled 4 days post-treatment Neg. Reported to induce a dose-related increase in SCEs at 0.5-4 mg/embryo; significant increase (p<0.05) at 4 mg/embryo (commercial product only)

but the increases were only 1.1or 1.2X higher than the solvent control or the spontaneous background rate. Also significantly reduced (4-8%) MIs at the high dose for both products Arias 2003

SCEs in chick embryos 0, 0.5, 1, 2, 4 mg/embryo 99% 2, 4-D & 0, 0.5, 1, 2, 4 mg/embryo 37% 2, 4-D as Agroxone sampled 4, 7 & 10 days post-treatment Neg. Results similar to above study (i.e., significant increase (p<0.05) in SCEs at 4 mg/embryo (commercial product) at 4, 7, & 10 days & at 4 mg/embryo (pure 2,4-D) at 7 & 10 days but only 1.1X for both products. Arias 2007

SCE & Comet test

CHO cells 2- 10 μg/mL 2, 4-D (unknown purity) 2- 10 μg/mL 2, 4-D DMA (60.2% 2, 4-D); treatment times: 24 & 36 hrs (SCE)

& 90 min. & 36 hrs (Comet test) –S9 only. Pos. No significant effects on cell cycling time or RI; reductions in MIs: 25-35% at 6 and 10 μg/mL at 24 hrs & 42-46% at 6 and 10 μg/mL at 36 hrs (2, 4-D) ≈ 38% (24 hrs) & ≈ 28% (36 hrs) at 6 and 10 μg/mL (2, 4-D DMA). Significant (p<0.01) & dose-related

increases in SCE at 2, 6 and 10 μg/mL (both products) regardless of time. Comet assay : Significant (p<0.01) &

dose-related increases in cells with tail comets at 2, 6 and 10 μg/mL (both products) at 90 min. in the absence of severe cytotoxicity ( ≥ 85% viability) & all cells, regardless of product or dose

had 100% of cells with tail comets at 36

hours. Gonzalez et al.,

2005

SCE human whole blood (WB) & plasma leukocytes (PL) 10 – 100 μg/mL 2, 4-D (unknown purity) 10- 100 μg/mL 2, 4-D DMA (unknown purity);

treatment time 72 hrs. Pos. Significant (p<0.05) reduction in cell cycling in WB at ≥ 25 μg/mL (both products) & in PL at 100 μg/mL (2, 4-D only). Significant (p<0.05) reduction in MIs & proliferative rates at ≥ 50 or 100 μg/mLin WB or PL, respectively (2, 4- D) at 100 μg/mL only in WB (2, 4-D DMA). SCEs significantly increased at 10, 25 & 50 μg/mL only in PL (2, 4- D) & 25, 50 & 100 μg/mL only in PL (2, 4-D DMA). Soloneski et al.,

2007

SCE mouse bone

marrow & mouse spermatogonia 0, 50, 100 or 200 mg/kg 98.95% 2, 4-D in

alkalinized H 2 O (pH 10.5); oral gavage Pos. No effects on MI, cell cycling or average generation time. Significant &

dose–related increases in SCEs (both cell types) at 100 & 200 mg/kg. MadrigalBujaidar et al.,

2001

### Page 156 of 186 ENDPOINT CONCENTRATION

RANGE RESULT REFERENCE

administration; 5 males/group

OTHER MECHANISMS

Cell

transformation in

SHE cells

Apoptosis in SHE

cells

RT-PCR gene expression ( c-myc gene) in SHE cells

Comet test in SHE

cells 0, 4.5, 11.5, 23 or 34 μM

≈ 0, 1, 2.5, 5 or 7.5μg/mL 2, 4-D (98%) 7 days

0, 4.5, 11.5, 23 μM 2, 4-D (98%) 5 & 24 hrs

0, 4.5, 11.5, 23 μM 2, 4-D (98%) 5 hrs

0, 4.5, 11.5, 23 μM 2, 4-D (98%) 5 & 24 hrs Pos. Dose-dependent & significant (p<0.01) increases in morphological cell transformation at 11.5 & 23 μM; cloning efficiency 14% at both doses. Neg. No significant incr ease at any dose

or time.

Pos. Significant (p<0.05 up-regulation

of c-myc gene expression at 11.5 & 23 μM; confirmed at protein level by Western immunoblotting

Pos. Dose-dependent & significant (p<0.01) increases in the number of DNA damaged cells at 11.5 & 23 μM (both time points). No dead cells were found at any dose or time Maire et al., 2007

Apoptosis in human hepatoma cells (HepG2) 0, 4, 8 or 16 mM ≈ 0, 0.9, 1.8 or 3.6 mg/mL 2, 4-D (unknown purity) treated

4- 48 hrs. No conclusions for apoptosis; severe cytotoxicity at all levels Tuschl and Schwab (2003)

a 

### Abbreviations: Mn = Micronucleus SCE = Sister Chromatid Exchange RI = Replicative index CHO = Chinese Hamster Ovary cells MI = Mitotic index SHE = Syrian Hamster Embryo cells

6. Conclusions

Based on the analysis of the new studies in the op en literature it can be concluded that neither 2,

4-D nor its derivatives are mutagenic in bacteria ( Salmonella typhimurium ); however, there is convincing evidence of gene mutations in yeast ( Saccharomyces cerevisiae D7), plants ( Arabidopsis thaliana ) and possibly insects ( Drosophila melanogaster ). No assays were located

for in vitro mammalian cell gene mutation studies. 2, 4-D and its derivatives were negative in mouse bone marrow micronucleus assays up to th e MTD and did not cause UDS in primary rat hepatocytes. Similarly, 2, 4- D was not genotoxic in the in vivo rat hepatocyte UDS assay. Three derivatives of 2, 4-D (2, 4-D IPA, 2, 4-D TI PA and 2, 4-D BEE) we re not mutagenic or clastogenic in CHO cells or ra t lymphocytes. However, 2, 4-D was not tested in these assay systems. Nevertheless, four earlier in vitro chromosome aberration st udies of 2, 4-D showed conflicting results for chromosome aberrations (see Table 3). Contrary to the negative in vivo findings, 2, 4-D induced structur al chromosome aberrations, mi cronuclei and nuclear buds in human lymphocytes and was clastogenic in mouse bone marrow when delivered by ip injection. There are several studies showing DNA damage, as measured by SCE induction (3 studies) and positive comet assays (2 studies) in CHO cells, SH E cells or human leukocytes. Additionally, 2, 4-D, prepared in alkalinized H 2 

### O and delivered by oral gavage gave positive results in the in

vivo mouse bone marrow and mouse spermatogoni a SCE assay. Finally, 2, 4-D induced malignant cell transformation (an in vitro test of carcinogenesis) a nd overexpression of the c-myc at both the mRNA and protein level. The induction in c-myc expression is indicative of cell proliferation and is associat ed with cell transformation, in vitro

.

### Page 157 of 186 7. Revisit of Existing Genetic Toxicology Data on 2, 4-D

Data included in the fourth carcinogenicity peer review of 2, 4- D are summarized in Table 3. These data support the conclusion that 2, 4-D is not mutagenic in bacteria. There is also

confirmation of sex-linked re cessive lethal induction in Drosophila larvae. As previously mentioned, results from the in vitro cytogenetic assays were conf licting (negative without S9 but positive with S9 in CHO cells and human ly mphocytes; negative in embryonic bovine and peripheral lymphocytes and in lymphocytes from 2, 4- D work ers). Two rat bone marrow cytogenetic assays, using ip injection as the expo sure route provided only e quivocal results. The

one mouse bone marrow micronucleus assa y (MRID Nos. 41409804, 41870101) is the same assay that was published by Char les et al. (1999b). Simila rly, the findings from the in vitro UDS assay with 2, 4-D was also previously presente d (Charles et al., 1999a ). A similar clouded picture emerged for in vitro DNA damage; four of five assays were negative (2 SCE assays in rat lymphocytes and human lymphocytes from 2,4- D workers; 1 UDS assay in primary rat hepatocytes; and 1 DNA damage assay in hum an fibroblasts). The findings from the in vitro UDS assay with 2, 4-D were previously presente d (Charles et al., 1999a). The remaining SCE assay, which was in CHO cells and was part of the chromosome aberrati on test performed by Galloway et al. (1987), gave results that confli cted with the cytogenetic data (i.e., positive without S9 but negative with S9).

8. Overall Conclusions

There is a rather extensive Genetic Toxicology database on 2, 4-D and most of the assays required to fulfill the FIFRA guidelines for pes ticides are available, negative and acceptable, with the exception of no gene mutation in mammalian cells assays. However, the only

conclusions that can be reached wi th assurance (i.e., confirmed or unambiguous results) is that 2, 4-D induces DNA damage both in vitro and in vivo and gene mutations and/or chromosome mutations in yeast, plants and insects. Thes e data should nevertheless, be interpreted with

caution for two reasons:

a. As stated earlier, DNA damage alone, whether in vitro or in vivo, cannot be interpreted as a causal event leading to mutagenicity or other adverse outcomes in rodents or humans because the ability of ce lls or whole animals to repair damage to

DNA cannot be overlooked.

b. Confirmed or clear evidence of mutagenicity comes from test systems (i.e., yeasts, plants and insects) that generally are not given the same consideration in a weight-ofthe-evidence mutagenicity evaluation of a pesticide. Thus, the relevance to human

health cannot be ascertained.

No recommendations for further testing are given at this time. It is doubtful, considering the uncertainty in the Genetic Toxicology database, th at additional testing will resolve the issue of whether 2, 4-D is a mutagen.

9. References

Bus, J.S. (2009). Personal communication: E-mail: 1999 Article on 2, 4-D. To Nancy McCarroll, OPP/HED/TEB From: Jim Bu s, Dow Chemical, dated 10/01/2009.

### Page 158 of 186 US Environmental Protection Agency (USEPA ), 1991. Pesticide assessment guidelines, Subdivision F, Hazard Evalua tion: Human and Domes tic Animals, Series 84, Mutagenicity, Addendum 9. Washington, DC: Office of Pesticide Programs, USEPA,

EPA Publication No. 540/09-91-122.

Table 3. Summary of Mutagenicity Studies with 2, 4-D Acid Presented to the Fourth

Cancer Peer Review Committee #

Test system Test object Concentration Results Reference

Ames Test Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 100 - 10000 μg per plate with S9; 66.7 - 6670 μg per plate

without S9 Negative Lawlor, 1990

MRID No.

41409801.

