CHEMICAL NAME PC Code: 030001 Toxicology Disciplinary Chapter for the Reregistration Eligibility Decision Document Date completed:

Prepared by: Linda L. Taylor, Ph. D. Reregistration Branch I Health Effects Division Reviewed by: Whang Phang, Ph. D. Reregistration Branch I Health Effects Division form: FINAL June 21, 2000 TABLE OF CONTENTS 1.0 HAZARD CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.0 REQUIREMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.0 DATA GAP( S) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.0 HAZARD ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.1 Acute Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.2 Subchronic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4.3 Prenatal Developmental Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 4.4 Reproductive Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.5 Chronic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.6 Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4.7 Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.8 Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 4.9 Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 4.10 Special/ Other Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 5.0 TOXICITY ENDPOINT SELECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 5.1 See Section 9.2 for Endpoint Selection Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 5.2 Dermal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 5.3 Classification of Carcinogenic Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 6.0 FQPA CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 6.1 Special Sensitivity to Infants and Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 6.2 Recommendation for a Developmental Neurotoxicity Study . . . . . . . . . . . . . . . . . . . . . . . 45 7.0 OTHER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 8.0 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 9.0 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Toxicity Profile Summary Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Acute Toxicity Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.2 Subchronic, Chronic and Other Toxicity Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Summary of Toxicological Dose and Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 3 1.0 HAZARD CHARACTERIZATION 2,4 Dichlorophenoxyacetic acid [ 2,4 D] and its amine salts [ diethanolamine ( DEA), dimethylamine ( DMA), isopropylamine ( IPA), and triisopropanolamine ( TIPA)] and esters [ butoxyethyl ester ( BEE), ethylhexyl ester ( EHE), and isopropyl ester ( IPE)] are not acutely toxic via the oral [ Toxicity Category III], dermal [ Toxicity Category III], and inhalation [ Toxicity Category III/ IV] routes of exposure. 2,4 D and its amine salts and esters are not skin irritants [ Toxicity Categories III and IV] and none is a skin sensitizer. The acid and amine salt forms of 2,4 D are considered severe eye irritants [ Toxicity Category I], but the ester forms are not eye irritants [ Toxicity Category III].

In general, 2,4 D undergoes limited metabolism primarily involving minor conjugation of the parent acid that is then excreted in the urine. No detectable metabolites of 2,4 D have been reported in the rat; i. e., only the parent acid is found in rat urine. In addition to 2,4 D itself, 2,4 D conjugates have been found in the urine of dogs, humans, mice, and hamsters following oral exposure. The mechanisms responsible for the renal clearance of 2,4 D have been investigated in several species also. This phenoxy herbicide is actively secreted by the proximal tubules in a manner similar to paraaminohippuric acid [ PAH], and this mechanism of renal clearance for 2,4 D is consistent with results seen with other phenoxy acids. It has been suggested that the observed dosedependent non linear pharmacokinetics of 2,4 D are primarily due to the saturation of this renal secretory transport system. Due to a limited capacity to excrete organic acids, the dog is more sensitive to the effects of 2,4 D than the rat with respect to repeated dosing.

Following subchronic oral exposure at dose levels of 2,4 D above the threshold of saturation for renal clearance, the primary target organs are the eye [ retinal degeneration, cataract formation], thyroid [ increased thyroid weight, increased T3/ decreased T4, and follicular cell hypertrophy], kidney [ brush border loss in proximal tubule], adrenals [ hypertrophy], and ovaries/ testes [ atrophy]. These changes are also observed following exposure to the amine salts and esters of 2,4 D. Systemic toxicity was not observed following repeated dermal exposure to 2,4 D, EHE, and TIPA at or above the limit dose or following repeated dermal exposure to BEE and IPA at the highest dose tested. Liver toxicity was observed following repeated dermal exposure to DEA, and one death [ female] occurred following dermal exposure to DMA at a high dose level.

There are no repeated inhalation exposure data available on 2,4 D. The only reliable way to characterize inhalation toxicity and to quantify inhalation risk is through the use of inhalation toxicity studies. In general, chemicals tend to be more toxic by the inhalation route than by the oral route due to rapid absorption and distribution, bypassing of the liver's metabolic protection ( portal circulation), and potentially serious portal ofentry effects, such as irritation, edema, cellular transformation, degeneration, and necrosis. An inhalation risk assessment that is based on oral data generally underestimates the inhalation risk because it cannot account for these factors. However, in the case of 2,4 D, based on the limited metabolism of 2,4 D via the oral route, the moiety to which the body would be exposed would be the same for both routes of exposure. With regard to portal of entry effects, these can only be assessed in an inhalation study. Therefore, a subchronic inhalation study is required for 2,4 D pending the submission of an acceptable waiver rationale.

Developmental toxicity, characterized mainly as an increased incidence of skeletal variations in the rat, was observed following exposure to 2,4 D and its amine salts and esters. Malformations in the rat were observed only at dose levels that were at or above the threshold of saturation of renal clearance. Similarly, developmental CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 4 toxicity was observed in the rabbit only following high dose exposures above the renal clearance level for 2,4 D [ abortions] and DEA [ increased number of litters with fetuses with 7th cervical ribs.

Reproductive toxicity, characterized as an increase in gestation length, was observed following exposure to 2,4 D at a dose level above the threshold of saturation of renal clearance. A repeat 2 generation reproduction study [ using the new protocol] is required to address concerns for endocrine disruption [ thyroid and immunotoxicity measures].

Neurotoxicity was demonstrated following exposure to 2,4 D at relatively high dose levels. Clinical signs of neurotoxicity [ ataxia, decreased motor activity, myotonia, prostration, lateral recumbency, and impaired/ loss of the righting reflex, cold to the touch] were observed in pregnant rabbits following exposure to 2,4 D and its amine salts and esters. Neuropathology [ retinal degeneration] was observed following 2,4 D exposure in several studies in female rats. Incoordination and slight gait abnormalities ( forepaw flexing or knuckling) were observed following acute dosing and increased forelimb grip strength was observed following chronic exposure to 2,4 D at dose levels that exceeded the threshold of saturation of renal clearance. A developmental neurotoxicity study in the rat is required on 2,4 D.

2,4 D is classified as a Group D chemical [ not classifiable as to human carcinogenicity]. Based on the overall pattern of responses observed in both in vitro and in vivo genotoxicity tests, 2,4 D was not mutagenic, although some cytogenic effects were observed.

2,4 D affects thyroid hormone homeostasis following oral exposure, and there is a concern for endocrine disruption. Effects on the gonads in rats [ decreased testes and ovarian weights, atrophy of the ovary, uterus and testis, degeneration of the seminiferous epithelium, decreased spermatozoa in epididymis, testicular degeneration, increase in gestation length] and dogs [ increased testes weight, juvenile testes and prostate, and hypospermatogenesis] are seen following exposure to 2,4 D and/ or its amine salts and esters. Although there are data on thyroid hormone levels in the adult animal, there are no data with respect to thyroid hormones in the young; therefore, there is no information on whether the young are more sensitive with respect to this endpoint. There is no developmental neurotoxicity [ DNT] study available on 2,4 D, and the HIARC determined previously that a DNT study is required [ HED Document No. 014234]. There have been no studies on 2,4 D that specifically assess its endocrine disruption potential, and there are no data on other hormonal effects. Thyroid effects [ increased thyroid weight, increased T3/ decreased T4, and follicular cell hypertrophy] have been observed in the rat following subchronic exposure to 2,4 D and its amine salts and esters and following chronic exposure to 2,4 D [ decreased T4, increased thyroid weight, thyroid masses, and follicular cell hyperplasia ( males; interim sacrifice only)/ hypertrophy ( females; interim sacrifice only)]. Additionally, there is some concern for immunotoxicity following exposure to 2,4 D. There are clear NOAELs for each of the above mentioned effects, which occur only at high dose levels; above the doses selected for overall risk assessment. Therefore, there are no residual uncertainties with regard to these effects. However, the HIARC concluded that a 2 generation reproduction study using the current protocol is required to address both the concern for thyroid effects [ comparative assessment between the young and adult animals] and immunotoxicity, as well as a more thorough assessment of the gonads and reproductive/ developmental endpoints. The Task Force should consult the Agency on the protocol for this study. 2.0 REQUIREMENTS The requirements ( CFR 158.340) for food and non food use for 2,4 dichlorophenoxyacetic acid [ 2,4 D] CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 5 are in Table 1. Use of the new guideline numbers does not imply that the new ( 1998) guideline protocols were used. Table 1.

Test Technical Required Satisfied 870.1100 Acute Oral Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . 870.1200 Acute Dermal Toxicity . . . . . . . . . . . . . . . . . . . . . . 870.1300 Acute Inhalation Toxicity . . . . . . . . . . . . . . . . . . . . 870.2400 Primary Eye Irritation . . . . . . . . . . . . . . . . . . . . . . . 870.2500 Primary Dermal Irritation . . . . . . . . . . . . . . . . . . . . 870.2600 Dermal Sensitization . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes yes yes 870.3100 Oral Subchronic ( rodent) . . . . . . . . . . . . . . . . . . . . . 870.3150 Oral Subchronic ( nonrodent) . . . . . . . . . . . . . . . . . . 870.3200 21 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.3250 90 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.3465 90 Day Inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes no yesb yes yes yes  no 870.3700a Developmental Toxicity ( rodent) . . . . . . . . . . . . . . . 870.3700b Developmental Toxicity ( nonrodent) . . . . . . . . . . . . 870.3800 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yesa 870.4100a Chronic Toxicity ( rodent) . . . . . . . . . . . . . . . . . . . . 870.4100b Chronic Toxicity ( nonrodent) . . . . . . . . . . . . . . . . . 870.4200a Carcinogenicity ( rat) . . . . . . . . . . . . . . . . . . . . . . . . 870.4200b Carcinogenicity ( mouse) . . . . . . . . . . . . . . . . . . . . . 870.4300 Chronic/ carcinogenicity . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes 870.5100 Mutagenicity C Gene Mutation  bacterial . . . . . . . . . 870.5300 Mutagenicity C Gene Mutation  mammalian . . . . . . 870.5xxx Mutagenicity C Structural Chromosomal Aberrations 870.5xxx Mutagenicity C Other Genotoxic Effects . . . . . . . . . . yes yes yes yes yes yes yes yes 870.6100a Acute Delayed Neurotoxicity ( hen) . . . . . . . . . . . . . 870.6100b 90 Day Neurotoxicity ( hen) . . . . . . . . . . . . . . . . . . . 870.6200a Acute Neurotoxicity Screening Battery ( rat) . . . . . . . 870.6200b 90 Day Neurotoxicity Screening Battery ( rat) . . . . . . 870.6300 Developmental Neurotoxicity . . . . . . . . . . . . . . . . . . no no yes yes yes

yes yes no 870.7485 General Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 870.7600 Dermal Penetration . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes Special Studies for Ocular Effects Acute Oral ( rat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subchronic Oral ( rat) . . . . . . . . . . . . . . . . . . . . . . . . Six month Oral ( dog) . . . . . . . . . . . . . . . . . . . . . . . no no no

aa repeat 2 generation reproduction study following new protocol is required to address concerns for endocrine disruption [ thyroid and immunotoxicity measures]; b a 28 day inhalation study is not satisfied CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 6 3.0 DATA GAP( S)

There are three datagaps: ( 1) repeat 2 generation reproduction study following new protocol to address concerns for endocrine disruption with thyroid and immunotoxicity measures; ( 2) developmental neurotoxicity study; and ( 3) 28 day inhalation toxicity study.

4.0 HAZARD ASSESSMENT 4.1 Acute Toxicity Adequacy of data base for acute toxicity: The data base for acute toxicity is considered complete. No additional studies are required at this time. The acute toxicity data indicate that 2,4 dichlorophenoxyacetic acid [ 2,4 D] is not acutely toxic via the oral [ Toxicity Category III], dermal [ Toxicity Category III], or inhalation [ Toxicity Category III] routes of exposure. 2,4 Dichlorophenoxyacetic acid is not a skin irritant [ Toxicity Category IV] nor a skin sensitizer but it is considered a severe eye irritant [ Toxicity Category I].

After oral [ single dose] administration of 2,4 D and its amine salts and esters, the clinical signs of toxicity observed consistently were ataxia, myotonia, and decreased limb tone. No dermal or systemic toxicity was observed in rabbits dosed dermally, and no deaths were observed after inhalation. Clinical signs of toxicity observed during inhalation exposure were decreased activity and closed eyes. All rats had dried white material on their fur, presumably test material. Signs observed at the end of and during the week after inhalation exposure were salivation, lacrimation, mucoid nasal discharge, labored breathing, dried red or brown material around eyes and nose, matted fur, and staining of the fur in the anogenital region. There were no significant findings post mortem.

The acute toxicity data on 2,4 dichlorophenoxyacetic acid are summarized below in Table 2.

Table 2. Acute Toxicity Data on 2,4 Dichlorophenoxyacetic acid Guideline No. Study Type MRID #( S) Results Toxicity Category 870.1100 Acute Oral 00101605 rat LD 50 = 699 mg/ kg III 870.1200 Acute Dermal 00101596 rabbits LD 50 > 2000 mg/ kg III 870.1300 Acute Inhalation 00161660 rat LC 50 > 1.79 mg/ L III 870.2400 Primary Eye Irritation 41125302 severe eye irritant I 870.2500 Primary Skin Irritation 42232701 non irritating IV 870.2600 Dermal Sensitization 00161659 not a dermal sensitizer

870.6200 Acute Neurotoxicity 43115201 NOAEL = 67 mg/ kg/ day

above studies satisfy the acute toxicity data requirements [ OPPTS 870.1100, 870.1200, 870.1300, 870.2400, 870.2500, 870.2600, 870.6200; formerly § 81 1 through § 81 6, § 81 8] for 2,4 dichlorophenoxyacetic acid. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 7 The acute toxicity data on several amine salts [ diethanolamine ( DEA; CAS No. 5742 19 8), dimethylamine ( DMA; CAS No. 2008 39 1), isopropylamine ( IPA; 5742 17 6), and triisopropanolamine ( TIPA; 32341 80 3)] and esters [ butoxyethyl ester ( BEE; 1929 7303), ethylhexyl ester ( EHE; 1928 43 4), and isopropyl ester ( IPE; 94 11 1)] of 2,4 Dichlorophenoxyacetic acid [ 2,4 D; CAS No. 94 75 1] are summarized below in Table 2a.

Table 2a. Acute Toxicity Data on Amine Salts and Esters of 2,4 Dichlorophenoxyacetic Acid Guideline No. Study Type MRID #( S) Results Toxicity Category 870.1100 Acute Oral DEA salt DMA salt IPA salt IP ester TIPA salt BEE ester EHE ester 41920901 00157512 00252291 41709901 41413501 40629801 41209001 rat LD50 = 910 mg/ kg rat LD50 = 949 mg/ kg rat LD50 = 2322/ 1646 mg/ kg rat LD50 = 1250 mg/ kg rat LD50 = 1220/ 1074 mg/ kg rat LD50 = 866 mg/ kg rat LD50 = 896 mg/ kg III III III III III III III 870.1200 Acute Dermal DEA salt DMA salt IPA salt IP ester TIPA salt BEE ester EHE ester 41920911 00157513 00252291 41709902 41413502 40629802 41209002 rabbits LD50 > 2000 mg/ kg rabbit LD50 > 2000 mg/ kg rabbits LD50 > 2000 mg/ kg rabbits LD50 > 2000 mg/ kg rabbits LD50 > 2000 mg/ kg rabbits LD50 > 2000 mg/ kg rabbits LD50 > 2000 mg/ kg III III III III III III III 870.1300 Acute Inhalation DEA salt DMA salt IPA salt IP ester TIPA salt BEE ester EH ester 41986601 00157514 40085501 40352701 41957601 40629803 42605202 rat LC50 > 3.5 mg/ L rat LC50 > 3.5 mg/ L rat LC50 = 3.8 mg/ L rat LC50 > 4.97 mg/ L rat LC50 = 5.35 mg/ L rat LC50 = 4.6 mg/ L rat LC50 > 5.4 mg/ L IV IV IV IV IV IV IV 870.2400 Primary Eye Irritation DEA salt DMA salt IPA salt IP ester TIPA salt BEE ester EHE ester 41920902 00157515 00252291 40352702 41413504 40629804 44725303 severe eye irritant severe eye irritant severe eye irritant not an eye irritant severe eye irritant not an eye irritant not an eye irritant I I III I III III 870.2500 Primary Skin Irritation DEA salt DMA salt IPA salt IP ester TIPA salt BEE ester EH ester 41920903 00157516 00252291 40352703 41413505 40629805 41209005 slight skin irritant slight skin irritant slight skin irritant very mild irritant slight skin irritant very mild irritant not a skin irritant IV IV IV IV IV IV IV CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Guideline No. Study Type MRID #( S) Results Toxicity Category 8 870.2600 Dermal Sensitization DEA salt DMA salt IPA salt IP ester TIPA salt BEE ester EH ester 41920904 41642805 41233701 40352704 41413506 40629806 41209006 not a dermal sensitizer not a dermal sensitizer not a dermal sensitizer not a dermal sensitizer not a dermal sensitizer not a dermal sensitizer not a dermal sensitizer

above studies satisfy the acute toxicity data requirements [ OPPTS 870.1100, 870.1200, 870.1300, 870.2400, 870.2500, 870.2600, 870.6200; formerly § 81 1 through § 81 6,] for several amine salts and esters of 2,4 dichlorophenoxyacetic acid.

4.2 Subchronic Toxicity Adequacy of data base for subchronic toxicity: The data base for subchronic oral and dermal toxicity is considered complete. No additional oral or dermal studies are required at this time. There is no subchronic inhalation study available, and one is required. The subchronic database provides evidence that the kidney [ increased kidney weight, histopathological lesions], thyroid [ decreased thyroxine, increased thyroid weight, hypertrophy of follicular cells], liver [ increased liver weight, increased ALT, and AST, histopathological lesions, including hypertrophy], and adrenal [ increased adrenal weight, histopathological lesions] are target organs in the rat following subchronic exposure via the oral route. No systemic toxicity was observed in rabbits following repeated exposure via the dermal route at dose levels up to the limit dose [ 1000 mg/ kg/ day]. In the dog, the kidney and thyroid are target organs [ elevated blood urea nitrogen and creatinine, decreased glucose, decreased T4, increased kidney and thyroid weights, cellular alterations of the proximal tubule]. Additionally, decreased testes weight and hypospermatogenesis/ juvenile testis were observed in both dog studies. Effects in dogs were observed at lower dose levels than those observed in rodents, and this is attributed to the dog's limited capacity to eliminate 2,4 D and other organic acids. Subchronic toxicity following exposure to the amine salts and esters of 2,4 D was similar to that observed following 2,4 D exposure.

870.3100 90 Day Oral Toxicity  Rat EXECUTIVE SUMMARY: In a subchronic oral toxicity study [ MRID 41991501], 10 Fischer 344 rats/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 96.1% a. i.] via the diet for 13 weeks at concentrations of 0, 1 mg/ kg/ day, 15 mg/ kg/ day, 100 mg/ kg/ day, and 300 mg/ kg/ day.

There were no treatment related deaths. Clinical signs of toxicity occurred mainly in the high dose females and included hunched posture [ 1 high dose female during weeks 13 14], depressed activity [ all high dose females during first week], and few/ no feces [ 4 high dose males and all high dose females during week 1; 5 high dose females during week 12; 1 high dose female during week 13]. There was a dose related increase in the incidence of pale/ opaque eyes in both sexes [ 1/ 10 controls, 2/ 10 at 1, 15, and 100 mg/ kg/ day and 4/ 10 at 300 mg/ kg/ day in males; 1/ 10 controls, 2/ 10 at 1 mg/ kg/ day, 1/ 10 at 15 mg/ kg/ day, 4/ 10 at 100 mg/ kg/ day, and 8/ 10 at 300 mg/ kg/ day in females]. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 9 Decreased body weights were observed throughout the study at the high dose level [ both sexes], with the magnitude of the deficit increasing with time [ males 85%/ 77% and females 79%/ 72% of control at weeks 6/ 13, respectively], and decreased body weight gains [ weeks 0 6: males 72%/ females 50% of control; overall: males 63%/ females 43% of control] were observed in both sexes at the highest dose level throughout the study. At the next highest dose level, decreased body weight [ 93% of control at 13 weeks; both sexes] and body weight gain [ overall males 91%/ females 89% of control] were observed. A corresponding decrease in food consumption was observed in both sexes at the two highest dose levels.

Complete cataract formation was observed in 7 high dose [ 300 mg/ kg/ day] females and in one female at the next highest [ 100 mg/ kg/ day] dose, and posterior subcapsular cataract was observed in 5 high dose females. Treatment related alterations in hematology [ statistically significant decreases in RBC, HGB, HCT, platelet count, absolute and corrected leukocyte counts, and lymphocyte counts] were observed at the 6 and/ or 13 week intervals at the high dose level [ both sexes], and decreased platelet counts were observed in both sexes at the next highest dose level at week 13. Alterations in clinical chemistry [ decreased thyroxine and triiodothyronine levels] were observed at 100 and 300 mg/ kg/ day at both intervals in one or both sexes.

Changes in absolute and/ or relative organ weights [ adrenals, brain, thymus, heart, kidneys, testes with epididymides ( males), ovaries ( females), pituitary, liver ( increased), thyroids/ parathyroids ( increased) were observed primarily at the high dose level [ both sexes], and many of these changes may be attributable to decreased body weight. Gross findings, mainly in the high dose group, included small testes and epididymis and opaque eyes [ females]. Treatment related histopathological changes were observed primarily in the high dose group and included centrilobular hepatocellular hypertrophy [ liver], bilateral retinal degeneration and cataract formation [ females], atrophy of the testes [ males], hypertrophy of the zona glomerulose [ adrenal cortex, both sexes] and follicular cells [ thyroid, females], atrophy of the thymus [ both sexes] and spleen [ both sexes], congestion and edema of the bone marrow [ both sexes], brush border loss in proximal tubular cells [ kidney, both sexes]. Many of the lesions correlated well with the alterations observed in hematology and clinical chemistry parameters and/ or organ weight data of the high dose groups.

The NOAEL is 15 mg/ kg/ day, based on decreased body weight/ body weight gain, alterations in some hematology [ decreased platelets ( both sexes)] and clinical chemistry [ decreased T3 ( females) and T4 ( both sexes)] parameters, and cataract formation in females at the LOAEL of 100 mg/ kg/ day.

This subchronic oral toxicity study is classified Acceptable/ Guideline, and the study satisfies the guideline requirement [ OPPTS 870.3100; § 82 1( a)] for a subchronic oral toxicity study in the rodent.

The following table summarizes the subchronic oral toxicity studies in rats available for 2,4 D and its amine salts and esters for comparison. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 10 Subchronic Oral Toxicity of 2,4 D and its Salts and Esters in Rats Guideline STUDY RESULTS 870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1, 15, 100, 300 mg/ kg/ day]

MRID: 41991501 [ 1991]

Classification: Acceptable/ Guideline 2,4 D ( 96.1% a. i.) Systemic Toxicity NOAEL = 15 mg/ kg/ day Systemic Toxicity LOAEL = 100 mg/ kg/ day, based on decreases in body weight/ gain, alterations in hematology and clinical chemistry parameters, and cataract formation in females;

At the highest dose tested [ HDT], effects listed above occurred more frequently and/ or to a greater extent, as well as histopathological lesions in eyes, liver, testes, adrenals, kidneys, thymus, bone marrow, spleen, thyroids, lungs.