Ames Test Salmonella typhimurium TA98, TA100, TA 1535, TA1537,

TA1538 0 - 1000 g/plate Negative Rashid et. al., 1984

Ames Test Salmonella typhimurium 0 to 3333 μg/plate Negative SolerNeidzieler, 1988

Ames Test Salmonella typhimurium TA 97, TA 98, TA 100, TA 1535, TA 1537, TA

1538 0 - 1000 g/plate (Although, weakly positive in TA97a at 250 μg/plate with S9) Negative Kappas, 1988

Ames Test Salmonella typhimurium

TA 1538 and TA 1535 0 - 2000 μg/plate Negative Rashid and Mumma, 1986

Ames Test Salmonella typhimurium TA 98, TA 100, TA 1535,

TA 1538 up to 5000 μ g /plate Negative Simmon et al.,

1977

E. Coli K12, WP2 0 - 2000 g/plate Negative Rashid and Mumma, 1986

E. Coli PQ 37 0 - 200 g/plate Negative MerschSundermann et al., 1989

SLRL

Mutation Drosophila melanogaster

larvae 0.l - 10,000 ppm Positive Kale et al., 1995

SLRL

Mutation Drosophila melanogaster

Adults 1000 - 10,000 ppm (feeding) 10,000 ppm (injection) Negative Zimmering et al., 1985

HamsterHGPRT V79 fibroblasts 10 - 100 μg/ml Inconclusive Pavlica et al., 1991

Chromosomal

Aberrations CHO Cells 500 - 920 μg/ml Negative

without S9 a Galloway et al., 1987

Chromosomal

Aberrations Embryonic bovine kidney and peripheral lymphocytes 1 - 1000 ppm Negative Bongso and Basrur, 1973

Chromosomal

Aberrations In vitro Human lymphocytes 0.125-0.35 mM

0.125-1.250 mM Negative

without S9

Positive without

S9 b Mustonen et al., 1986

Chromosomal

Aberrations Human lymphocytes (occupational exposure) (0.03 - 0.04 mg/m 3 ) c Negative Mustonen et al., 1986

### Page 159 of 186 Test system Test object Concentration Results Reference

Chromosomal

Aberrations In vivo Charles River Rat (bone marrow) 0 - 350 mg/kg i.p./4 or 24 hr; 3 replicates. Equivocal d Turkula and Jalal, 1987

Chromosomal

Aberrations In vivo Wistar Rat (bone marrow) 0, 17.5, 35 or 70 mg/kg/day/ i.p/2 times Equivocal e Adhikari and Grover, 1988

SCE Rat lymphocyte 100 mg/kg Negative Mustonen et al.,

1989

SCE CHO cells 50 - 299 μ g/ml Positive without

S9 Galloway et al., 1987

SCE CHO cells 500 - 4200 μ g/ml Negative with

S9 Galloway et al., 1987

SCE Human lymphocyte (occupational exposure) Not reported Negative Linnainmaa,

1983

Micronucleus ICR mouse (bone marrow) 40 - 400 μ g/kg mg/kg $ Negative Ivett, 1990;

MRID No. 41409804,

41870101

Unscheduled DNA synthesis Fischer 344 primary rat hepatocytes 0.969

- 2890 μg/ml 2.43 – 96.9 μg/ml; cytotoxic at ≥ 96.9 μg/ml $ Negative Cifone, 1990;

MRID No.

41409807
DNA damage Human Fibroblasts (PM2 DNA) 0 - 100 nmol/l Negative Clausen et al., (1990)

a = Positive with S9 at 10.5 hrs after treatment, but not at 19 hrs. b = Attributed to "unidentified clastogens"(i.e., contaminants) by investigators.

c = Present in urine at 0.09 -1.14 m/ L (non smokers); 0.11-1.56 (smokers).

d = Positive < 75 mg/kg but only in one replicate e = Positive at top two doses but results were similar to DMSO solvent control

# 

### Extracted from the Carcinogenicity Peer Review (4th) of 2, 4-Dichlorophenoxyacetic acid (2, 4- D); TXR #: 0050017, Jan. 29, 1997 (Cover Memo); July 17, 1996 (Actual document).

$ 

### When these data were revisited, it was noted that the dosage unit ( μ g/kg) or concentration range (μg/ml) was incorrect.

Bin: NC

(Study ) Citation : Oakes, D.J. and Pollak, J.K. (2000). The in vitro Evaluation of the Toxicities of ThreeRelated Herbicide Formulations Containing Ester Derivatives of 2, 4, 5-T and 2, 4-D Using SubMitochondrial Particles. Toxicology 151: 1-9. [Human Studies Considerations Apply: No] Principal Endpoint : [ Bin: NC] Methods Summary: not applicable Results Summary: sub-mitochrodrial particle test is useful as an initial screening assay.

CONCLUSION: essential to establish the toxicities of complete formulations, rather than just evaluating the toxicities of the active components.

COMMENT/ HED : This is an in vitro study (sub-mitochrodrial particle test), which cannot be extrapolated directly to the complex in vivo situation. Such assays cannot account for pharmacokinetic and pharmacodynamic changes that occur to xenobiotics in whole animals. Studies in whole animals are more relevant, and there are numerous studies on 2, 4-D that have identified effect levels and no effect levels. Apoptosis is considered a vital component of various processes including normal cell turnover, proper

### Page 160 of 186 development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Apoptosis plays a crucial role in developing and maintaining health by eliminating old cells, unnecessary cells, and unhealthy cells. There is no indication of how the doses that resulted in cytotoxicity and induction of apoptosis in mitochondria relate to the dose levels attained in cells

in the intact animal.

CITED BY: Beyond Pesticides cited this study as showing 2, 4-D to be “cytotoxic and induces apoptosis

via direct effect on mitochondrial membranes”. However, no review of this paper was provided by Beyond Pesticides, and the cited paper references other studie s that demonstrated the e ffects on mitochondria that

BP noted.

### Page 161 of 186 

# Appendix C: Exposure Data Summaries

### Page 162 of 186 Title: Biomonitoring of 2,4-dichlorophenoxyacetic acid exposure and dose in farm families

Journal Citation: Envir. Health Perspectives, V115, No. 3, March 2007 (pps 370-376) Author(s): Alexander, B.H., Mandel, J.S., Baker, B.A., Burn s, C.J., Bartels, M.J., Acquavella,J.F., Gustin, C. Study Type: PB – Population Based Monitoring Methods Summary: Farm families were recruited from licensed a pplicators in Minnesota and South Carolina. Families were recruited from licensed applicators in Minn esota and South Carolina. There were 43 applicators, 34 spouse, and 22 children. None of them had mixed or applied 2,4-D in previous week. The families were instructed to follow their normal routines as related to pesticide applicati on. The farmer and spouse were asked to complete an enrollment questionnaire before and following the study appli cation. Daily 24-hr urine samples were collected one day before through 3 days after an application of 2,4-D, char acterizing exposure profiles. Eligibility for the study required that a) the family lived on a farm, b) th e family consisted of the farmer, a spouse, and at least one child 4-17 years of age, c) the farmer was planning to personally apply 2,4-D to at le ast 10 acres of cropland as part of the normal operation, and d) the family members were willing to collect all of their urin e for 5 days. Eligible family members collected all urine during five 24-hr intervals, 1 day before through 3 days after an application of 2,4-D. Exposure profiles were characterized with 24-hr urine 2,4-D concentrations, which then were related to potential pred ictors of exposure. Systemic dose was estimated using the urine collections from the a pplication day through the third day after application. Results Summary: Median urine 2,4-D concentrations at baselin e and day after applica tion were 2.1 and 73.1 μ g/L for applicators, below the limit of detection, and 1.2 μ g/L for spouses, and 1.5 and 2.9 μ g/L for children. The younger children (4–11 years of age) had higher median post-a pplication concentrations than the older children ( ≥ 12 years of age) (6.5 vs. 1.9 μ g/L). The geometric mean systemic doses (micrograms per kilogram body weight) were 2.46 (applicators), 0.8 (spouses), 0.22 (all children), 0.32 (child ren 4–11 years of age), and 0.12 (children ≥ 12 years of age). Exposure to the spouses and children was primarily determined by direct cont act with the application process and the number of acres treated. Multivariate models identified glove use, repairing equipment, and number of acres treated as predictors of exposure in the applicators.

Conclusions: Investigators observed considerable heterogeneity of 2,4-D exposure among farm family members, primarily attributable to level of contact with the app lication process. Awareness of this variability and the actual magnitude of exposures are important for developing exposure and risk characteri zations in 2,4-D–exposed agricultural populations. The FFES is a comprehensive exposure study, but is not without limitations. Overall, the compliance with the urine collection protocol was good, but a few of the 24- hr urine collections were incomplete. Although the 2,4-D concentrations may be well represented in these incomplete samples, the systemic dose will be underestimated. The applications in this study were intended to represent the real -world scenarios that a farm family would encounter year in and year out; however, the study was limited to a single app lication. The applicators were asked to follow their usual practices to capture real-world exposure scenarios, but it is possible that some altered prac tices because the field observer was present. Changes in equipment or agricultural practices in the past or future may alter the actual exposure to the family. This study was not able to address how much the e xposure will vary over one or mo re seasons and whether the peak exposures are representative of exposures for that individu al or the distribution of expected exposure from applying

this chemical.

Comments: This study is part of the industry FFES (Farm Fam ily Exposure Study) conducted in South Carolina and Minnesota using similar methods to the Ag Health Study. The use of the results of this study in the Task Force Comments on the Petition appears reasonable. No environmen tal media sampling within the subject homes was reported

and the results are consistent with other studies of this nature (i.e., the highest exposures are associated with applications)

.

### Page 163 of 186 Title: Predictors of herbicide exposure in farm applicators.

Journal Citation: Int. Arch Occup. Environ. Health (2002) 75: 406-414 Author(s): Arbuckle, T.E., Burnett, R., Cole, D., T eschke, K., Dosemeci, M., Bancej, C., Zhang,

J. Study Type: PB – Population Based Monitoring Methods Summary: A subset if the participants in a re trospective cohort study of Ontario farm families volunteered for a pestic ide exposure assessment study. Immediately prior to and after handling phenoxy herbicides (including 2,4-D) for the first time during the season, 126 applicators provided pre-exposure spot urine sa mples and a subsequent 24 hour urine sample. They also completed a questionnaire on pesticide use and handling practices for the 1 st day of application. Results Summary: Assuming that the presence of 2,4-D in the urine was a measure of true exposure and that questionnaire indications of 2, 4-D use were the exposure classification subject to error, then the questionnaire’s prediction of exposure had a sensitivity of 56.7% and specificity of 86.4%. The comparable values for MCPA were sensitivity and speci ficity of 91.6% and 67.4%, respectively. In multi-variate models, th e variables pesticide formulation, protective clothing/gear, application equipment, handling practice, and personal hygiene were significant predictors of urinary herbicide levels in the firs t 24 hours after application (or spraying) had been initiated (adjusted R 2 = 44% for MCPA and 39% for 2,4-D). Investigators also indicated the only protective factor identified in the 2,4-D multiv ariate model was the w earing of rubber gloves during mixing or loading of the herbicide.

Conclusions: The investigators concluded that alt hough similar domains of factors were associated with exposure in both models, the specific factors identified and the signs of the coefficients were sufficiently different between th e final models for each herbicide that additional investigations appear to be warranted to dete rmine the sources of the differences and assess the validity of the models and their ability to be generalized.

Comments: The investigation in this paper was conduc ted as part of the Ontario farm family retrospective cohort study. In this study, ur inary concentrations for applicators only were reported for 2,4-D and MCPA. No exposure resu lts were reported for spouses or children. A total of 126 families participated in the exposure monitoring component of this study. In Table 2 results were presented for applicators who repor ted application of 2,4-D (N=43) and MCPA (N=89). Comparatively, as would be expected, those who reported applications had much higher urinary concentrations of each chemical comp ared to those who did not report application involvement. The arithmetic means for 2,4-D were 27.6 and 2.6 μg/L for applicators and those not involved in application, respectively. Th e maximum reported 2,4-D concentrations were 410.0 and 66.0 μg/L for applicators and those not involved in application, respectively. The use of the results of this study in the Task For ce Comments on the Petition appears reasonable with regard to the efficacy of protective glove use. No environmental media sampling within the subject homes was reported.

### Page 164 of 186 Title: Increased lymphocyte replicative index follo wing 2,4-dichlorophenoxyacetic acid herbicide

exposure.
Journal Citation: Cancer Causes and Control 11: 373-380 (2000) Author(s): . Figgs LW, Holland NT, Rothmann N, Zahm SH, et al. Study Type: EPI/PB – Epidemiology & Population Based Monitoring Methods Summary: The objective of this study was to evaluate peripheral blood lymphocyte proliferation (replicative index: RI) and mi cronuclei frequency (MF) among 2,4-D herbicide applicators. Twelve applicators (one 17 years old) spraying only 2,4-D provided a blood and urine specimen upon enrollment, several urine samples during the spraying season, and a blood specimen at the end of the study. Nine controls also pr ovided blood and urine specimens. Standard methods were used to quantify impacts and 2,4-D residues. Applicator RI and MF were compared before and after spraying and with controls. Results Summary: . Applicators contributed 45 urine specimens with concentrations ranging from 1.0 to 1700 μg 2,4-D/g creatinine/L urine that logarithmically increased as spray time increased. Applicator RI increased after spraying (p=0.016) , independent of tobacco and alcohol use, and demonstrated a weak dose-response with increasi ng urinary 2,4-D levels (p=0.15). Among 2,4-D applicators pre-exposure complete blood c ounts and lymphocyte immunophenotypes were not significantly different from post-exposure measurements.

Conclusions: Urinary 2,4-D concentration, an exposure biomarker, may be associated with lymphocyte replicative index, a cell proliferation biomarker. Investigators also indicated the results of this study do not support a genotoxic pathway.

Comments: In the NRDC petition this study is cited in a manner that reflects the conclusions drawn by the investigators except it was not explicitly stated by the investigators that there is an increased risk of chronic toxicity due to the a pparent exceedance of clearance mechanisms. The 2,4-D task force also cited this study correctly but included characterization elements which were not raised by NRDC including indicating the author s described the study as preliminary, a pilot investigation, and the first to show a relationship between RI and urine levels. The task force also cited the investigator indication that the link between 2,4-D function and lymphocyte replicative index is unclear and that the mechanism, our repor ts and other recent investigations do not support a genotoxic pathway.

### Page 165 of 186 Title: Review of 2,4-dichlorophenoxyacetic ac id (2,4-D) Epidemiology and Toxicology

Journal Citation: Critical Reviews in Toxicology 32(4): 233-257 (2002) Author(s): Garabrant, D.H. and Philbert, M.A. Study Type: EPI – Epidemiology [various tox] Methods Summary: A literature review meta-analysis was comp leted using a variety of animal data and epidemiological evidence to determine if 2,4-D or its salts or esters damage DNA under physiologic conditions or induce oncogenic or carcinogenic effects. Results Summary: Detailed review of the genotoxic effects of 2, 4-D, its salts, and esters showed that 2,4- D does not have any genotoxic/mutagenic potential. It was also found that 2,4-D does not form reactive electrophile metabolites which are associated with carcinogenicity. Human observation studies found an association between exposure to 2,4-D and neurol ogic effects, including persistent peripheral polyneuropathy, demyelination and ganglion degeneration in CNS, re duced nerve conduction velocities, and myotonia. However, the doses associated with these effects are unknown. There is no convincing evidence in the literature that 2, 4-D is associated with human reproductive toxicity. Case-control and cohort epidemiology studies provide scant evidence that supports the conclusion that exposure to 2,4-D is associated with soft tissue sarcoma (STS), non-Ho dgkin’s lymphoma (NHL), or Hodgkin’s disease (HD).

Conclusions: Investigators concluded overall the availabl e evidence from epidemiologic studies is not adequate to conclude that any form of cancer is ca usally associated with 2,4-D exposure. There is no human evidence of adverse reproductive outcomes related to 2,4-D. The available data from animal studies of acute, subchronic, and chronic exposure to 2,4-D, its salts, and esters show an unequivocal lack of systemic toxicity at doses that do not exceed renal cl earance mechanisms. There is no evidence that 2,4-D in any of its forms activates or transforms the immune system in animals at any dose. At high doses, 2,4-D damages the liver and kidney and irritates mucous membranes. Although myotonia and alterations in gait and behavioral indices are observed after overwhelming doses of 2,4-D in the microgram/kg/day range. It is unlikely that 2,4-D has any neurotoxic potential at doses below those required to induce systemic toxicity.

Comments: This study was conducted for the 2,4-D Industry Task Force II on Research Data to provide an update of the evidence on human toxicity and cancer ri sks related to 2,4-D on research completed between

1995 and 2001. The use of the results of this stud y in the Task Force Comments on the Petition appears reasonable. No environmental media sampling or di rect exposure monitoring from the considered studies were reported.

### Page 166 of 186 Title: Birth Defects, Season of Conception, and Sex of Children Born to Pesticide Applicators Living in the Red River Valley of Minnesota, USA

Journal Citation: Environ Health Perspect 110(suppl 3):441–449 (2002). Author(s): Vincent F. Garry, Mary E. Harkins, Leanna L. Eric kson, Leslie K. Long-Simpson, Seth E. Holland, and Barbara L. Burroughs Study Type: EPI – Epidemiology Methods Summary: A cross-sectional study of 695 families and 1,532 children conducted during 1997 to 1998 in the Red River Valley of Minnesota was reporte d with reference to a previous study conducted in 1989-1991. Birth defects, season of conception and the sex of children born to pesticide applicators were evaluated. Associations with varied types of pesticides were considered. All reported anomalies were recorded according to the International Classification of Disease, 9 th Revision. Confounding variables such as maternal smoking, drinking, age and chronic diseases (such as diabetes and hypertension), were examined. Where possible, familial genetic history, pr egnancy medication use, and non-medicinal drug use (vitamins) was assessed in families with birth defects. Re garding pesticide use, subjects were re-interviewed 6 months later, where possible, by written questionnaire. This was used to document common pesticide use by pesticide class, acreage treated, type of crop, and us e of personal protective gear, as well as to validate use of pesticides by class. Where appropriate, regression analysis, tw o-sided t-tests, and analysis of variance methods were employed. Variables were mother’s age, smoking status, alc ohol use, and season of conception. Information regarding specific medication used during pregnancy and subjects’ diet we re not considered in the survey. Residence at a rural site (towns with populations < 3,000) or on a farm during childhood (<18 years of age) was considered in some statistical analysis. Results Summary: In this current study, in the first year of life th e birth defect rate was 31.3 births per 1,000, with 83% of the total reported birth defects confirmed by medical records. Inclusion of children identified with birth or developmental disorders within the first 3 years of life and later led to a rate of 47.0 per 1,000 (72 children from 1,532 live births). Conceptions in spring resulted in significantly more children with birth defects than found in any other season (7.6 vs. 3.7%). Twelve families had more than one child with a birth defect (n = 28 children). Forty-two percent

of the children from families with recurrent birth defects were conceived in spring, a signifi cantly higher rate than that for any other season. Three families in the kinships defined contributed a first-de gree relative other than a sibling with the same or similar birth defect, consistent with a Mende lian inheritance pattern. The remaining nine families did not follow a Mendelian inheritance pattern. The sex ratio of childre n with birth defects born to applicator families shows a male predominance (1.75 to 1) across speci fic pesticide class use and exposure cate gories exclusive of fungicides. In the fungicide exposure category, normal female births significantl y exceed male births (1.25 to 1). Similarly, the proportion of male to female children with birth defects is signifi cantly lower (0.57 to 1; p = 0.02). Adverse neurologic and neurobehavioral developmental effects clustered among the children born to applicators of the fumigant phosphine (odds ratio [OR] = 2.48; confidence interval [CI], 1.2–5.1). U se of the herbicide glyphosate yielded an OR of 3.6 (CI, 1.3–9.6) in the neurobehavioral category.

Conclusions: Investigators concluded that the present and previous studies these studies point out that a ) herbicides applied in the spring may be a factor in the birth defects observed and b ) fungicides can be a significant factor in the

determination of sex of the children of the families of the RRV. Thus, two distinct classes of pesticides seem to have adverse effects on different reproductive outcomes. Biol ogically based confirmatory studies are needed. The investigators also indicated use of the phosphonamino he rbicides (glyphosate, Roundup) was overrepresented in the adverse birth and developmental effect group. No other comm only used pesticide (i.e., 2,4-D) compared by major organ and/or functional system was uniquely associated with specific adverse birth or developmental effects.

Comments: The use of the results of this study in the 2,4- D Task Force Comments on the Petition appears reasonable in that the investigators only identified a possible relationshi p with the herbicide glyphosate and not with the use of the chlorophenoxy acid herbicides such as 2,4-D. No environm ental media sampling or direct exposure monitoring were discussed or reported.