870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1.5, 22, 145, 436 mg/ kg/ day] { 1, 15, 100, 300 mg/ kg/ day}

MRID: 41928101 [ 1991]

Classification: Acceptable/ Guideline BEE ester of 2,4 D ( 94.6% ; acid equivalent

65%) Systemic Toxicity NOAEL = 22 { 15} mg/ kg/ day Systemic Toxicity LOAEL = 145 { 100} mg/ kg/ day, based on slight increase in body weight/ gain, food consumption, alterations in some hematological and clinical chemistry parameters, changes in thyroid hormone concentrations [ females] and histopathological lesions [ males] in thyroids.

At the highest dose tested [ HDT; 436 { 300} mg/ kg/ day], one/ sex were sacrificed moribund, females lost weight during 2nd and 3rd months and both exhibited lesions in the thyroid, liver, and kidney.

870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1.5, 27.1, 147.6, 442.8 mg/ kg/ day] { 1, 18.4, 100, 300 mg/ kg/ day}

MRID: 41994001 [ 1991]

Classification: Acceptable/ Guideline DEA salt of 2,4 D ( 73.8%; acid equivalent 50.0%???) Systemic Toxicity NOAEL = 1.5 { 1} mg/ kg/ day Systemic Toxicity LOAEL = 27.1 { 18.4} mg/ kg/ day, based on decreased body weight gain, food consumption, increased relative kidney weight [ females], histopathological lesions in kidneys of both sexes.

At the highest dose tested [ HDT; 442.8 { 300}], 3 of each sex died between weeks 6 9; effects observed on liver [ increased weight ( females), hypertrophy, individual cell necrosis], thyroid [ follicular cell hypertrophy, decreased T4], testes [ decreased weight, degeneration of seminiferous epithelium, decreased spermatozoa in epididymis, decreased secretory content ( prostate and seminal vesicles)] and ovaries [ decreased weight, atrophy] at the two highest dose levels. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Subchronic Oral Toxicity of 2,4 D and its Salts and Esters in Rats Guideline STUDY RESULTS 11 870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1.5, 22.6, 150, 452 mg/ kg/ day] { 1, 15, 100, 300 mg/ kg/ day}

MRID: 41896701 [ 1991]

Classification: Acceptable/ Guideline EH ester of 2,4 D ( 98% ; 63.25% acid equivalent) Systemic Toxicity NOAEL = 22.6 { 15} mg/ kg/ day Systemic Toxicity LOAEL = 150 { 100} mg/ kg/ day, based on alterations in clinical chemistry [ decreased total protein ( both sexes), calcium and albumin ( males), globulin and T4 ( females), increased ALT ( females)].

At the highest dose tested [ HDT 452 { 300} mg/ kg/ day], deaths in both sexes between weeks 7 11, clinical signs [ hunched posture, thin appearance, languid behavior, ataxia, anorexia, squinted eyes, opaque eyes ( females)], decreased body weight gain, negative BWG during weeks 6 13, bilateral cataracts ( females), retinal degeneration ( males), alterations in hematology, numerous organ weight changes, many of which can be attributed to decreased BW, histopathological lesions [ centrilobular hypertrophy, bilateral retinal degeneration and cataract formation, atrophy of testes, prostate, seminal vesicles, hypertrophy of zona glomerulose ( adrenal of both sexes), thyroid follicular cells hypertrophy, lymphoid hypoplasia of thymus ( both sexes) and spleen, hypoplasia of bone marrow, vacuolation of tubular cells and brush border loss in proximal tubular cells (, females) kidney.

870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1.2, 18.1, 120, 361 mg/ kg/ day] { 1, 15, 100, 300 mg/ kg/ day}

MRID: 41896702 [ 1991]

Classification: Acceptable/ Guideline DMA salt of 2,4 D ( 66.18% ; 55.5% acid equivalent) Systemic Toxicity NOAEL = 18.1 { 15} mg/ kg/ day Systemic Toxicity LOAEL = 120 { 100} mg/ kg/ day, based on decreased body weight gain, food consumption in females, alterations in hematology parameters [ females], decreased T4/ T3 [ females] increased kidney weight in males and decreased ovarian weights in females.

At the highest dose tested [ HDT { 300} mg/ kg/ day], changes in several organ weights, histopathological changes [ bilateral retinal degeneration and cataracts ( females), centrilobular hepatocellular hypertrophy ( females), atrophy of testes, hypertrophy of thyroid follicular cells ( both sexes), brush border loss in proximal tubular cells in kidney ( both sexes), and hypoplasia of spleen ( females) and bone marrow ( both sexes)]. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Subchronic Oral Toxicity of 2,4 D and its Salts and Esters in Rats Guideline STUDY RESULTS 12 870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1, 19, 127, 380 mg/ kg/ day] { 1, 15, 100, 300 mg/ kg/ day}

MRID: 42021401 [ 1991]

Classification: Acceptable/ Guideline IPA salt of 2,4 D ( 45.1%; acid equivalent 35.6%) Systemic Toxicity NOAEL = 19 { 15} mg/ kg/ day Systemic Toxicity LOAEL = 127 { 100} mg/ kg/ day, based on decreased body weight gain, food consumption, histopathological lesions in kidneys ( both sexes), adrenal ( females), and thyroid ( females).

At the highest dose tested [ HDT; 380 { 300} mg/ kg/ day], females displayed alterations in hematology, both sexes displayed alterations in clinical chemistry, increased liver and thyroid weights, decreased testes and ovarian weights, lesions in eyes, liver, thyroids, kidneys, adrenals, spleen, thymus, and lungs, testes.

870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 1.23, 18.4, 123, 369 mg/ kg/ day] { 1, 15, 100, 300 mg/ kg/ day}

MRID: 43515901 [ 1995]

Classification: Acceptable/ Guideline IP ester of 2,4 D ( 97.1% a. i.; 81.6% acid equivalent) Systemic Toxicity NOAEL = 15 mg/ kg/ day Systemic Toxicity LOAEL = 100 mg/ kg/ day, based on decreased body weight/ body weight gain [ females), increased incidence of retinal degeneration, decreased T4 ( both sexes), increased TSH ( females).

At the highest dose tested [ HDT; { 300} mg/ kg/ day], both sexes displayed alterations in hematology and clinical chemistry, increased liver and thyroid weights, decreased brain, testes, ovarian, thymus, adrenal, and spleen weights, and hepatocellular hypertrophy, atrophy and aspermatogenesis in testes, thymus atrophy, hypocellular bone marrow.

870.3100 [ § 82 1a] Subchronic oral toxicity  Fischer 344 rats [ 2, 28, 187, 560 mg/ kg/ day] { 1, 15, 100, 300 mg/ kg/ day}

MRID: 42021402 [ 1991]

Classification: Acceptable/ Guideline TIPA salt of 2,4 D ( 72.2% ; acid equivalent 38.7%) Systemic Toxicity NOAEL = 28 { 15} mg/ kg/ day Systemic Toxicity LOAEL = 187 { 100} mg/ kg/ day, based on alterations in some hematology and clinical chemistry parameters, histopathological lesions in adrenal ( females), kidney ( both sexes), and eye ( females).

At the highest dose tested [ HDT, 560 { 300} mg/ kg/ day], decreased body weight gain, in addition to effects listed above, increased liver weight, decreased testes and ovarian weights, histopathological lesions in thyroids, testes, lung, spleen, thymus, bone marrow. LOAEL = Lowest Observable Adverse Effect Level; NOAEL = No Observable Adverse Effect Level;

{ acid equivalent}

870.3150 90 Day Oral Toxicity  Dog EXECUTIVE SUMMARY: In a subchronic oral toxicity study [ MRID 42780001], 4 beagle dogs/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 96.7%] via the diet for 13 weeks at concentrations of 0, 0.5 mg/ kg/ day, 1.0 mg/ kg/ day, 3.75 mg/ kg/ day, and 7.5 mg/ kg/ day. The dose levels were selected based on the results of a 4 week range finding study [ MRID 42780004] in which decreased body weight, food consumption, alterations in clinical chemistry parameters, and distended gall bladders were observed at

10 mg/ kg/ day. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 13 There were no deaths, and no treatment related clinical signs were observed. Decreased body weights were observed in males at the highest dose level [ 85% of control at week 14] and in females at the lowest and highest dose levels [ 91% of control] at week 14. Body weight gains were decreased throughout the study in both sexes at the two highest dose levels but there was no dose response [ overall gain: males 52% and 55% of the control value/ females 53% and 58% of the control value at 3.75 and 7.5 mg/ kg/ day, respectively]. Females at the two lowest dose levels also displayed decreased body weight gains [ overall gain: 74% and 79% of control at 0.5 and 1.0 mg/ kg/ day, respectively] compared to the control. Decreased food consumption was observed in males at the two highest dose levels and in all female groups compared to their respective controls, but there was no consistent dose response.

Ophthalmology findings were comparable among the groups for both sexes. There were no apparent treatmentrelated alterations in hematology among the groups for either sex, with the exception of decreased platelet counts. Decreased platelet counts [ 73% and 87% of control] were observed in males at the high dose level at both [ week 4 and week 13, respectively] time points. Elevations in blood urea nitrogen [ two highest dose levels, both sexes], creatinine [ three highest dose levels, both sexes], and alanine aminotransferase [ two highest dose levels, both sexes] and decreased glucose levels [ two highest dose levels, females] were observed at week 4 and/ or week 13; however, a dose response was not always apparent. Although no corroborative histopathological changes were observed in the liver or kidneys, similar changes in several of these clinical chemistry parameters have been observed in other studies on both 2,4 D and the 2,4 D salts and esters in rats and in an earlier subchronic toxicity study in dogs with 2,4 D [ MRID 41737301]. In this latter study, microscopic lesions were observed in the kidneys of both sexes at 10 mg/ kg/ day.

Males at the two highest dose levels displayed a dose related decrease in testes weight [ absolute, relative to body, and relative to brain], although statistical significance was not attained [ n= 4]. Decreased adrenal weight [ absolute and relative to brain] was observed in both sexes at the highest dose level, decreased liver weight [ absolute and relative to brain] was observed in both sexes at the highest dose level, and decreased heart weight [ absolute and relative to brain] was observed in both sexes at the two highest dose levels, but there was no dose response. Microscopically, hypospermatogenesis/ juvenile testis was observed in 1 control, 1 mid high [ 3.75 mg/ kg/ day], and 2 high dose [ 7.5 mg/ kg/ day] males, and inactive, juvenile prostate was observed in one male in the control, 0.5 mg/ kg/ day, 1 mg/ kg/ day, and 3.75 mg/ kg/ day dose groups and in all 4 high dose males.

The NOAEL is 1.0 mg/ kg/ day. The LOAEL of 3.75 mg/ kg/ day is based on decreased body weight gain ( both sexes) and food consumption ( males), as well as alterations in clinical chemistry parameters [ increased BUN, creatinine, and alanine aminotransferase] in both sexes, and decreased testes weight.

This subchronic oral toxicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 3150; § 82 1( b)] for a subchronic non rodent oral toxicity study.

EXECUTIVE SUMMARY: In a subchronic oral toxicity study [ MRID 41737301], 5 beagle dogs/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 96.1%] via capsules for 13 weeks at concentrations of 0, 0.3 mg/ kg/ day, 1.0 mg/ kg/ day, 3.0 mg/ kg/ day, and 10 mg/ kg/ day.

Two males [ weeks 4, 13] and one female [ week 9] at the high dose level were sacrificed moribund. Treatmentrelated clinical signs [ thin, languid appearance, anorexia, tremors] were observed at the high dose level in the CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 14 three dogs sacrificed in extremis. Decreased body weights were observed in males at the two highest dose levels [ 83% and 80% of control at 3 and 10 mg/ kg/ day, respectively] and in females at the high dose level [ 86% of control] at week 13. Body weight gains were decreased throughout the study in males at the two highest dose levels [ weeks 0 13: 56% and 44%, respectively]. The mid and high dose females displayed decreased bodyweight gains also [ weeks 0 13: 84% and 52% of control, respectively]. Decreased food consumption was observed in both sexes at the highest dose level and in males at the next highest dose level throughout the study.

Decreased RBC, hemoglobin, hematocrit, and platelet count values were observed in both sexes at the highest dose level, although statistical significance was not always attained [ n= 4 5]. Treatment related alterations in clinical chemistry parameters were observed mainly at the two highest dose levels in both sexes at various times during the dosing period [ increased blood urea nitrogen ( both sexes), creatinine ( both sexes), alanine aminotransferase ( high dose females; males not statistically significant); decreased T4 ( both sexes at high dose; not statistically significant), and glucose ( high dose, both sexes]. High dose females displayed slightly increased kidney and thyroid weights [ absolute, relative to body and brain weights] compared to the control group. Decreased testis weight was observed at the two highest dose levels, and two of the 3 surviving high dose males and one of the 2 high dose males sacrificed moribund displayed hypospermatogenesis and giant cell formation. Microscopic lesions [ cellular alterations, proximal tubule] were observed in the kidney of both sexes at the highdose level [ 3 of 5 males at 3 mg/ kg/ day, 3/ 3 surviving males at 10 mg/ kg/ day, 1 of 4 surviving females at 10 mg/ kg/ day, and all dogs sacrificed moribund].

The NOAEL is 1.0 mg/ kg/ day. The LOAEL of 3.0 mg/ kg/ day is based on decreased body weight/ bodyweight gain and food consumption ( males), alterations in clinical chemistry parameters [ increased BUN ( both sexes), creatinine ( both sexes)], and decreased testis weight ( males).

This subchronic oral toxicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 3150; § 82 1( b)] for a subchronic non rodent oral toxicity study.

The following table summarizes the subchronic oral toxicity studies in dogs on 2,4 D and two esters [ EHE and IPE] and one amine salt [ DMA] for comparison.

Subchronic Oral Toxicity of 2,4 D and its Salts and Esters in Dogs Guideline STUDY RESULTS 870.3150 [ § 82 1b] Subchronic oral ( capsule) toxicity  dogs [ 0, 0.3, 1.0, 3.0, and 10 mg/ kg/ day]

MRID: 41737301 [ 1990]

Classification: Acceptable/ Guideline 2,4 D ( 96.1% a. i.) Systemic Toxicity NOAEL = 1 mg/ kg/ day Systemic Toxicity LOAEL = 3 mg/ kg/ day, based on decreased body weight/ body weight gain and food consumption ( males), alterations in clinical chemistry parameters [ increased BUN ( both sexes), creatinine ( males)], and decreased testis weight in males.

At the highest dose tested [ HDT, 10 mg/ kg/ day], mortality [ 2 males, 1 female sacrificed moribund], hematology alterations, and microscopic lesions of the kidneys and testes [ hypospermatogenesis] were observed. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Subchronic Oral Toxicity of 2,4 D and its Salts and Esters in Dogs Guideline STUDY RESULTS 15 870.3150 [ § 82 1b] Subchronic oral ( diet) toxicity  dogs [ 0, 0.5, 1.0, 3.75, and 7.5 mg/ kg/ day]

MRID: 42780001 [ 1993]

Classification: Acceptable/ Guideline 2,4 D ( 96.1% a. i.) Systemic Toxicity NOAEL = 1 mg/ kg/ day Systemic Toxicity LOAEL = 3.75 mg/ kg/ day, based on decreased body weight gain ( both sexes) and food consumption ( males), as well as alterations in clinical chemistry parameters [ increased BUN, creatinine, and alanine aminotransferase] in both sexes, and decreased testes weight and sl ightly higher incidence of hypospermatogenesis/ juvenile testis and inactive/ juvenile prostate were observed.

870.3150 [ § 82 1b] Subchronic oral [ diet] toxicity  dogs { 0, 1, 3.75, and 7.5 mg/ kg/ day}

MRID: 42780003 [ 1993]

Classification: Acceptable/ Guideline EH ester of 2,4 D ( 98% ; 63.25% acid equivalent) Systemic Toxicity NOAEL = < 1 mg/ kg/ day Systemic Toxicity LOAEL = 1 mg/ kg/ day, based on increased BUN and creatinine values at all dose levels at study termination At the highest dose tested [ HDT 7.5 mg/ kg/ day], there was a slightly higher incidence of hypospermatogenesis/ juvenile testis and inactive/ juvenile prostate compared to the control dogs 870.3150 [ § 82 1b] Subchronic oral [ capsule] toxicity  dogs { 0, 0.3, 1.0, 3.0, and 10.0 mg/ kg bw/ day}

MRID: 43515501 [ 1994]

Classification: Acceptable/ Guideline IP ester of 2,4 D ( 97.1% a. i.) Systemic Toxicity NOAEL = 1 mg/ kg/ day Systemic Toxicity LOAEL = 3 mg/ kg/ day, based on increased blood urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase values.

At the highest dose tested [ HDT 10 mg/ kg/ day], males displayed decreased BW/ BWG, decreased absolute and relative testes and ovarian weights and decreased absolute adrenal ( male) weights .

870.3150 [ § 82 1b] Subchronic oral [ diet] toxicity  dogs { 0, 1, 3.75, and 7.5 mg/ kg/ day}

MRID: 42780002 [ 1993]

Classification: Acceptable/ Guideline DMA salt of 2,4 D ( 66.18% ; 55.5% acid equivalent) Systemic Toxicity NOAEL = 1 mg/ kg/ day Systemic Toxicity LOAEL = 3.75 mg/ kg/ day, based on decreased body weight gain and food consumption, as well as alterations in clinical chemistry parameters [ increased BUN, creatinine] in both sexes.

At the highest dose tested [ HDT; 7.5 mg/ kg/ day], decreased platelet counts and slightly higher incidence of hypospermatogenesis/ juvenile testis and inactive/ juvenile prostate were observed in the males. LOAEL = Lowest Observable Adverse Effect Level; NOAEL = No Observable Adverse Effect Level; { acid equivalent}

870.3200 21/ 28 Day Dermal Toxicity B Rat EXECUTIVE SUMMARY: In a 21 day dermal toxicity study [ MRID 41735304], 5 Hra: ( NZW) SPF rabbits/ sex/ group were administered the 2,4 dichlorophenoxyacetic acid [ 96.1%] via dermal application [ neat material ( powder) spread evenly on 4" x 4" gauze pad and moistened with 0.5 mL distilled water [ 6 hours/ day, 7 days/ week for 21 days at concentrations of 0 [ distilled water], 10, 100, and 1000 mg/ kg/ day. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 16 Treatment had no adverse effect on survival, clinical signs, mean body weight, hematology, clinical chemistry, urinalysis, gross pathology, or histopathology. At the high dose level, increased kidney weights [ absolute and relative] were observed in both sexes, but statistical significance was attained in the females only, and there were no corroborating changes in clinical chemistry parameters or histopathology in the kidneys. 2,4 D was only slightly irritating to the skin of the rabbits, inducing very slight erythema and epidermal scaling, with the females displaying a higher incidence than males.

The NOAEL for dermal and systemic toxicity is 1000 mg/ kg/ day, the highest dose tested.

This 21 day dermal toxicity study is classified ACCEPTABLE/ Guideline and satisfies the guideline requirement [ § 82 2; 870.3200] for a 21 day dermal toxicity study.

The following table summarizes the subchronic dermal studies on 2, 4 D and its amine salts and esters for comparison.

Subchronic Dermal Toxicity of 2,4 D and its Salts and Esters Guideline STUDY RESULTS 870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 10, 100, 1000 mg/ kg/ day]

MRID: 41735301, 41735304 [ 1990]

Classification: Acceptable/ Guideline 2,4 D ( 96.1% a. i.) Dermal Toxicity NOAEL 1000 mg/ kg/ day, HDT Dermal Toxicity LOAEL = > 1000 mg/ kg/ day, no adverse effects observed at the limit dose Systemic Toxicity NOAEL = 1000 mg/ kg/ day Systemic Toxicity LOAEL = > 1000 mg/ kg/ day, no adverse effects observed at the limit dose 870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 50, 150, 500 mg/ kg/ day] { 32, 96, 321 mg/ kg/ day}

MRID: 41407901 [ 1990]

Classification: Acceptable/ Guideline BEE ester of 2,4 D ( 94.6% ;

65% acid equivalent) Dermal Toxicity NOAEL = 500 mg/ kg/ day [ HDT Dermal Toxicity LOAEL = not determined.

Systemic Toxicity NOAEL = 500 mg/ kg/ day Systemic Toxicity LOAEL = no effects observed at HDT [ 500 mg/ kg/ day]

870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 15, 148, 443 mg/ kg/ day] { 9, 92, 276 mg/ kg/ day}?

MRID: 41920905 [ 1991]

Classification: Acceptable/ Guideline DEA salt of 2,4 D ( 73.09%;

45.6% acid equivalent) Dermal Toxicity NOAEL 15 mg/ kg/ day Dermal Toxicity LOAEL = 148 mg/ kg/ day, based on microscopic dermal lesions.

Systemic Toxicity NOAEL = 148 mg/ kg/ day Systemic Toxicity LOAEL = 443 mg/ kg/ day, based on liver toxicity [ increased AST, ALT, AP, liver weight, liver lesions]. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Subchronic Dermal Toxicity of 2,4 D and its Salts and Esters Guideline STUDY RESULTS 17 870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 18, 180.1, 540.5 mg/ kg/ day] { 10, 100, 300 mg/ kg/ day}

MRID: 41735303, 41735306 [ 1990]

Classification: Acceptable/ Guideline DMA salt of 2,4 D ( 66.18% ; 55.5% acid equivalent) Dermal Toxicity NOAEL 18 mg/ kg/ day Dermal Toxicity LOAEL = 180.1 mg/ kg/ day, based on microscopic dermal lesions Systemic Toxicity NOAEL = 180.1 mg/ kg/ day Systemic Toxicity LOAEL = 540.5 mg/ kg/ day, based on one female death.

870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 16.3, 162.8, 1627.5 mg/ kg/ day] { 10, 100, 1000 mg/ kg/ day}

MRID: 41735302, 41735305 [ 1990]

Classification: Acceptable/ Guideline EH ester of 2,4 D ( 98% ; 62.8% acid equivalent) Dermal Toxicity NOAEL 16.3 mg/ kg/ day Dermal Toxicity LOAEL = 162.5 mg/ kg/ day, based on histopathological lesions of the skin.

Systemic Toxicity NOAEL = 1627.5 mg/ kg/ day [ HDT] Systemic Toxicity LOAEL = not determined, based on no effects at greater than the limit dose.

870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 50, 125, 350 mg/ kg/ day] { 39, 98, 275 mg/ kg/ day}

MRID: 41407903 [ 1990]

Classification: Acceptable/ Guideline IPA salt of 2,4 D ( 50.2%;

39% acid equivalent) Dermal Toxicity NOAEL 50 mg/ kg/ day Dermal Toxicity LOAEL = 125 mg/ kg/ day, based on microscopic dermal lesions.

Systemic Toxicity NOAEL = 350 mg/ kg/ day [ HDT] Systemic Toxicity LOAEL = not determined, based on no effects at HDT.

870.3200 [ § 82 2] 21 Day dermal toxicity  rabbits [ 100, 350, 1000 mg/ kg/ day { 55, 193, 553 mg/ kg/ day}

MRID: 41407902 [ 1989]

Classification: Acceptable/ Guideline TIPA salt of 2,4 D ( 72.2% ;

37% 38% acid equivalent) Dermal Toxicity NOAEL = 1000 mg/ kg/ day [ HDT] Dermal Toxicity LOAEL = not determined, based on no effects at the limit dose.