### Page 167 of 186 Title: Pesticide Appliers, Biocides, and Birth Defects in Rural Minnesota

Journal Citation: Environmental Health Perspectives V 104, No. 4, April 1996 (394-399) Author(s): Garry, V.F.; Schreinmachers, D.; Harkins, M.E.; and Griffith, J. Study Type: EPI – Epidemiology Methods Summary: This ecologic study evaluated the hypothesis sugg ested previously by the investigators that the offspring of pesticide applicators might have increased risk s of birth anomalies. This was investigated by linking 4,935 births to 34,772 state-licensed, private pesticide applicators in Minnesota occurring between 1989 and 1992 to the Minnesota state birth registry containing 210,723 live bi rths in the same timeframe. SAS software; Chi-square tests were used to estimate the statistical significance of observed differences in the proportion of birth anomalies between pesticide applicators and the general population, and between populations living in three identified crop regions. Odd ratios were developed to estimate risk. Th e Mantel-Haenszel method was used to analyze crude and age-adjusted data, and to measure overall association. Logistic regression was used to estimate the effects of potential pregnancy risk factors. Results Summary: The birth defect rate for all birth anomalies was significantly increased in children born to private applicators. Specific birth defect categor ies, circulatory/respiratory, urogenital, and musculoskeletal/integuemental showed significant increases. For the general population and for applicators, the birth anomaly rate differed by crop- growing region. Western Minnesota, a major wheat, sugar beet, and potato growing region, showed the highest rate of birth anomalie s per 1000 live births: 30.0 for private appliers versus 26.9 for the general population of the same region. The lowest rates, 23.7/1000 for private applicators versus 18.3/1000 for the general population, occurred in noncrop regions. The highest frequency of use of chlorophenoxy herbicides and fungicides also occurred in western Minnesota. Births in the general population of western Minnesota showed a significant increase in birth anomalies in the same three birth anomaly categories as applicators and for central nervous system anomalies. This increase was most prono unced for infants conceived in the spring. The seasonal effect did not occur in other regions. The male/female sex ratio for the four birth anomaly categories of interest in high phenoxy herbicide/fungicide use is 2.8 for applicators versus 1.5 for the general population of the same region (p=0.05). In minimal use regions, this ratio is 2.1 for a pplicators versus 1.7 for the general population. Pesticide applicators had significantly more children with an anom aly than non-applicators (p<0.001) in the corn/soybeangrowing region. The most consistent results were obtained for 2,4-D and Methyl-4-chlorophenoxyacetic acid (MCPA). The frequency of anomalies in high-use areas si gnificantly increased (OR=1.86; 95% CI, 1.69- 2.05) for combined births with central nervous system, circulatory/ respiratory, urogenital, and musculoskeletal anomalies (OR = 1.51; 95%CI, 1.40-1.62).

Conclusions: Investigators concluded that the pattern of excess frequency of birth anomalies by pesticide use, season, and alteration of sex ratio sugge sts exposure-related effects in appli cators and the general population of the crop-growing region of western Minnesota. Birth defect s increased in 1) offspring born to licensed private applicators, 2) offspring born to the general populati on residing in high use chlorophenoxy herbicide/fungicide regions, and 3) infants conceived in spring in western Minnesota.

Comments: The use of the results of this study in the NRDC Petition and in the comments from Beyond Pesticides that reflect the specific conclusions described in the study but NRDC and Beyond Pesticides ignored the comments provided in later publications by the same investigator wh ich were cited by the 2,4-D task force comments on the petition which generally indicate lower incidence rates. Th e 2,4-D task force commented the study of Garry and coworkers (1996) assessing potential associations of herbicide use and birth defect outcomes in an agricultural region of Minnesota was later described by this same research team as “a first effort” that suffered from a “limited reporting timeframe” (Garry et.al., 2002). In the latter report, the 2,4- D task force indicated the authors concluded that a more detailed cross-sectional analysis of th is area showed no statistically signifi cant correlations between 2,4-D use and excess adverse birth or neurodevelopmental effects. No environmental media sampling or direct exposure monitoring were discussed or reported. It is also not clear how the trends in the general population of the Minnesota birth registry compare to the general population of the United States nor was any comparison provided pertaining to other high use areas for 2, 4-D. Analysis was done at the gr oup level, rather than individual level. Individuals were assigned an exposure classification that might not represent thei r true exposure. This study also did not mention if the maternal factors that can cause birth defects (such as ma ternal infections, vaccinations, drug/medicine used) were

### Page 168 of 186 considered.

Title: Biomarker Correlations of Urinary 2,4-D Levels in Foresters: Genomic Instability and Endocrine Disruption

Journal Citation: Environmental Health Perspectives V109/No. 5 (May 2001) pp. 495-500 Author(s): Vincent F. Garry, Robert E. Tarone, Ilan R. Kirsch , Jorge M. Abdallah, Donald P. Lombardi, Leslie K. Long, Barbara L. Burroughs, Dana B. Barr, and James S. Kesner Study Type: PB/EPI – Population Based Monitoring/Epidemiology Methods Summary: Forest pesticide applicators were selected for consideration in this case-control study of genomic stability and reproductive hormone levels related to the use of chlorophenoxy herbicides. Aberrations in G-banded chromosomes, reproductive hormone leve ls, and polymerase chain reaction-b ased V(D)J rearrangement frequencies were examined. At the end of the peak of the 2,4-D app lication season, first-voided morning urine and morning blood specimens were obtained from applicators. A second blood specimen was obtained from exposed subjects, only within 6 weeks of the beginning of the following season’s application work and used to compare to the earlier data set. For reproductive hormone analysis, luteiniz ing hormone (LH), follicle-stimulating hormone (FSH), and testosterone concentrations (total and free) in serum or plasma obtai ned from blood specimens donated by study participants were measured. For chromosome studies, chromosome analysis and PCR-based V (D) J trans-rearrangement assay were performed. Subjects were selected to participate based on th e following criteria: a) no chronic disease, b) no chronic medication use, c) have applied 2,4-D more than 5 days per year (for the exposed group), d) did not apply pesticides this past year (for the control group). Analysis of varian ce methods was used to test for changes in hormone level according to 2,4-D application method. Pearson correlati on coefficients were used to quantify the relationship between urinary 2,4-D level and changes in hormone leve l across all application methods. Wilcoxon rank-sum tests were computed to determine if chromosome aberration freque ncies varied according to level of herbicide exposure. Poisson regression analyses were used to examine the rela tionship between aberration frequencies and urinary 2,4-D

levels. Pearson correlation coefficients were used to quantify the relationship between urinary 2,4-D level and V (D)J rearrangement frequencies. Results Summary: Categorized by applicator method, men who u sed a hand-held, backpack sprayer in their applications showed the highest average level (453.6 ppb) of 2,4-D in urine. Serum luteinizing hormone (LH) values were correlated with urinary 2,4-D leve ls, but follicle-stimulating hormone and fr ee and total testosterone were not. At the height of the application season; 6/7 backpack spraye rs, 3/4 applicators who used multinozzle mechanical (boom) sprayers, 4/8 aerial applicators, and 2/5 skidder-radiarc (closed cab) applicators had two or more V(D)J region rearrangements per microgram of DNA. Only 5 of 15 mi nimally exposed (control) foresters had two or more rearrangements, and 3 of these 5 subjects demonstrated det ectable levels of 2,4-D in the urine. Only 8/24 DNA samples obtained from the exposed group 10 months or more after their last chlorophenoxy use had two rearrangements per microgram of DNA, suggesting that th e exposure-related effects observed were reversible and temporary. Although urinary 2,4-D levels were not corre lated with chromosome aberration frequency, chromosome aberration frequencies were correlated with the total volume of herbicides applied, including products other than 2,4- D. One issue the investigators attempted to define was wh ether or not it was not possible to separate the impacts of adjuvant use. The sample size in this study was very sm all and most applications use adjuvants which aid in the retention and absorption of pesticides on treated plants. Th erefore to the extent to wh ich the cumulative chromosome effect observed can be related to adjuvant use is uncertain . Another issue considered by investigators was to elicit whether or not the volume of pesticide used and/or applicati on equipment used. It is clear more significant effects are noted with higher exposure scenarios.

Conclusions: Investigators concluded that herbicide applicators with high urinary levels of 2,4-D (backpack and boom spray applications) exhibited elevated LH levels. They al so exhibited altered genomic stability as measured by V(D)J rearrangement frequency, which appears reversible months after peak exposu re. However, there is uncertainty

associated with these results because a lthough they are highly detailed, the limited sample size warrants cautious interpretation of the data.

### Page 169 of 186 Comments: The use of the results of this study in the comment s from Beyond Pesticides appears to be reasonable and it reflects the specific conclusions described by the investigators. However, it was used to make the argument that 2,4- D is an endocrine disruptor which is being evaluated sep arately and the results are pending. Several uncertainties should be noted including no environmental media sampling were reported and he sa mple size is small as noted by the investigators and should be considered in the interpreta tion of the results. Biomonitoring results could be used quantitatively if desired to calculate risk.

Title: Factors Affecting the Exposure of Ground-rig Applicators to 2,4-D Dimethylamine Salt

Journal Citation: Arch. Environ. Contam. Toxicol. 15, 677-686 (1986) Author(s): Grover, R.; Cessna, A.J.; Muir, N.I.; Riedel, D.; Franklin, C.A.; Yoshida, K. Study Type: PL – Planned Exposure Monitoring Methods Summary: A total of 30 individual spray opera tions using 2,4-D dimethylamine salt were monitored. These operations involved fa rmers carrying out spray operations with tractordrawn ground rigs, involving handling, transfe rring, mixing, and applying to wheat. These

individual events lasted 55 to 870 minutes and invol ved 1 to 11 tankloads, and application of 6.7 to 88.3 kg 2,4-D acid equivalent. Between 16 to 194 hectares were treated. Air sampling using a typical personal sampling pump method was used. Dermal exposure was me asured using patches

and a handwash method. Patches were worn unde r 2 work clothing and on the exterior. Some applicators wore protective gloves. Most appli cators in the study used tractors with no cabs but

13 of 30 had some sort of enclosure. Urine was al so collected and analyzed for 2,4-D residues. A total of nine subjects participated in the resear ch and all but one were monitored multiple times with one worker being monitored during 7 spray ope rations. The investigators used the results of this study to evaluate various factors which ma y impact exposures (e.g., area or amount sprayed). Results Summary: Hand exposures accounted for 80 to 90 percent of the potential cumulative exposure. The 2,4-D on the rest of the bod y ranged from 10 to 20 percent of the potential cumulative exposure. Urinary 2,4-D excretion accounted for 1 to 2 percent of the potential cumulative exposure. The median air concentration was 2.1 μg/m 3 with a range of <0.01 to 101 μg/m 3 . Dermal (non-hand) dose values had a median of 27.9 μg/kgBW with a minimum of 7.5 μg/kgBW and a maximum of 278 μg/kgBW. Ha nd dose values had a median of 120 μg/kgBW with a minimum of 0.1 μg/kgBW and a maximum of 552 μg/kgBW. Correlations were evaluated for various exposure factors. It should be noted the investigators considered all results together with no consideration as to whether or not gloves we re used to a cab was present or not on each

tractor.

Conclusions: Investigators concluded the total calcula ted amount of 2,4-D deposited on the body (minus the hands) and the total amount excreted in the urine were highly correlated with the number of tank fills, area sprayed, amount sprayed, and duration of operation.

Comments: There is no mention in the study of consid eration by an institutional review board. This study was conducted prior to the initiati on of the Pesticide Handlers Exposure Database (PHED). This study reflects the monitoring tec hnology and approaches of that timeframe but the results still appear consistent with current thinki ng in that hands are large contributors to overall exposures especially when mixing/loading activities ar e evaluated. The use of the results of this study in the comments from the 2,4-D task for ce appears to be reasonable and it reflects the specific conclusions described by the investig ators. No environmental media sampling was completed. Direct exposure monitoring was re ported and could be cons idered for quantitative use. However, it should be noted that PHED is currently used for routine assessments of this type

rather than individual studies.

### Page 170 of 186

### Page 171 of 186 Title: A Case–Control Study of Non-Hodgkin Lymphoma and Exposure to Pesticides

Journal Citation: CANCER March 15, 1999 / Volume 85 / Number 6 (pp. 1353-1360) Author(s): Hardell, L. and Ericsson, M. Study Type: EPI – epidemiology Methods Summary: A population-based case–control study in nor thern and middle Sweden was performed. Exposure data were ascertained by comprehensive ques tionnaires, and the questionnaires were supplemented by telephone interviews. In total, 404 cases and 741 cont rols answered the questionnaire. Univariate and multivariate analyses were performed with SAS. Cases w ith a histopathologic diagnosis of NHL were identified from the regional cancer registries. The cases were divide d into different groups acco rding to histopathology, i.e. B-cell lymphoma aggressive (n= 157), indolent (n= 185), T-cell lymphoma (n=18), and other (n=6) or unspecified types (n=38). Exposure to each herbicide (MCPA, 2,4-D/2,45-T, Glypho sate, etc.) was analyzed separately. Results Summary: Increased risk for NHL was found for subjects e xposed to herbicides (odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0 –2.5) and fungicid es (OR, 3.7; 95% CI, 1.1–13.0). Among herbicides, the phenoxyacetic acids dominated (OR, 1.5; 95% CI, 0.9 –2.4) ; and, when subclassified, one of these, 4-chloro-2- methyl phenoxyacetic acid (MCPA), turn ed out to be significantly associated with NHL (OR, 2.7; 95% CI, 1.0– 6.9). Odds ratios were lower for 2,4- D than for the other phenoxyacid herbicides. 2,4-D and 2,4,5-T exposure slightly increased the risk of NHL (OR= 1.3, CI 0.7-2.3) . For several categories of herbicides, it was noted that only exposure during the most recent d ecades before diagnosis of NHL was ass ociated with an increased risk of NHL. Exposure to impregnating agents and insecticides was, at most, only weakly related to NHL.

Conclusions: Investigators concluded exposure to herbicides in total, including phenoxyacetic acids (i.e., 2,4-D had lowest reported OR values in this chemical class) during the decades before NHL diagnosis resulted in increased risk for NHL. Thus, the risk following exposure was related to the latency period. Fungicides also increased the risk for NHL when combined, but this group consisted of several different agents, and few subjects were exposed to each type of fungicide.

Comments: This study was population based, using the Swedis h Cancer Registry to identify the cases. No environmental or personal sampling was completed. Th e Swedish compulsory reporting system for malignant diseases makes it likely that almost all incident cases in th e study area during the 4 years of inclusion were used. To avoid any selection of cases associated with prognos is, both living and deceased cases with NHL were included in this case–control study. To assess exposure in an equal manner for both cases and controls and to minimize recall bias, deceased controls were used for d eceased cases. Regarding farm ers, the questionnaire data had to be supplemented over the tele phone for all subjects due to the detailed, written instructions for the interviews. Thereby, exposure data had a significant uncer tainty associated with it (e.g ., type of chemical used, years and number of days for exposure, methods of use). It should also be noted that the combination of 2,4-D and 2,4,5-T, which constituted Agent Orange in U.S. wa rfare in Vietnam, was the most predominantly used herbicide combination in Swedish forestry. In this st udy, however, the analysis was restricted to exposure during the last two decades preceding the diagnosis. Sin ce 2,4,5-T was banned in Sweden in 1977 no subjects in this study had their first exposure to this substance during the 10-year period preceding NHL diagnosis. Thus, it seems to be difficult to demonstrate any lymphomage nic effect from 2,4,5-T in subjects with lymphoma diagnosis during later years. Fina lly, improved requirements for personal protective equipment are used in current practices which would be expected to alter resu lting OR (Odds Ratio) values. The citation of this study by Beyond Pesticides reflects the conclusions of the investigators but it does not account for the uncertainty associated with the exposure predictors acknowledge d by the investigators in this study.

### Page 172 of 186 Title: Distributions and Determinants of Pre-emerge nt Herbicide Exposures Among Custom Applicators

Journal Citation: Ann. Occup. Hyg. Vol 45, No. 3, pp 227-239 (2001) Author(s): Hines, C.J.; Deddens, J.A.; Tucker, S.P.; and Hornung, R.W. Study Type: PL – Planned Exposure Monitoring Methods Summary: Custom applicators intensively apply herbicides to corn and soybean fields each spring. The primary objective of this study was to characterize the e xposure distributions of the herbicides alachlor, atrazine,2,4-D 2-ethylhexyl ester (2,4-D EH), and metolachlor among a group of applicators during the spring preemergent spray season. A secondary objective was to evalua te determinants of exposure and to estimate withinand between-worker variance components. Fifteen appli cators were sampled using a systematic design that included spray and non-spray days and multiple measurements (five to seven) on each applicator. Air, patch, and handwash samples were collected on 89 applicator-days. Applicator-days were classifi ed into three categories: target herbicide sprayed, non-target herbicide sprayed, and no herbicide sp rayed. The median age for applicators was 40 years (range 23-58). The median number of years of experience as an applicator was 8 (range 1-22), the median number of years worked for the current comp any was 5 (range 1-28). Of the 89 applicator-days, herbicide spraying was done for 30 days; 23 of these days in cluded use of one or more of the target herbicides. Mixed-model regression analysis was used. Results Summary: For all exposure metrics, adjusted mean herb icide exposures were significantly higher on days when target herbicides were sp rayed as compared to non-spray days. For 2,4-D EH only, adjusted mean exposures on non-target herbicide spray days were signifi cantly higher than on non-spray days. For all exposure metrics, the within-worker variability (GSDW 2.1–5.6) was greater than the between-worker variability (GSDB 1.2–2.7).

Conclusions: Investigators concluded wearing gloves significantly reduced adjusted mean hand exposure for all herbicides (4–20 fold) and adjusted mean thigh exposure fo r three herbicides (8–53 fold) on days the herbicides were sprayed; however, wearing gloves significantly incr eased adjusted mean atrazine hand and thigh exposures (9 and 7 fold, respectively) on days that non-atrazine he rbicides were sprayed. Few of the other covariates were

consistent determinants of exposure. Glove use can offer protection but reusing the same glove can be a source of exposure. Therefore, frequent hand washing and wearing new gloves should be encouraged.

Comments: This study evaluated several determinants of wo rker exposure and also produced distributions of exposure under varied field use circumstances. It did not exam ine concurrent health effects. The use of the results of this study in the comments from the 2,4-D task for ce appears to be reasonable they reflect the specific conclusions described by the investig ators in that the study illustrated th at protective gloves significantly reduce exposures. This has been widely demonstrated in many circumstances given proper glove use. No environmental media sampling was completed. Direct exposure monito ring was also reported and could be considered for quantitative use. However, it should be noted that PHED is currently used for routine assessments of this type

rather than individual studies in most circumstances.

According to the author, applicators were not selected by a strictly random process. Therefore, these results may not apply to all custom applicators.

### Page 173 of 186 Title: Exposure of Forestry Ground Workers To 2,4-D, Picloram, and Dichlorprop

Journal Citation: Environmental Toxicology and Chemistry (Vol. 6) pp. 209-224 (1987) Author(s): T.L. Lavy; L.A. Norris; J.D. Mattice; and D.B. Marx Study Type: PL – Planned Exposure Monitoring Methods Summary: Urine samples from 80 forest workers applying 2,4-D plus dichlorprop or picloram by ground techniques were monitored to de termine the dose of chemical absorbed. Four

crews of 20 workers each were studied to compare the exposures during application by backpack, injection bar, hypohatchet or a hack and squirt method. Total urine output was analyzed to compare the absorbed dose to each worker under tw o operational modes. In the first (T1) the workers wore their usual clothing and followed the wo rk habits they ordinarily used on their job. In the second (T2), conducted 6 days after the fi rst, workers were given new leather gloves and boots and asked to take all feasible precautions (e .g., wash hands, bathe, wash clothes, avoid sprayed areas) to prevent herbicide exposure and to wash immediately if they come in contact

with the chemical. Results Summary: For all application methods except bac kpacks the T-2 treatment decreased the absorbed dose. During both T-1 and T-2 the clothing of backpack sprayers often became saturated with spray, dew and perspiration, and these workers received a hi gher absorbed dose of 2,4-D (0.04 – 0.24 mg/kg/bw) than did workers in other crews. The absorb ed dose of dichlorprop ranged from undetectable to 0.18 mg/kg. Hypoha tchet workers received a greater dose than did injection bar or hack-and-squirt workers. The absorbed dose of picloram ranged from undetectable to 0.02 mg/kg/bw. If equal dermal penetration of 2,4-D and picloram is assumed, this represents much less dermal absorption th an would have been predicted from the relative amounts of 2,4-D and picloram in Tordon 101-R.