Systemic Toxicity NOAEL = 1000 mg/ kg/ day [ HDT] Systemic Toxicity LOAEL = not determined, based on no effects at the limit dose. LOAEL = Lowest Observable Adverse Effect Level; NOAEL = No Observable Adverse Effect Level 870.3465 90 Day Inhalation B Rat Adequacy of data base for Inhalation Toxicity: The data base for subchronic inhalation toxicity is considered incomplete. A 28 day inhalation study in the rat is required.

4.3 Prenatal Developmental Toxicity Adequacy of data base for Prenatal Developmental Toxicity: The data base for prenatal developmental toxicity is considered complete. No additional studies are required at this time. Developmental toxicity was observed in the rat, as evidenced by the increased incidence of skeletal malformations and variations observed at the same dose level that resulted in maternal toxicity, which manifested as decreased body weight gain. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 18 Similar developmental toxicity as seen in the rat was not observed in the rabbit. The only developmental toxicity observed in rabbits was abortion, which occurred in 2 does at the highest dose tested.

870.3700a Prenatal Developmental Toxicity Study  Rat EXECUTIVE SUMMARY: In a developmental toxicity study [ MRID 00130407; 00130408], pregnant Fischer 344 rats [ 35/ group] were administered 2,4 dichlorophenoxyacetic acid [ 97.5%] via gavage at dose levels of 0 [ corn oil], 8 mg/ kg/ day, 25 mg/ kg/ day, and 75 mg/ kg/ day from gestation day [ GD] 6 through gestation day 15.

There were no treatment related deaths. Two [ one control and one low dose] dams delivered prematurely on gestation day 19, and in both instances the offspring produced were of similar size and development as those from full term delivery. Clinical signs were comparable among the groups. Body weights were comparable among the groups throughout the study, but dams at the high dose level displayed a decrease in body weight gain during the dosing period [ 79% of control for GD 6 15; 57% of control for GD 6 10], although statistical significance was not attained. The corrected body weight gain was comparable among the groups. Food consumption data were not reported.

There was a slight decrease in pregnancy rate with increasing dose [ 85%, 85%, 80% and 77%]. The numbers of corpora lutea, implantations, and live fetuses were comparable among the groups, and there were no dead fetuses. The number of resorptions, as well as pre and post implantation losses, were not adversely affected by treatment. The number of the dams with 100% resorptions was 2, 0, 1, and 1 [ control, low, mid, and highdose groups, respectively]. One control, 2 low, 4 mid, and 2 high dose dams had late resorptions. Fetal body weight and crown rump length were comparable among the groups, as was the sex ratio.

There were no statistically significant or treatment related differences in the incidence of fetal external or visceral malformations. There was an increased incidence of skeletal malformations/ variations at the high dose level that included the presence of 7th cervical ribs [ 4 fetuses in 3 litters vs none in the control], the presence of 14th rudimentary ribs [ 4 fetuses in 3 litters vs none in the control], malaligned sternebrae [ 15 fetuses in 10 litters vs 7 fetuses in 7 litters of the control], reduced ossification of the vertebral arches [ 6 fetuses in 5 litters vs 2 fetuses in 1 litter of the control], and unossified sternebrae # 5 or # 6 [ 73 fetuses in 22 litters ( 3.32 fetuses/ litter) vs 62 fetuses in 24 litters ( 2.58 fetuses/ litter)]. Although none of the increases attained statistical significance, they were attributed to treatment since some of the findings [ maligned sternebrae, 14th rudimentary ribs, and reduced ossification of vertebral arches] were also observed in the F1b pups of dams fed 2,4 D at 80 mg/ kg/ day [ actual dose  75 mg/ kg/ day] in the 2 generation reproduction study in the same strain of rat. Additionally, skeletal variations of the ribs [ 2nd wavy ribs, lumbar ribs] and missing sternebrae were observed in another developmental toxicity study using a different strain of rat [ Sprague Dawley] at a comparable dose of 87.5 mg/ kg/ day [ 2,4 D].

The maternal toxicity NOAEL is 25 mg/ kg/ day, and the maternal toxicity LOAEL is 75 mg/ kg/ day, based on decreased body weight gain.

The NOAEL for developmental toxicity is 25 mg/ kg/ day, and the developmental toxicity LOAEL is 75 mg/ kg/ day, based on the increased incidence of skeletal abnormalities.

This developmental toxicity study is classified Acceptable/ Guideline, and it satisfies the guideline requirement CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 19 [ OPPTS 870.3700; § 83 3( a)] for a developmental toxicity study in the rodent.

The following table provides summaries of the rat developmental toxicity studies on 2,4 D and its amine salts and esters for comparison.

Developmental Toxicity of 2,4 D and its Esters and Salts in Rats Guideline STUDY RESULTS 870.3700 [ § 83 3a] Developmental Toxicity  Fischer 344 rats [ 0, 8, 25, and 75 mg/ kg/ day]

MRID 00130407, 00130408 [ 1983]

Classification: Acceptable/ Guideline 2,4 D ( 97.5% a. i .) Maternal NOAEL: 25 mg/ kg/ day Maternal LOAEL: 75 mg/ kg/ day, based on decreased bodyweight gains. Survival was not affected by treatment.

Developmental NOAEL: 25 mg/ kg/ day Developmental LOAEL: 75 mg/ kg/ day, based on skeletal abnormalities.

870.3700 [ § 83 3a] Developmental toxicity  CD ® [ Sprague Dawley derived] rats [ 0, 25, 75, 185 mg/ kg/ day] { 10.2, 50.8, 101.6 mg/ kg/ day} MRID 41527101; 41527104 [ 1990]

Classification: Acceptable/ Guideline BEE ester of 2,4 D ( 95.6%; acid equivalent 65.1 65.8%) Maternal NOAEL: 75 mg/ kg/ day Maternal LOAEL: 185 mg/ kg/ day, based on decreased bodyweight gain on days 6 16, decreased food consumption, RBC, increased reticulocytes. There were no deaths.

Developmental NOAEL: 75 mg/ kg/ day Developmental LOAEL: 185 mg/ kg/ day, based on an increased incidence of skeletal variations [ incompletely ossified supraoccipital, squamosal( s), maxilla, and 4th sternebrae, and 14 rib pairs, short ribs, rudimentary first lumbar rib( s), and unilateral 14th rib( s)]

870.3700 [ § 83 3a] Developmental toxicity  Sprague Dawley Crl: CD BR VAF/ Plus rats [ 0, 15, 75, and 150 mg/ kg/ day] { 10.2, 50.8, and 101.6 mg/ kg/ day} MRID 41920906; 41986602 [ 1990]

Classification: Acceptable/ Guideline DEA salt of 2,4 D ( 73.09%; acid equivalent

49.5%) Maternal NOAEL: 15 mg/ kg/ day Maternal LOAEL: 75 mg/ kg/ day, based on decreased bodyweight gain. There were no deaths.

Developmental NOAEL: 15 mg/ kg/ day Developmental LOAEL: 75 mg/ kg/ day, based on an increased incidence of skeletal variations/ malformations [ reduced ossification of skull, 14th rudimentary rib( s), 7th cervical rib( s), bent rib( s)].

870.3700 [ § 83 3a] Developmental Toxicity  Crl: CD ® BR VAF/ Plus ® rats [ 15, 60.2, and 120.4 mg/ kg/ day] { 12.5, 50, and 100 mg/ kg/ day}

MRID 41735201 [ 1990]

Classification: Acceptable/ Guideline DMA salt of 2,4 D ( 66.18%; acid equivalent 55.5%) Maternal NOAEL: 12.5 mg/ kg/ day Maternal LOAEL: 50 mg/ kg/ day, based on decreased bodyweight gain and food consumption. There were no deaths.

Developmental NOAEL: 50 mg/ kg/ day Developmental LOAEL: 100 mg/ kg/ day, based on decreased fetal body weight, increased incidence of wavy ribs and/ or incompletely ossified ribs. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Developmental Toxicity of 2,4 D and its Esters and Salts in Rats Guideline STUDY RESULTS 20 870.3700 [ § 83 3a] Developmental Toxicity  Sprague Dawley Crl: CD BR VAF/ Plus rats [ 15, 45, 135 mg/ kg/ day] { 10, 30, and 90 mg/ kg/ day}

MRID 42304601; 42304602 [ 1992]

Classification: Acceptable/ Guideline EH ester of 2,4 D ( 95%; acid equivalent 63.25%) Maternal NOAEL: 10 mg/ kg/ day Maternal LOAEL: 30 mg/ kg/ day, based on decreased bodyweight gain [ GD 6 9] and an increased incidence of clinical signs [ perivaginal staining]. There were no deaths.

Developmental NOAEL: 10 mg/ kg/ day Developmental LOAEL: 30 mg/ kg/ day, based on an increased incidence of delayed sternebrae ossification.

870.3700 [ § 83 3a] Developmental Toxicity  Sprague Dawley Crl: CD ® BR rats [ 0, 12.3, 36.7, and 123 mg/ kg/ day] { 10, 30, and 100 mg/ kg/ day}

MRID 43523101; 43523001 [ 1995]

Classification: Acceptable/ Guideline IP ester of 2,4 D ( 97.1%; 78.9% acid equivalent) Maternal NOAEL: 10 mg/ kg/ day Maternal LOAEL: 30 mg/ kg/ day, based on decreased bodyweight gain and food consumption during the dosing period.

At the highest dose tested [ 100 mg/ kg/ day], one death, clinical signs [ rocking, lurching, or swaying, decreased defecation, clear matting on the ventral abdominal and thoracic areas, red material on the urogenital area, and ptosis], and one litter with 100% resorptions were observed. decreased body weight gain [ GD 6 9] and an increased incidence of clinical signs [ perivaginal staining]. There were no deaths.

Developmental NOAEL: 30 mg/ kg/ day Developmental LOAEL: 100 mg/ kg/ day, based on decreased fetal body weight, an increased incidence of skeletal malformations [ 7th cervical ribs and 14th rudimentary ribs], and a decreased percent of female fetuses.

870.3700 [ § 83 3a] Developmental Toxicity  CD ® [ Sprague Dawley derived] rats [ 0, 22, 65, and 190 mg/ kg/ day] {

17, 51, 150 mg/ kg/ day}

MRID 41527103; 41527106 [ 1990]

Classification: Acceptable/ Guideline IPA salt of 2,4 D ( 50.2%; acid equivalent 39.5%) Maternal NOAEL: 65 { 51} mg/ kg/ day Maternal LOAEL: 190 { 150} mg/ kg/ day, based on decreased body weight gain [ GD 6 9], food consumption [ GD 6 11]. ], and there was one death [ GD 7].

Developmental NOAEL: 65 { 51} mg/ kg/ day Developmental LOAEL: 190 { 150} mg/ kg/ day, based on a slight increase in the incidence of skeletal malformations/ variations and external malformations.

870.3700 [ § 83 3a] Developmental Toxicity  CD ® [ Sprague Dawley  derived] rats [ 0, 32.5, 100, and 325 mg/ kg/ day] { 17.4, 53.6, 174.2 mg/ kg/ day}

MRID 41527102; 41527105 [ 1978]

Classification: Acceptable/ Guideline TIPA salt of 2,4 D ( 72.2%; acid equivalent 38.7?%) Maternal NOAEL: not determined Maternal LOAEL: 32.5 mg/ kg/ day, based on decreased bodyweight gain on days 6 9. At HDT [ 325 mg/ kg/ day], there were 2 deaths [ GD 9, 11] and 2 moribund sacrifices [ GD 9, 12].

Developmental NOAEL: 32.5 mg/ kg/ day Developmental LOAEL: 100 mg/ kg/ day, based on a significant increase in the incidence of skeletal malformations and fetal incidence of total skeletal variations. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 21 [ active ingredient; a. i.]; { acid equivalent; a. e.}

870.3700b Prenatal Developmental Toxicity Study  Rabbit EXECUTIVE SUMMARY: In a developmental toxicity study [ MRID 41747601], artificially inseminated female New Zealand White rabbits [ 20/ group] were administered 2,4 Dichlorophenoxyacetic acid [ 96.1%] at dose levels of 0 [ aqueous 0.5% methylcellulose], 10 mg/ kg/ day, 30 mg/ kg/ day, and 90 mg/ kg/ day from gestation day 6 through gestation day 18. NOTE: All dose concentrations were corrected for the 96.1% purity of the test material.

There were no treatment related deaths. Two high dose does aborted [ days 21 and 24]. Treatment related clinical signs of toxicity were observed in the two does that aborted [ days 21 and 24, after 13 doses each] and included ataxia in both [ days 16 19 and after day 13], and decreased motor activity, loss of righting reflex, extremities that were cold to the touch, and dried feces in doe that aborted on day 21. Body weights were comparable among the groups throughout the study, but body weight gains were decreased at the high dose level [ 73% of control] during the dosing period [ days 6 19; statistical significance was not attained]. During days 7 8, the low and mid dose groups showed no body weight gain, and the high dose group displayed a negative body weight gain [ 0.01 grams] compared to the control [+ 0.01 gram]. During days 15 19, the high dose group displayed no body weight gain, and corrected body weight gain was decreased at the high dose level [ 77% of control; statistical significance was not attained] also. Food consumption was comparable among the groups.

Pregnancy rates were comparable among the groups. Comparable numbers of corpora lutea, implantations, and live fetuses were observed among the groups, and there were no dead fetuses. One control doe had 100% resorptions. The number of resorption and pre and post implantation losses were comparable among the groups also. Gravid uterine weights were comparable among the groups.

Mean fetal body weight was comparable among the groups. At the high dose level, there was a significant increase in the percent of live male fetuses [ 71.2%] compared to the control [ 52.8%] and other dose groups [ low: 54.4%; mid: 59.4%]. At the high dose level, the fetal incidence [ 3 fetuses of one litter; p< 0.01] of hindlimbs turned inward was increased compared to the control ( 0) and other treatment groups ( 0), and the same fetuses displayed domed head [ hydrocephaly]. This finding is not considered treatment related. There were no apparent differences in the incidence of external, visceral, or skeletal variations, anomalies, retardations, or malformations among the groups.

The maternal toxicity NOAEL is 30 mg/ kg/ day, based on abortions, decreased body weight gain, and clinical signs of toxicity [ decreased motor activity, ataxia, loss of righting reflex, extremities cold to the touch] at the maternal toxicity LOAEL of 90 mg/ kg/ day.

The developmental toxicity NOAEL is 30 mg/ kg/ day, based on abortions at the developmental toxicity LOAEL of 90 mg/ kg/ day.

This developmental toxicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870.3700/ § 83 3 ( b)] for a developmental toxicity study in rabbits. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 22 The following table provides summaries of the rabbit developmental toxicity studies on 2,4 D and its amine salts and esters for comparison.

Developmental Toxicity of 2,4 D and its Esters and Salts in Rabbits Guideline STUDY RESULTS 870.3700 [ § 83 3b] Developmental Toxicity  rabbits [ 0, 10, 30, and 90 mg/ kg/ day]

MRID 41747601 [ 1990]

Classification: Acceptable/ Guideline 2,4 D ( 96.1% a. i .) Maternal NOAEL: 30 mg/ kg/ day Maternal LOAEL: 90 mg/ kg/ day, based on clinical signs [ ataxia, decreased motor activity, loss of righting reflex, cold extremities], abortion ( 2), decreased body weight gains. Survival was not affected by treatment.

Developmental NOAEL: 30 mg/ kg/ day Developmental LOAEL: 90 mg/ kg/ day, based on abortions.

870.3700 [ § 83 3b] Developmental Study  rabbits [ 0, 15, 45, 110 mg/ kg/ day] { 10, 30, 75 mg/ kg/ day}

MRID 42158703; 42158706 [ 1990]

Classification: Acceptable/ Guideline BEE ester of 2,4 D ( 95.6%; acid equivalent 65.8 Maternal NOAEL: 15 mg/ kg/ day Maternal LOAEL: 45 mg/ kg/ day, based on mortality/ morbidity, clinical signs [ decreased activity, prostration, myotonia, transient lateral recumbency, cold to touch, perineal soiling/ blood, red urine], and decreased body weight gain. Deaths: mid dose [ sacrificed day 21, another died day 24]; high dose [ 4 sacrificed ( days 14, 15, 18, 21), 4 died ( days 11,12,13,19].

Developmental NOAEL: 110 mg/ kg/ day [ HDT].

870.3700 [ § 83 3b] Developmental Toxicity  rabbits [ 0, 15, 30, and 60 mg/ kg/ day] { 10.2, 20.3, 40.6 mg/ kg/ day}

MRID 42055501, 42013501 [ 1991]

Classification: Acceptable/ Guideline DEA salt of 2,4 D ( 73.09%; acid equivalent 49.5% Maternal NOAEL: 15 mg/ kg/ day Maternal LOAEL: 30 mg/ kg/ day, based on decreased bodyweight gain and food consumption. At HDT, one doe died, slight increase in resorptions/ post implantation loss].

Developmental NOAEL: 30 mg/ kg/ day Developmental LOAEL: 60 mg/ kg/ day, based on the number of litters with fetuses with 7th cervical rib( s).

870.3700 [ § 83 3b] Developmental Toxicity  rabbits [ 12, 36.1, 108.4 mg/ kg/ day] { 10, 30, and 90 mg/ kg/ day}

MRID 42224001 [ 1991]

Classification: Acceptable/ Guideline DMA salt of 2,4 D ( 66.18%; acid equivalent 55.5%) Maternal NOAEL: 30 mg/ kg/ day Maternal LOAEL: 90 mg/ kg/ day, based on mortality/ morbidity, clinical signs of toxicity [ decreased motor activity, ataxia, impaired/ loss of righting reflex, myotonia], and decreased body weight gain and food consumption. At HDT, 2 found dead [ day 10], 2 found dead day 18, one sacrificed day 17. Clinical signs occurred by day 7.

Developmental NOAEL: 90 mg/ kg/ day [ HDT]. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter Developmental Toxicity of 2,4 D and its Esters and Salts in Rabbits Guideline STUDY RESULTS 23 870.3700 [ § 83 3b] Developmental Toxicity  rabbits [ 15.1, 45.2, and 113 mg/ kg/ day] { 10, 30, and 75 mg/ kg/ day}

MRID 42304603, 42304604 [ 1992]

Classification: Acceptable/ Guideline EH ester of 2,4 D ( 95%; acid equivalent 63.25%) Maternal NOAEL: 30 mg/ kg/ day Maternal LOAEL: 75 mg/ kg/ day, based on mortality/ morbidity, clinical signs of toxicity [ decreased motor activity, ataxia, impaired/ loss of righting reflex, bradypnea], and decreased body weight gain in does that died. Two does were sacrificed moribund [ days 15, 16]. Since no deaths were observed at 50 mg/ kg/ day in the rangefinding study, the one mid dose death at day 21 was not considered treatment related Developmental NOAEL: 75 mg/ kg/ day [ HDT].

870.3700 [ § 83 3b] Developmental Toxicity  rabbits [ 13, 38, and 95 mg/ kg/ day] { 10, 30, 75 mg/ kg/ day}

MRID 42158702; 42158704 [ 1991]

Classification: Acceptable/ Guideline IPA salt of 2,4 D ( 50.1%; acid equivalent 39.6%) Maternal NOAEL: could not be determined. Maternal LOAEL: 13 { 10} mg/ kg/ day, based on decreased body weight gain throughout the dosing period. At mid and high dose levels [ 38 { 30} and 95 { 75} mg/ kg/ day], there were 2 mid dose [ days 16, 24] and 3 high dose [ days 12, 20, 21] death and 4 high dose does were sacrificed moribund [ days 12, 13, 14, 20] and clinical signs [ lateral recumbency, cold extremities, myotonia, perineal staining, blood in urine].

Developmental NOAEL: 95 { 75} mg/ kg/ day [ HDT].

870.3700 [ § 83 3b] Developmental Toxicity  rabbits [ 19, 56, and 140 mg/ kg/ day] { 10, 30, 75 mg/ kg/ day}

MRID 42158701; 42158705 [ 1991]

Classification: Acceptable/ Guideline TIPA salt of 2,4 D ( 73.1%; acid equivalent 39.2%) Maternal NOAEL: 19 { 10} mg/ kg/ day Maternal LOAEL: 56 { 30} mg/ kg/ day, based on mortality/ morbidity, clinical signs of toxicity [ lateral recumbency, myotonia, perineal staining, blood in urine], and decreased body weight gain. Deaths: one mid dose doe [ day 18]; 3 highdose does were sacrificed moribund [ days 12, 14, 15].

Developmental NOAEL: 140 { 75} mg/ kg/ day [ HDT]

[ active ingredient]; { acid equivalent}

4.4 Reproductive Toxicity Adequacy of data base for Reproductive Toxicity: The existing data base meets the guideline for reproductive toxicity. However, a 2 generation reproduction study using the current protocol is required to address both the concern for thyroid effects [ comparative assessment between the young and adult animals] and immunotoxicity, as well as a more thorough assessment of the gonads and reproductive/ developmental endpoints. At dose levels above the saturation of renal clearance, reproductive toxicity was observed in the rat, as evidenced by the increased duration of gestation of the F0 dams producing the F1b litters.

870.3800 Reproduction and Fertility Effects  Rat EXECUTIVE SUMMARY: In a 2 generation reproduction study [ MRID 00150557; 00163996], 30 male/ 30 CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 24 female F0 Fischer 344 rats/ sex/ group were administered 2,4 D [ 97.5% a. i.] via the diet for 105 days prior to mating and through gestation and lactation of two litters and for 30 days after weaning the last litter at target dose levels of 0, 5, 20, and 80 mg/ kg/ day. Rats were mated, one male with one female. The resulting F1a litters were weaned at day 28 post partum. After a 2 week rest period, the F0 parental rats were re bred using different male/ female combinations to produce the F1b litters, from which 30 males/ 30 females/ group were selected to become the F1 parents. The F1 generation [ 30 rats/ sex/ group] was administered the test material at target dose levels of 0, 5, and 20 mg/ kg/ day [ high dose level dropped due to excess toxicity; there were an insufficient number of F1b pups] in utero and continuously via the milk or feed for 125 days postnatally and prior to mating and through gestation and lactation of two litters [ F2a and F2b] and for 30 days after weaning the last litter.

There were no apparent treatment related deaths, and clinical signs were comparable among the groups throughout the study. During the pre mating dosing period, body weights of the F0 parental animals were slightly lower [ males 95% 97% ( by week 6)/ females 95% 96% ( by week 13) of control] at the high dose level for both sexes. Body weight gains of the F0 high dose males were decreased initially [ weeks 2 3 ( 86% of control] and overall [ weeks 0 13 and weeks 0 40 ( 93% of control], as were those of the high dose females [ weeks 0 1 ( 79% of control); weeks 0 13 ( 92% of control) and weeks 0 40 ( 94% of control)] compared to the controls.

The high dose F0 dams displayed a significantly lower body weight throughout [ F1A litter] gestation ( 94% 95% of control) and by gestation day 20 during F1b pregnancy [ 90% of control]. The high dose F0 dams displayed significantly reduced body weight gains compared to the controls during both gestation periods, with the greater deficit being observed during the second gestation period [ F1a litters: days 0 7 ( 67%* of control); days 13 20 ( 95% of control; days 0 20 ( 87% of control); F1b litters: days 0 7 ( 70%* of control); days 13 20 ( 59%** of control); days 0 20 ( 67%** of control)]. The high dose F0 dams displayed decreased body weight on day 7 of lactation [ both litters; 92% 93% of control], but body weights were significantly increased compared to the controls at day 28 of lactation [ F1a ( 108%/ F1b 111% of control]. Body weight gains were significantly reduced during lactation days 1 7 for both litters [ F1a ( 40% of control); F1b ( 6% of control)]. Overall, however, the high dose dams displayed positive body weight gain during lactation days 1 28 compared to negative bodyweight gains in the control and other dose groups.