Conclusions: Investigators evaluated the utility of usi ng improved hygiene practices with fresh gloves and boots as a means of reducing exposures. In cases where exposures impinge mainly on

the feet and hands the T-2 conditions described by the authors coupled with enhanced hygiene appear to reduce exposures. As noted by the au thors, T-2 conditions and enhanced hygiene were not as effective with backpack applicators wher e much of the exposure occurs around the neck and upper chest/back area and they were noted to have been working in dense vegetation areas which had been previously treated (i.e., 2 to 7 mete rs high). Investigators also calculated various margins of safety with the lowest one being 272 for the highest exposed b ackpack applicators in T-2 conditions (based on geometric mean and NO EL of 24 mg/kg/d). It was concluded that having a calculated MOS less than the level of concern was attributable to very low dermal absorption since the backpack app licators had very high levels of contact with the 2,4-D spray solution as evidenced by their wet clothing and observed activities.

Comments: This study evaluated several determinants of worker exposure and also produced distributions of exposure under varied field use circ umstances. The use of the results of this study in the comments from the 2,4-D t ask force appears to be reasonable in that they reflect the specific conclusions described by the investig ators. No environmental media sampling was completed. The direct exposure monitoring could be considered for quantitative use. However, PHED is currently used for routine assessments of this type rather than individual studies but some values for the monitored forestry operations are not addressed by PHED.

### Page 174 of 186 Title: Study of Reproductive Function In Pe rsons Occupationally Exposed to 2,4-D

Journal Citation: Mutation Research, 262 (1991) pp. 47-50 Author(s): Lerda, D. and Rizzi, R. Study Type: PB&EPI – Population Based Monitoring & Epidemiology Methods Summary: In this experimental study, sperm quality of fifty-seven male subjects, thirty two 2, 4-D exposed (farmers, mean ages 38 years) and 25 non-exposed (controls, mean ages 35 years) were assessed. These evaluations were completed in March through July 1989. Farmers working with 2, 4-D but without history of urologic abnormalities, severe testicular injury, and recent illness, which can affect spermatogenesis, were selected. Factors such as socio-economic status, cigarette smoking, drug use, radiati on exposure, general health, and sexual history were also considered in both control and exposed groups.

The exposure periods were August to September 1988 with effects being de termined in March 1989 and possible recovery being evaluated in July 1989 . Controls were evaluated in Apr il 1989. Sperm quality was assessed by four parameters: volume, sperm count, motility, and mo rphology. The samples of sperm in unexposed and exposed groups were coded in order to avoid subjectivity in analysis. A ll sperm parameters were evaluated 3 times per individual and mean values were calculated. The leve ls of 2,4-D in urine were also determined. It is not clear when the urine samples were co llected relative to exposure events. The locale in which this study was

conducted was not clear nor was a descri ption provided regarding the cultural practices used by the test subjects who applied 2,4-D in this study . Results Summary: The results indicate a mean urinary level of 2, 4-D of 9.02 mg/liter in the 32 farmers selected for this study. There were increas ed percentages of abnormal sperm mor phology, mobility, and sperm count in the exposed group than in controls. The Chi-square t est showed a significant difference between control and exposed groups. Sperm volume did not show significant differences between the exposed subjects, compared to that of the controls. Following a short recovery peri od (after 4 months), asthenospermia (poor sperm motility), motility, and necrospermia (dead sperm) disappeared, while teratospermia (def ects of sperm morphology)

remained.

Conclusions: Investigators concluded that 2,4-D exposure can affect spermatogenesis and male fertility in farmers. Significant levels of asthe nospermia, necrospermia and teratospermia were found in exposed workers as compared to unexposed controls. Over time, asthenos permia and necrospermia diminished but the abnormal spermatozoa (teratospermia) continued.

Comments: This study evaluated male reproductive effects in conjunction with the collection of urine from 32 applicators of 2,4-D. The investigators concluded 2,4-D can produce harmful reproductive effects. In the petition NRDC cited the results of this study in a manner reflective of the conclusions of the investigators. Beyond Pesticides also cited the results of this st udy in a manner reflective of the conclusions of the investigators. No environmental media sampling was completed. The direct exposure monitoring could be considered for quantitative use. However, PHED is curre ntly used for routine assessments of this type rather than individual studies. No explanation of the ci rcumstances of how exposures occurred was included in the discussions in the paper so those aspects of the study coul d not be considered. Additionally, the locale of this study was not described so it is not clear that the prac tices evaluated in this study are reflective of current cultural practices in the United States. Finally, requireme nts for endocrine screening of 2,4-D have just been released by the Agency under its updated strategy and this study should be considered in conjunction with any results from those requirements.

Urine and sperm samples should have been taken at the beginning of study to compare with post exposure samples, although many of the subjects had already worked with 2,4-D at that time. An experimental study such as this can provide the evidence of cause and effect, but there is still a chance of unrecognized confounders. For example, this study did not mention in detailed which drugs were considered in drug history. There are several drugs and chemicals which can affect the male reproduc tive system (e.g., antihypertensives, chemotherapy).

### Page 175 of 186 Title: Insect Repellents, Phenoxyherbicide Exposure, and Non-Hodgkin’s Lymphoma

Journal Citation: JOEM • Volume 47, Number 8, August 2005 (pp. 806-815) Author(s): Helen H. McDuffie, PhD; Punam Pahwa, PhD; Diane Robson, BSc; James A. Dosman, MD; Shirley Fincham, PhD; John J. Spinelli, PhD; John R. McLaughlin, PhD Study Type: EPI – epidemiology Methods Summary: A hypothetical explanation was tested of contra dictory results in studies of phenoxyherbicides and non-Hodgkins lymphoma (NHL), that the exposure of rubber gloves recommended for use by farmers when mixing or applying pesticides simultaneously to 2,4-D (2,4-dichlorophe noxyacetic acid), DEET (N,N-diethyl-m-toluamide), and ultraviolet rays increased their permeability to 2,4-D. A case (NHL n =513)/control (n=1506) study among men was conducted using age; province of residence; exposure to insect repellents containing DEET, phenoxyherbicides, or dicamba; and gloves when handling pesticides. This population-based case–control epidemiologic study was completed using mail questionnaires, modified from previous studies in Kansas and Ne braska, for the entire study gr oup and structured pesticide exposure telephone interviews for those who indicated exposure to 10 hours or more per year of pesticide exposure, and a 15% random sample of the remaining cases and controls. Case subjects were ascertained from provincial cancer registries except in Quebec where hospital ascertainmen t was used. The control subjects were men aged 19 years or older who were residents in the same province as the appropriate case and were free of the cancers of interest and selected at random within age constraints from the provincial Health Insurance reco rds (four provinces), computerized telephone listings (one province) or voters’ lists (one province). Demographic data, detailed lifetime occupation and occupational exposure history, indoor pesticide application, and usage pattern of use of insect repellents on skin or clothing, home/garden use by days per year, average acres sprayed per year, details of occupational h ygiene practice (such as use of gloves and protective clothing) data were collected. Descriptive analyses of each variable, in cluding frequencies, ranges, means, standard deviations, and median values for cases and controls were done separately

. Conditional logistic regression was used to compute odds ratios (OR), and conditional logi stic regression subanalyses of farm residents us ing the variables related to phenoxyherbicide exposure, insect repellents, use of rubber gloves, and potentia l association with NHL. No environmental or human exposure monitoring data were collected in this study. Results Summary: Using conditional logistic regression, the stratum w ith reported exposure to mecoprop, to DEET and the use of rubber gloves had higher odds ratios (3.86; 95% confiden ce interval _ 1.57–9.49) compared with strata with other combinations (e.g., 2,4-D with OR = 1.77 and range from 0.90 –3.45). The investigators acknowledged that they did not determine the exact type of rubber gloves used, which may vary in their permeability or whether gloves were specifically used while mixing or applying 2,4-D or other phenoxyherbicides of interest. The temporal relationship between the mixing or application of pesticides and the use of DEET is unknown. Lack of information in each of these instances would tend to

bias the results toward the null. They also acknowledged the case– control design which they used has inherent limitations, including recall bias and selection bias.

Conclusions: Investigators concluded the etiologic complexity of NHL was demonstrated. The investigators indicated in future research sample sizes must be large enough to statisti cally address the issues of exposure to multiple pesticides, eithe r simultaneously or concurrently and other effect modifiers or confounders, which include pe rsonal hygiene habits during and after handling pesticides and other agricu ltural chemicals, personal and family me dical history, and cigarette-smoking practices. Biomonitoring of selected herbicides or their meta bolites in biological tissues/fluids among large numbers of subjects in conjunction with questionnaires was also recomme nded as a useful addendum in the identification of those exposed who may be at elevated risk of developing NHL.

Comments: NRDC indicated in the petition that there is a rela tionship between NHL and phenoxyacid herbicide use, DEET use and the use rubber gloves. NRDC also indicated that this study suggests that herbicides could be penetrating the skin even when gloves are worn and this could be leading to development of NHL in humans. There are several critical uncertainties which have not been addressed by NRDC that were described by the investigators. For example, the types of gloves used by subjects were not ascertained and the timeframe of DEET application re lative to 2,4-D use was not described. Exposure that occurs even with glove use is anticipated based on existing monitoring data (e.g., PHED). Investigators also suggested that several exposure related aspects should be further investigated because of the uncertainty.

### Page 176 of 186

### Page 177 of 186 Title: Effect of the Mosquito Repellent DEET and Longwave Ultraviolet Radiation On Permeation of the Herbicide 2,4-D and th e Insecticide DDT in Natural Rubber Gloves

Journal Citation: Am. Ind. Hygiene Assoc. J. 53(7): 436-441 (1992) Author(s): Moody, R.P.; Nadeau, B. Study Type: PL – Planned exposure monitoring Methods Summary: A laboratory based permeation cell test was conducted to determine the effect of the commonly used insecticide DEET on the permeability of 100 percent latex rubber gloves (Becton Dickson N36-124). These gloves coul d be worn by applicators as part of a chemical protective clothing ensemble. Glove permeation analysis was conducted with an automated in vitro diffusion analysis (AIDA) method employing an in-house, flow through permeation cell design. Results Summary: Permeation of 14C ring labeled 2,4-D in natural rubber glove material was 2.4±1.81 percent at 48 hours after treatment of the glove with 2,4-D applied with DEET; this was not significantly different (Student’s t-test; p< 0.05) from 3.2±3.46 percent permeation of 2,4-D

observed without DEET. Results were similar for DDT in that no differences were noted when DEET was used. Scanning electron microscopy of the natural rubber glove material, however, demonstrated disruption of the surface ultrastruct ure following a 24 hour treatment with DEET. The AIDA analysis also suggested that exposure of the glove material to longwave ultraviolet radiation enhanced the glove permeability to 2,4-D (6.2±0.73% [+UVA]; 0.3±0.14% [-UVA]).

Conclusions: Investigators concluded that because chem ical protective clothing is commonly exposed to solar UVA this study raises concerns about the efficacy and safety of protective equipment in general.