Food consumption [ g/ rat/ day] during the pre mating period was slightly lower [ 94% 95% of control] in the high dose females during a few weeks, but on a g/ kg/ day basis, both sexes at the high dose level displayed a slight increase [ 104% of control] in food consumption compared to the controls. During the first week of the two week rest period following the weaning of the first litter, the F0 dams displayed a significant decrease in food consumption [ 83% 84% of control]. Food consumption was decreased at the high dose level during both gestation periods [ F1a during first 2 weeks ( 91% 93% of control); F1b during third week ( 82% of control)]. A significant decrease in food consumption was observed throughout lactation [ both litters] at the high dose level [ F1a litter ( 58% of control for days 1 28); F1b litter ( 71% 83% of control)]. At necropsy, no treatmentrelated adverse effects were observed at any dose level, although the F0 females displayed increased kidney weights at all dose levels but there was no dose response.

There were no apparent, treatment related, adverse effects on body weights or body weight gains of the F1 parental animals during the pre mating dosing period at the two remaining dose levels, although the mid dose [ 20 mg/ kg/ day; the highest dose in the F1 generation] males displayed an initial decrease in body weight gain CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 25 [ weeks 35 36 ( 91%** of control) and weeks 36 37 ( 89%** of control)]. At 20 mg/ kg/ day, there were no significant differences in body weights in the F1 dams during gestation [ F2a litters 95% 99%; F2b litters 95% 96% of control] or body weight gains F2a litters 85% ( days 7 13); F2b litters 83% ( days 0 7); 86% ( days 13 20); 90% ( days 0 20) of control], and comparable body weights/ gains were observed during lactation [ both litters]. Food consumption was comparable among the groups [ both sexes] throughout the study. At necropsy, no treatment related adverse effects were observed at either dose level, although the F1 males and females displayed slightly increased kidney weights at the 20 mg/ kg/ day dose level, and the females at this dose level displayed a slight increase in liver weight.

F0 Generation. No apparent adverse effect was observed on fertility. Pre coital intervals were comparable among the groups. The duration of gestation was significantly increased in the high dose [ 80 mg/ kg/ day] F0 females producing the F1b pups [ 22.5 days vs 21.9 days]. The gestation survival index was comparable among the groups for the F1a pups but significantly decreased for the F1b litters [ 31.7% vs 97.8%]. There was a significant decrease in the number of F1a female fetuses at the high dose level [ 39% vs 54%]. The number of F1b pups born dead/ dying by day 1 [ 110] was significantly increased at the high dose level compared to the control [ 5]. F1a litter size was slightly lower at the high dose level compared to the control [ 9.0 vs 10.1], but F1b litter size was significantly lower than the control [ 5.1** vs 9.5]. F1a pup viability was comparable throughout weaning, but F1b pup viability was significantly lower throughout the weaning period. There was a significant decrease in F1b pup survival to lactation day 4 at the high dose level [ 86.3%] compared to the control [ 100%] and other dose levels [ 98% and 99.6%], as well as survival to lactation day 28 [ 71.4% vs 100% ( control) and other dose groups 99.4% and 100%]. Decreased pup body weight [ F1a males 89%/ females 90% of control ( day 1), 75%/ 81% of control ( day 28); F1b males 78%/ females 85% of control ( day 1), 73%/ 76% of control ( day 28)] and body weight gains [ F1a males 68%/ females 70% of control ( days 1 4), 75%/ 80% of control ( days 4 28); F1b males 26%/ females 43% of control ( days 1 4), 76%/ 78% of control ( days 4 28)] were observed at the high dose level, with the F1b litters displaying the greater effect. At the mid dose level, there was a slight decrease in body weight [ F1a males 93%/ females 94% of control ( day 28); F1b males 84%/ females 87% of control ( day 28)] and body weight gains [ F1a males 92%/ females 93% of control ( days 4 28); F1b males 83%/ females 85% of control ( days 4 28)], with the deficits being greater in the F1b litters.

Skeletal anomalies and reduced ossification were observed in the high dose F1b pups [ 80 mg/ kg/ day] that were dead at birth [ only dose level examined].

F1 Generation. No apparent adverse effect was observed on fertility at either dose level. Pre coital intervals and gestation lengths were comparable among the groups. The gestation survival index and the viability index were comparable among the groups for both the F2a and F2b litters. Litter size, body weights, and the sex ratio were comparable among the groups in both the F2a and F2b litters.

Degenerative changes in the tubules of the cortical region [ high dose F0 males] and outer medullary regions [ mid and high dose F0 males, mid dose F1 males ( highest dose tested in this generation)] of the kidneys were found in a subsequent histopathological examination. The original reviewer noted that these effects on the kidney were not found originally but during a subsequent re examination of the tissues, casting doubt on the quality of the histopathological examination of the reproductive organs. However, the RfD/ QA Peer Review Committee determined that, based on the lack of effects on reproductive organs in the chronic and subchronic studies at similar or higher dose levels, reevaluation of these tissues [ testes and ovaries] is not necessary [ HED Document CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 26 No. 011908, dated 5/ 9/ 96].

The NOAEL for parental toxicity is 5 mg/ kg/ day ( target dose; actual dose range 3.8 13.5 mg/ kg/ day) and the parental LOAEL is 20 mg/ kg/ day ( target dose; actual dose range 14 48 mg/ kg/ day), based on decreased female body weight/ body weight gain ( F1) and male renal tubule alteration ( F0 and F1).

The NOAEL for reproductive toxicity is 20 mg/ kg/ day ( target dose; actual dose range 18 35 mg/ kg/ day), and the LOAEL for reproductive toxicity is 80 mg/ kg/ day ( target dose; actual dose range 69 114 mg/ kg/ day), based on an increase in gestation length.

The NOAEL for offspring toxicity is 5 mg/ kg/ day ( target dose; actual dose range 7.2 13.5 mg/ kg/ day), and the LOAEL for offspring toxicity is 20 mg/ kg/ day ( target dose; actual dose range 26 48 mg/ kg/ day), based on decreased pup body weight [ F1b]. At 80 mg/ kg/ day ( target dose; actual dose range 76.1 133 mg/ kg/ day), there was an increase in pup deaths.

This 2 generation reproduction study is classified Acceptable/ guideline. This study satisfies the guideline requirement ( OPPTS 870.3800; § 83 4) for a 2 generation reproduction study. However, as noted above, a 2 generation reproduction study using the current protocol is a data requirement for 2,4 D. The latter study is required to address both the concern for thyroid effects [ comparative assessment between the young and adult animals] and immunotoxicity, as well as for a more thorough assessment of the gonads and reproductive/ developmental endpoints.

4.5 Chronic Toxicity Adequacy of data base for chronic toxicity: The data base for chronic toxicity is considered complete. No additional studies are required at this time. At doses above saturation of renal clearance, the kidney [ increased kidney weight, proximal tubule degeneration], thyroid [ increased thyroid weight, decreased T4, hyperplasia, hypertrophy], testes [ decreased testes weight, atrophy], ovaries [ decreased ovarian weight], and eyes [ lens opacity, cataracts, retinal degeneration] are target organs in the rat following chronic exposure to 2,4 D. In the dog, decreased brain weight [ females], increased incidence of kidney lesions, and aspermatogenesis and testes degeneration were observed.

870.4100a ( 870.4300) Chronic Toxicity B Rat EXECUTIVE SUMMARY: In a combined chronic toxicity/ carcinogenicity study [ MRID 43612001; 44284501], 50 Fischer 344 rats/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 96.4%] via the diet for up to 24 months at concentrations of 0, 5 mg/ kg/ day, 75 mg/ kg/ day, and 150 mg/ kg/ day. The achieved doses were 4.77, 73.15, and 144.98 mg/ kg/ day [ males] and 4.89, 73.11, and 143.52 mg/ kg/ day [ females], respectively. Additionally, 10 rats/ sex/ group were sacrificed at 12 months [ interim sacrifice]. NOTE: The interim sacrifice data were reported in MRID 43293901, HED Document No. 011614, along with the chronic neurotoxicity screening battery substudy.

There were no treatment related deaths or clinical signs of toxicity. Decreased body weight was observed CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 27 throughout the study at the high dose level in both sexes [ males 92% 96%/ females 74% 90% of control] and at the mid dose level in females [ 86% 90% of control]. At week 13, decreased body weight was very slight in the high dose males [ 96% of control] and somewhat greater in the high dose females [ 90% of control]. At study termination, both sexes displayed decreased body weight at the high dose level [ males 92%/ females 74% of control] with the females displaying a greater effect than males. The mid dose females also displayed a decrease in body weight at study termination [ 86% of control]. Body weight gains were decreased throughout the study in females [ 3 month interval ( 86% and 74% of control); 6 month interval ( 88% and 71% of control), and overall ( 77% and 52% of control)] at the mid and high dose levels, respectively. Similarly, high dose males displayed decreased body weight gains throughout the study [ 83% 87% of control]. Consistent with the decreased bodyweight gains was a decrease in food consumption, which was observed at the mid dose level in females [ 3.9%] and in both sexes at the high dose level [ males ( 4.7%)/ females ( 11.6%)].

Ophthalmology findings at study termination consisted of increased incidences of constricted blood vessels, fundus and hyper reflective, fundus in the high dose males and an increased incidence of lens opacity in the highdose females compared to the control and lower dose groups. Decreased RBC, HCT, and HGB values were observed at various time points in the mid and high dose females, and platelet counts were decreased at various time points in both sexes at the mid and high dose levels. Elevations in creatinine were observed in both sexes at the mid and high dose levels throughout the study, except at termination when comparable levels were observed among the male groups. Increased aspartate aminotransferase [ mid and high dose males], alanine aminotransferase [ mid and high dose males], and alkaline phosphatase [ mid and high dose, both sexes], and decreased glucose levels [ mid dose females, high dose both sexes], cholesterol [ mid and high dose, both sexes], and triglycerides [ mid dose females and high dose both sexes] were observed throughout the study, although a dose response was not always apparent. There was a dose related decrease in T4 values throughout the study in both sexes at the mid and high dose levels, and the females displayed the greater effect except at study termination.

Thyroid weights were increased in the mid dose females and in both sexes at the high dose level at the 12 month interim sacrifice. At study termination, thyroid weights were increased in both sexes at the mid and high dose levels, although the mid dose males did not attain statistical significance and the increase at the mid dose level [ both sexes] was greater than at the high dose level. Decreased testes weights were observed at the high dose level at both the interim and terminal sacrifices and at the mid dose level at study termination, although statistical significance was not attained at the mid dose. Decreased ovarian weights were observed at the high dose level at both sacrifice times and in the mid dose females at study termination. The decreases in testes and ovarian weights are consistent findings in other studies on 2,4 D and its salts/ esters. Kidney weights were increased only at the interim sacrifice in males at the mid and high dose levels [ dose related].