Comments: This study describes the permeability ch anges in a single glove material after exposure to UVA and also DEET coupled with 2,4-D. The manner in which this study is cited by NRDC is not appropriate because they indicate that the gloves are highly permeable when the glove is exposed to DEET and UVA. The investig ators reported that with DEET the permeation rate of the glove material was similar with 2, 4-D whether or not DEET was present. The latex material tested did indicate increased permeability to UVA. NRDC indicates it then follows that enhanced exposures would occur. This conclusi on is not warranted because it is not clear the application of UVA in this study mimics real wo rld conditions (e.g., intens ity of light beam). Also, only one type of latex rubber glove mate rial was evaluated and its composition was not clear (e.g., UV stabilization or not?). Curre nt production methods likely use UVA and UVB stabilizers in the manufacturing process. It is also easy to recommend other types of glove materials which as UVA/UVB impervious. Finally, it should be noted that in PHED exposures under real world conditions with actual glove use are measured and used to develop risk estimates. While chamber permeability tests of glove materials are useful they do not necessarily accurately predict protection factors for gloves which can also be impacted by design and seam sealing methods. The Agency concurs with the comments of the 2,4-D task force on the NRDC use of this study.

### Page 178 of 186 Title: Adult and children’s exposure to 2,4-D from multiple sources and pathways

Journal Citation: Journal of Exposure Science and Envir onmental Epidemiology (2008) 18, 486–494 Author(s): Marsha K. Morgan, Linda S. Sheldon, Kent W. Thomas, Peter P. Egeghy, Carry W. Croghan, Paul A. Jones, Jane C. Chuang and Nancy K. Wilson Study Type: PB/PL – Population Based Monito ring/Planned Expos ure Monitoring Methods Summary: This study investigated the 2,4-dichloroph enoxyacetic acid (2,4-D) herbicide exposures of 135 preschool-aged children and their adult caregivers at 135 homes in North Carolina (NC) and Ohio (OH). Participants were randomly recruited from six NC and six OH counties. Monitoring was performed over a 48-h period at the participants’ homes. Envi ronmental samples included soil, outdoor air, indoor air, and carpet dust. Personal samples collected by the adult caregivers con cerning themselves and their children consisted of solid food, liquid food, handwipe , and spot urine samples. A ll samples were analyzed for 2,4-D (free acid form) by gas chromatography/mass spectrometry. Results Summary: 2,4-D was detected in all types of environm ental samples but most often in carpet dust samples, with detection frequencies of 83% and 98% in NC and OH, respectiv ely. The median level of 2,4-D in the carpet dust samples was about th ree times higher in OH homes compared to NC homes (156 vs. 47.5 ng/g, P<0.0002). For personal samples, 2,4-D was more fre quently detected in the handwipe samples from OH participants (>48%) than from NC participants (<9%). Ha ndwipe levels at the 95th percentile were about five times higher for OH children (0.1 ng/cm2) and adults (0 .03 ng/cm2) than for the NC children (0.02 ng/cm2) and adults (<0.005 ng/cm2). 2,4-D was detected in more than 85% of the child and adult urine samples in both states. The median urinary 2,4-D concentration was more than twice as high for OH children compared to NC children (1.2 vs. 0.5 ng/ml, P<0.0001); however, the medi an concentration was identical at 0.7 ng/ml for both

NC and OH adults. The intraclass correlation coefficien t of reliability for an individual’s urinary 2,4-D measurements, estimated from the unadjusted (0.31–0.62 ) and specific gravity-adjusted (0.37–0.73) values, were somewhat low for each group in this study. The variability in urinary 2,4-D measurements over the 48-h period for both children and adults in NC and OH suggests that several spot samples we re needed to adequately assess these participants’ exposures to 2,4-D in residential settings.

Conclusions: Investigators concluded results from this study sh owed that children and their adult caregivers in NC and OH were likely exposed to 2,4-D through several pathways at their homes. In addition, our findings suggest that the OH children might have been exposed to higher levels of 2,4-D through the dermal and nondietary routes of exposure than the NC children and the NC and OH adults.

Comments: This study was designed using the CTEPP (Childre n’s Total Exposure To Pers istent Pesticides and Other Persistent Organic Pollutants) multi-media approach . The key difference is was that in this case potential exposures were quantified for children who did not attend daycare and their adult caregiver’s exposure was also evaluated. The investigators concluded 2,4-D urinary le vels were low compared to established reference doses established by IRIS. This study was cited appropriately by the 2,4-D task force including in its description of possible body burden values that could be experienced in households (i.e., NC and OH families were sampled). Additionally, the reported house dust levels were compared by the 2,4-D task force to previous values defined by Nishioka (1996) cited by NRDC. Additionally, inv estigators indicated levels did not indicate a health concern compared to IRIS and existing BE values. The New York Departments of Environmental

Conservation and Health both indicated that EPA should c onsider this study in its 2,4-D risk assessment. The values could be considered quantitatively in an assessment but the results would always be compared to those calculated using applicable existing me thods (e.g., SOPs For Residential Expos ure Assessment). Finally, this study was cited in the NRDC supplement to the petition. It was cited properly and NRDC indicated residues

can be tracked into indoor environments. NRDC also indicated the Agency did not account for this exposure pathway. NRDC indicates the data from this study c ould be used quantitatively for risk assessment purposes. In fact, in its updated SOPs for Residential Exposur e Assessment the Agency has proposed a method for completing such assessments. It could be incorporated as appropriate.

### Page 179 of 186 Title: Measuring Transport of Lawn-Applied Herbicide Acid s from Turf to Home: Correlation of Dislodgeable 2,4-D Turf Residues with Carpet Dust and Carpet Surface Residues

Journal Citation: Environ. Sci. Technol. , 1996, 30 (11), pp 3313–3320 Author(s): Marcia G. Nishioka, Hazel M. Burkholder, Marielle C. Brinkman, and Sydney M. Gordon Study Type: PL – Planned Exposure Monitoring Methods Summary: Transport of lawn-applied herbicides into the hom e via walking over treated turf, defined here as track-in, was measured at five different times out to 1 week after application. At five discrete times after herbicide application, study pa rticipants walked through a treated turf plot and then over a section of carpet to simulate the transfer that migh t occur with a homeowner walking across turf (e.g., to a mailbox). Dislodgeable turf residues were collected prio r to walking; dislodgeable carpet surface residues and carpet dust were collected after walking. Residues of turf-applied 2,4-D and dicamba were measured in carpet dust and on the carpet surface after track-in. The protocol was approved by the Battelle Human Subjects Review Committee in compliance with government regulations. Results Summary: Both carpet dust levels and carpet surface dislodg eable residue levels were highly correlated with turf dislodgeable residue levels. Turf dislodgeable residues were 0.1

− 0.2% of turf application levels. Transfer of herbicides from turf to carpet dust was 3% of the turf dislodgeable residues. Transfer from turf to carpet surface was 0.3% of the turf dislodgeable residues. Herbicide 2, 4-D was measured in the carpet dust of suburban homes at

the 0.1

− 5 μ g/g levels that are predicted by this efficiency of mass transfer. New tools used to collect these samples included the polyurethane foam roller dislodgeable resi due sampler and the high-volume solid surface sampler.

Conclusions: Investigators concluded they expect to find 2,4-D in residential carpet dust up to 1 year after turf application at concentrati ons of approximately 0.5 μ g/g and at carpet loadings of approximately 0.3 μ g/m 2 . These estimates are in agreement with the range of 2,4- D found in nine residential dust samples: 0.1-5 μ g/g and 0.02

− 1.1

μ g/m 2 . These 2,4-D levels suggest track-in as a contributi ng transport mechanism, with the number of children correlated with both carpet loading ( r 2 = 0.49; p = 0.04) and with dust concentration ( r 2 = 0.35; p = 0.09) as measured in regression analyses. The investigators also indicated in a recently reported study, the urine from matched populations of children in two Ar kansas towns was analyzed for 2,4-D and 2,4-D was detected in urine of 18% of the children from the town containing a 2,4-D manufacturing plant and was present in the urine of 23% of the children in the town with no manufacturing plant. The simi larity of exposure rates a nd detection levels indicated an exposure source other than the manufacturing plant, though the source was unknown. Given the fact that children's hand-to-mouth activity promotes ingestion of c ontaminated carpet dust, we might assume that chronic indoor exposure will follow a lawn application and may result in measurable urinary levels. Certainly this assumption needs to be fully investigat ed by analysis of matched samples of house dust and urine from resident children prior to and after turf application. [Note: No me ntion was made concerning potential dietary or drinking water sources.]

Comments: This study evaluated 2,4-D residues in both carpe t dust and as transferable residues on turf using the PUF roller method in a simulated carp et sampling approach. Both NRDC and Beyond Pesticides cited the conclusions by the investigators that 2,4-D persists indoor s for up to one year. This value was not empirically derived through measurements but instead it was conclude d by the investigators based on a series of assumptions and sampling which only occurred out to 1 week after an a pplication of 2,4-D. The Agency does not concur with these conclusions by the investigators. The 2,4-D task force correctly indicates that the levels were calculated by the investigators and then compared them to empirically de rived dust concentrations by Morgan (2008) which were lower than those calculated by the investigators. The 2, 4-D task force did not discuss the temporal considerations associated with the interpretation in this comparison. Also, it should be noted that the Agency does not concur with the conclusions by the investigator s because the assumptions do not account for any degradation processes nor is the short sampling timeframe of 1 week discu ssed as an uncertainty in the conclusions.

### Page 180 of 186 Title: Urinary Excretion of 2,4-D In Commercial Lawn Specialists

Journal Citation: Appl. Ind. Hyg. 3:119-121; 1986 Author(s): Yeary, R.A. Study Type: PL – Planned Exposure Monitoring Methods Summary: Occupational exposure to the he rbicide 2,4-D was measured in 45 commercial lawn care specialists who had been spraying the herbicide on lawns on a daily basis, excluding Sundays, for a period of at least 3 weeks. Exposure was determined by collecting all urine voided during a 24 hour period, and analysis of 2,4-D was made by gas chromatography. The 24 hour urinary excretion of 2,4-D was considered to be a reasonable estimate of the daily absorbed dose. When handling concentrated pr oduct workers wore latex gloves, an apron or coveralls, goggles or faceshield, and rubber boots. When spraying dilute mixtures workers wore rubber boots, long pants, and short-sleeved shirts. Glove use was optional. Results Summary: The urinary concentrations of 2,4-D ranged from non-detectable at a sensitivity of 0.01 ppm to 2.29 ppm with a median of 0.18 ppm. The quantity excreted on a body weight basis was 0.0032 ± 0.0010, 0.0063 ± 0.0018, 0.00035 ± 0.0005, and 0.00138 ± 0.0005 mg/kg respectively at 4 work locations.

Conclusions: Investigators concluded these values are considerably less than the WHO/FAO acceptable daily dietary intake of 0.3 mg/kg for 2,4-D residues in food.

Comments: This study evaluated worker exposure and also produced distributions of exposure in

varied field locations. The use of the results of this study in the comments from the 2,4-D task force appears to be reasonable in that they reflect the specific conclusions described by the investigators. However, it should be noted that, as written, the 2,4-D task force summary of the study could be misleading because they indi cate exposures did not exceed a level of 6 μ g/kg/day which was really the maximum mean value from particular site with the highest average exposures. Exposures on a individual basis actually ranged about 10 times higher that this value as noted in the highest urinary concentration reported of 2.29 ppm. No environmental media sampling was completed. The direct exposure m onitoring could be considered for quantitative use. However, PHED is currently used for rou tine assessments of this type rather than individual studies but some values for the monitored forestry operations are not addressed by PHED.

### Page 181 of 186 

# Appendix D:

Pet Incident Summaries

### Page 182 of 186 Title: Canine Exposure To Herbicide Treated Lawns and Urinary Excretion of 2,4- Dichlorophenoxyacetic Acid

Journal Citation: Cancer Epidemiology, Vol. 3, 233-237 (April/May 1994) Author(s): Paula M. Reynolds, John S. Reif,1 Howard S. Ramsdell, and John D. Tessari Study Type: Pet Exposure Methods Summary: A recent study by Hayes et aL (J ı NatI. Cancer. Inst., 83: 1 226-i 231 , 1991) found an increased risk of malignant Iymphoma associated with exposure to 2,4- dichlorophenoxyacetic acid (2,4-D) in pet dogs. A study was conducted to determine the extent to which dogs absorb and excrete 2,4-D in urin e after contact with treated lawns under natural

conditions. Results Summary: Among 44 dogs potentially exposed to 2,4-D-treated lawns an average of 10.9 days after application, 2,4-D concentrations greater than or equal to 10.0 μg/l were found in 33 dogs (75%) and concentrations of ≥ 50 μg/l were found in 17 (39%). Among 15 dogs with no known exposure to a 2,4-D-treated lawn in the pr evious 42 days, 4 (27%) had evidence of 2,4-D in urine, 1 at a concentration of ≥ 50 μg/l . The odds ratio for the association between exposure to a 2,4-D-treated lawn and the detection of ≥ 50 μg/l 2,4-D in urine was 8.8 (95% confidence interval, 1 .4-56.2). Dogs exposed to lawns treat ed within 7 days before urine collection were more than 50 times as likely to have 2,4-D at concentrations ≥ 50 μg/l than dogs with exposure to a lawn treated more than 1 week previously (odds ratio = 56.0; 95% confidence interval, 10.0- 312.2). The highest mean concentration of 2,4-D in urine (21 3 mg/l) was found in dogs sampled within 2 days after application of the herbicide.

Conclusions: Investigators indicated these findings demonstrate that dogs living in and around residences with recent 2,4-D lawn treatment ab sorb measurable amounts of the herbicide for several days after application and thus may constitu te a useful animal model for evaluation of the effects of herbicides on the induction of lymphoid cancer.

Comments: This study was considered in the co mments of Beyond Pesticides and was cited appropriately except that the comparison made to pr evious data could not be verified. It should be noted that the NRDC petition does not make an y mention of exposure to pets or via pets.

### Page 183 of 186 Title: Case-Control Study of Canine Malignant Lymphoma: Positive Association With Dog Owner’s Use of 2,4-Dichlorophenoxyacetic acid herbicides

Journal Citation: J National Cancer Institute 1991; 83:1226-1231 Author(s): Hayes, H.M., Tarone, R.E., Cantor, K.P., Jessen, C.R., McCurnin, D.M., Richardson,

R.C. Study Type: Pet Exposure Methods Summary: Hospital-based case control study of companion dogs which examined developing canine malignant lymphoma associated with the use of chemicals in and around the home. A self administered owner questionnaire a nd/or phone interviews were completed for 491 cases, 466 nontumor controls, and 479 tumor contro ls. Dogs diagnosed between January 1984 and February 1988 were identified from 3 veterinary teaching hospitals (MI, IN, CO). Tumor controls included only animals excluding diseases that could be related to chemical exposures. A total of 588 patients met eligibility criteria for malignant lymphoma and 491 responded (84% participation rate). Results Summary: Dogs living in households where 2, 4-D was applied more frequently had significantly higher odds ratios that control s ituations (Odds Ratio=1.3). Risk of canine malignant lymphoma rose to twofold excess with fo ur or more yearly owner applications of 2,4-

D. No difference was observed in the results based whether applications were completed by homeowners or professional applicators. A positiv e trend (P<0.02) was observed for the yearly number of applications. Other possible factor s (e.g., flea product use, cigarette use, carpet presence) were evaluated but found not to be a factor. Known susceptible breeds (e.g., boxers, mastiffs) were also addressed. The investigators commented on how recall biases were addressed in the study but they acknowledged a major weakness is a lack of precise exposure data.

Conclusions: The investigators concluded that the study in consistent with results in humans where modest relationships between lymphoma inci dence and 2,4-D use is observed. They also indicate the investigation began with an a prio ri hypothesis that exposure to 2,4-D and canine malignant lymphoma might be observed and that the findings support this supposition. They indicate further investigation is warranted.

Comments: No environmental or personal samp ling was completed and the author s acknowledge significant uncertainty pertaining to a lack of exposure data. It is also not clear how germane the results of this study are given th e timeframe over which this study was conducted compared to modern use patterns associated with 2,4-D. For example, certain manufacturing contaminants were eliminated from 2,4-D products so it could be plausible that exposures to older types of products could have occurred in the po sitive cases considered. The citation of this study by Beyond Pesticides reflects the conclusions of the investigators but it does not account for the uncertainty associated with the exposure predicto rs acknowledged by the investigators in this study.

It should also be noted that in the follow-up 1995 paper to this investigators note significant

levels of criticisms that are addressed in the subsequent document described below. Please refer to that summary of Hayes et al 1995 for additional information.

### Page 184 of 186 Title: On the Association Between Canine Maligna nt Lymphoma and Opportunity for Exposure to 2,4-Dichlorophenoxyacetic Acid

Journal Citation: Environmental Research 70, 119-125 (1995) Author(s): Hayes, H.M., Tarone, R.E., Cantor, K.P Study Type: Pet Exposure Methods Summary: This article was written in response to specific criticisms leveled at the Hayes et al 1991 article described above. Se veral specific considerations were addressed including how control groups were treated, wh ether responses were via mail or phone, potential hospital selection bias, difficulties associated w ith exposure classification including frequency of use, and knowledge related to the specific use of 2,4-D products. Results Summary: The authors addressed each of the applicable criticisms. The results of Hayes et al 1991 were supported in their analysis. The authors clarified the conclusions to better address uncertainty issues (see below).

Conclusions: Selection and recall bias can never be elim inated and every attempt was made to do so. In this additional analysis the use of a la wn service and its implications on the results was further examined and it does not appear to impact results. The investigators also commented on the magnitude of the associated odds ratios. The investigators also acknowledged that the reported values could be explained by bias or confou nding factors. It was also indicated that the results should be interpreted with caution becau se of low exposures. The investigators also concur with several commenters that the study does not prove that 2,4-D exposure in a home environment is a cause of malignant lymphoma in companion dogs. They again indicate it does

warrant further research on the issue.

Comments: The authors addressed several specific critic isms of the 1991 study in an appropriate manner. The citation of this study in the Be yond Pesticides comments to the NRDC petition is correct. However, it is somewhat misleadi ng because they do not reiterate the level of uncertainty voiced by the investig ators in their comments about th e applicability of the results and the call in both Hayes 1991 and 1995 for additional research to investigate this relatively weak relationship between 2,4-D exposur e and canine malignant lymphoma.

### Page 185 of 186 

# Appendix E:

Meta Information

### Page 186 of 186 Title: Regenerative 21 st Century Farming: A Solution to Global Warming

Journal Citation:

http://www.ifoam.org/growing_organic/1_arguments_for_oa/environm ental_benefits/pdfs/Rodale_Research_Paper_Regenerative_Agricult ure.pdf Author(s): Dr. Timothy J. LaSalle, CEO; Dr. Paul He pperly, Director of Research and Fulbright Scholar; Rodale Institute © Study Type: Sustainable Agricultural Systems Analysis Methods Summary: This paper discusses organic alternatives to 2,4-D. Results Summary: Organic alternatives to 2,4-D are illustrated.

Conclusions: Investigators conclude there are viab le organic alternatives to 2,4-D.

Comments: This study was cited properly in the Beyond Pesticides Comment. It does not discuss human health aspects of exposure to 2,4- D but only discusses herbicide use as it relates to conventional large scale farming practices.

Title: Handbook of Successful Ecological Lawn Care

Journal Citation: Edaphic Press 1995, ISBN 9780963605313 Author(s): Paul D. Sachs Study Type: Sustainable Agricultural Systems Analysis Methods Summary: This paper discusses organic alternatives to 2,4-D. Results Summary: Organic alternatives to 2,4-D are illustrated.

Conclusions: Investigators conclude there are viab le organic alternatives to 2,4-D.

Comments: This study was cited properly in the Beyond Pesticides Comment. It does not discuss human health aspects of exposure to 2,4-D but only discusses organic alternatives

herbicide use in lawncare.

Title: The Herbicide Resistance Action Committee (HRAC). 2005. Classification of Herbicides According to Mode of Action

Journal Citation: http://www.hracglobal.com/Publications/Classifi cationofHerbicideModeof Action/tabid/222/Defa ult.aspx Author(s): Dr. Hubert Menne , Bayer CropScience AG Frankfurt Germany Study Type: Rating System to categorize herbicide mode of action Methods Summary: This paper discusses herbicide mode of action Results Summary: This paper discusses herbicide mode of action

Conclusions: This paper discusses herbicide mode of action

Comments: This study was cited properly in the Beyond Pesticides Comment in that it does

describe how herbicide modes of action can be categorized. It does not discuss human health aspects of exposure to 2,4-D or the need to co nduct a cumulative human health risk assessment that Beyond Pesticides raises partially based on this information.

i NAFTA Dermal Absorption Group Position Paper on Use of In Vitro Dermal Absorption Data in Risk Assessment (2009).