Gross pathology findings included decreased fat in high dose females at both sacrifice times, multifocal pale foci in the lungs [ interim: 1 mid dose, 10 high dose females; terminal: 4 high dose males, one control, 4 mid, 40 high dose females], and lens opacity in high dose females at termination. Microscopically, there were increased incidences of lesions in the bone marrow [ decreased hematopoiesis in high dose females at the interim sacrifice], eyes [ retina degeneration in 1 male and 9 females at high dose], kidney [ proximal tubule degeneration in midand high dose males and females], liver [ altered tinctorial properties in mid dose females and both sexes at high dose], lungs [ multifocal alveolar histiocytosis in mid dose females and both sexes at high dose], adipose tissue [ atrophy in mid dose female, both sexes at high dose], testes [ atrophy at high dose], and thyroid [ hyperplasia CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 28 high dose males; hypertrophy and epithelial cells high dose females] at the interim sacrifice. At study termination, there were increased incidences of cataracts and retina degeneration of the eyes in both sexes at the high dose level, and the severity of the retina degeneration was increased also. In the liver, there was an increased incidence of increased size of the hepatocytes with altered tinctorial properties in both sexes at the high dose level. In the lungs, both the incidence and severity of inflammation were increased at the high dose level in both sexes, and the incidence of atrophy of the adipose tissue was increased at the high dose level in both sexes at termination. Tumor incidence was not affected by treatment.

The NOAEL is 5 mg/ kg/ day. The LOAEL of 75 mg/ kg/ day is based on decreased body weight gain ( females) and food consumption ( females), alterations in hematology [ decreased RBC ( females), HGB ( females), platelets ( both sexes)] and clinical chemistry parameters [ increased creatinine ( both sexes), alanine and aspartate aminotransferase ( males), alkaline phosphatase ( both sexes), decreased T4 ( both sexes), glucose ( females), cholesterol ( both sexes), and triglycerides ( females)], increased thyroid weights ( both sexes at study termination), decreased testes and ovarian weights, and microscopic lesions in the lungs ( females). At the high dose level, there were microscopic lesions in the eyes, liver, adipose tissue, and lungs. There was no treatment related increase in the incidence of any tumor.

This chronic toxicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 4100 ( a); § 83 1 ( a)] for a chronic toxicity study in the rat.

NOTE: The study was performed to address whether the finding of an increased incidence of astrocytomas of the brain found in the 1986 rat study [ MRID 00160876] was attributable to 2,4 D. The HED Carcinogenicity Peer Review Committee [ CPRC; TXR No. 0050017] concluded that the doses used in the 1986 rat study were not adequate to assess the carcinogenic potential of 2,4 D. In the 1986 study, there was a significant trend for astrocytomas in male rats but no pair wise significance. The incidence in both the treated and control males exceeded the historical control incidence for this tumor. Additionally, the CPRC concluded that the high dose level in the 1986 study was not adequate. The current repeat chronic toxicity/ carcinogenicity study in rats was performed at higher dose levels. There was one astrocytoma in the brain of males at the high dose vs none in the control. There was one tumor each in the control and high dose female group. However, all of the brains of the rats in the low and mid dose groups were not examined. The HED Carcinogenicity Peer Review Committee [ CPRC; TXR No. 0050017] requested that the slides of the low and mid dose brains of the males be evaluated. These additional data were submitted [ MRID 44284501]. It was determined that there was no increased incidence of astrocytomas at any dose in the new study.

870.4100b Chronic Toxicity  Dog EXECUTIVE SUMMARY: In a chronic oral toxicity study [ MRID 43049001], 5 beagle dogs/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 96.7%] via the diet for 52 weeks at concentrations of 0, 1 mg/ kg/ day, 5 mg/ kg/ day, and 7.5 mg/ kg/ day. The target high dose level of 10 mg/ kg/ day was reduced to 7.5 mg/ kg/ day during week 8, due to loss of body weight/ failure to gain weight early in the study.

There were no treatment related deaths or clinical signs. One female at 5 mg/ kg/ day was sacrificed on Day 130 in a moribund condition; however, death was attributed to an abdominal inflammatory condition. Decreased body weight was observed throughout the study at the high dose level in both sexes, with females displaying CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 29 the greater effect. By week 3, high dose females displayed a 13% deficit in body weight compared to the control females. At the high dose level at 8 weeks when the dose level was lowered, both sexes at the high dose level displayed decreased body weight compared to the controls [ males 90%/ females 82% of control]. At week 13, the high dose males were 94% of control and the high dose females were 82% of control. At study termination, both sexes displayed decreased body weight at the mid [ males 89%/ females 87% of control] and high[ males 87%/ females 75% of control] dose levels compared to the controls. The low dose females also displayed a decrease [ 87% of control] in body weight at study termination. Body weight gain was decreased throughout the study in females at all dose levels [ weeks 1 13 ( 75%, 70%, and 35% of control); 1 52 ( 73%, 64%, and 36% of control) at the low, mid, and high dose levels, respectively]. Similarly, males displayed decreased bodyweight gains throughout the study ( except the low dose group during weeks 1 13) compared to the control, although there was no dose response [ weeks 1 13 ( 100%, 70%, 74% of control); 1 52 ( 78%, 63%, 67% of control]. Food consumption was decreased throughout most of the study, mainly at the high dose level [ both sexes], but statistical significance was not attained.

Ophthalmology findings were comparable among the groups for both sexes. There were no apparent treatmentrelated alterations in hematology or urinalysis among the groups for either sex. Elevations in blood urea nitrogen, creatinine, and alanine aminotransferase, and decreased glucose levels [ mid dose males, high dose both sexes] were observed throughout the study [ mid and high dose levels, both sexes], although a dose response was not always apparent, especially in the males. Alkaline phosphatase was decreased in both sexes at the highdose level at week 4. Similar alterations in these clinical chemistry parameters have been observed in subchronic studies in rats and dogs on 2,4 D, and its amine salts and esters.

There was a dose related decrease in absolute brain weight in females, with statistical significance being attained at the mid and high dose levels. Males at the high dose also displayed decreased brain weight, but statistical significance was not attained. In general, the organ weight effects observed can be attributed to the lower body weight, although the decreased testes weights at the high dose level and decreased ovarian weights at the midand high dose levels are consistent findings in other studies on 2,4 D and its salts/ esters. Microscopically, liver [ perivascular, chronic active inflammation ( males 1/ 5, 1/ 5, 3/ 5, 4/ 5; females 0/ 5, 0/ 4, 4/ 5, 3/ 5 with increasing dose) and sinusoidal lining cell pigment ( females; 1/ 5, 2/ 4, 5/ 5, 4/ 5 with increasing dose)] and kidney [ minimal increase in the frequency and average severity of pigment in the tubular epithelium ( males 2/ 5, 4/ 5, 5/ 5, 5/ 5; females 1/ 5, 1/ 4, 5/ 5, 5/ 5 with increasing dose)] lesions were observed at the mid and high dose levels in both sexes. Aspermatogenesis and degeneration were observed in one male at the high dose level compared to none in the other groups.

The NOAEL is 1 mg/ kg/ day. The LOAEL of 5 mg/ kg/ day is based on decreased body weight gain ( both sexes) and food consumption ( females), as well as alterations in clinical chemistry parameters [ increased BUN, creatinine, and alanine aminotransferase, decreased glucose] in both sexes, decreased brain weight in females, and histopathological lesions in the liver and kidneys.

This chronic oral toxicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 4100; § 83 1( b)] for a chronic, non rodent, oral toxicity study.

4.6 Carcinogenicity CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 30 Adequacy of data base for Carcinogenicity: The data base for carcinogenicity is considered complete. No additional studies are required at this time. There was no increase in the incidence of any tumor type in either sexes or species.

870.4200a Carcinogenicity Study  rat EXECUTIVE SUMMARY: see under Chronic Toxicity above.

This chronic toxicity/ carcinogenicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 4300; § 83 5] for a combined chronic toxicity/ carcinogenicity study in the rat.

870.4200b Carcinogenicity ( feeding)  Mouse EXECUTIVE SUMMARY: In a carcinogenicity study [ MRID 40061801], 50 B 6 C 3 F 1 CRL BR mice/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 97.5%] via the diet for 104 weeks at concentrations of 0, 1 mg/ kg/ day, 15 mg/ kg/ day, and 45 mg/ kg/ day. Additionally, 10 mice/ sex/ group were sacrificed at 52 weeks [ interim sacrifice].

No treatment related effect was observed on survival in either sex, and there were no treatment related clinical signs of toxicity. Males at the high dose level displayed a slight decrease [ 94% of control] in body weight at study termination, but body weight was comparable among the female groups. Body weight gain during the first week of exposure was decreased at the high dose level in both sexes [ males 50%/ females 12%]. Thereafter, the females displayed comparable body weight gains among the groups. The high dose males displayed decreased body weight gains during the 0 13 week [ 91% of control] and 0 104 week [ 84% of control, p< 0.05] intervals, and the low [ 91% of control, p< 0.05] and mid [ 89% of control, 0.05] dose males displayed lower body weight gains overall [ weeks 0 104] also. There was no apparent adverse effect of treatment on food consumption in either sex.

Hematology parameters were comparable among the groups [ both sexes]. There was no ophthalmoscopic assessment, and thyroid toxicity was not assessed in this study.

Increased kidney weights were observed in the high dose females at both the 52 week and terminal sacrifices. Kidney weights of the high dose males were slightly increased at the terminal sacrifice only. Adrenal weights were decreased at all dose levels in the males at the 52 week sacrifice, but there was no dose response. At study termination, the mid and high dose males displayed a significant increase in adrenal weight.

Gross pathology findings were comparable among the groups [ both sexes]. Microscopically, there was an increased incidence of lesions in the kidney of males at the mid and high dose levels [ histomorphologic alteration in the renal tubule epithelium, characterized as a cytoplasmic homogeneity due to the reduction of cytoplasmic vacuoles normally present] in unscheduled deaths and at both the interim and terminal sacrifices. This kidney lesion was not observed in females. No microscopic lesions were observed in the adrenal. There was no treatment related increase in any tumor type in either sex. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 31 The NOAEL is 1 mg/ kg/ day. The LOAEL of 15 mg/ kg/ day is based on treatment related increase in kidney weights in both sexes and microscopic renal lesions in males. There was no treatment related increase in the incidence of any tumor type.

This carcinogenicity study is classified Unacceptable because the high dose [ 45 mg/ kg/ day] is considered inadequate to assess the carcinogenic potential of 2,4 D. It was previously classified Core Minimum in the original DER; however, the HED Carcinogenicity Peer Review Committee [ HED TXR No. 0050017] concluded that a repeat carcinogenicity study in mice be performed at higher dose levels to adequately assess the carcinogenic potential of 2,4 D. The current study does not satisfy the guideline requirement [ OPPTS 870. 4200b; § 83 2] for a carcinogenicity study in the mouse. The repeated study [ MRIDs 43879801 and 43597201] employed dosages from 5 120 mg/ kg/ day for males and 5 300 mg/ kg/ day for females. The study has been reviewed under a separate DER [ TXR No. 011934], and the results indicated no treatment related increase in any tumor incidence relative to controls.

EXECUTIVE SUMMARY: In a carcinogenicity study [ MRID 43879801 and 43597201], 50 B 6 C 3 F 1 CRL BR mice/ sex/ group were administered 2,4 dichlorophenoxyacetic acid [ 96.4%] via the diet for 104 weeks at concentrations of 0, 5 mg/ kg/ day [ both sexes], 62 [ males]/ 150 [ females] mg/ kg/ day, and 120 [ males]/ 300 [ females] mg/ kg/ day. Additionally, 10 mice/ sex/ group were sacrificed at 52 weeks [ interim sacrifice].

There were no treatment related deaths or clinical signs of toxicity in either sex. Body weight, body weight gain, and food consumption were comparable among the male groups throughout the study. Females at the high dose level displayed a slightly lower body weight at the 3 and 6 month intervals [ 96% of control]. Body weight gains were decreased significantly at all dose levels in the females at the 3 month interval [ 93%, 94%, and 86% of control at the low, mid, and high dose, respectively], and the high dose females continued to display a reduced body weight gain at the 6 month [ 91% of control] and 12 month [ 91% of control] intervals. There were no consistent changes in food consumption in the female groups.

Ophthalmology and hematology parameters [ RBC, HGB, HCT, platelets] were comparable among the groups [ both sexes]; no thyroid parameters were monitored.

There was a dose related increase in kidney weights in both sexes. In males, increased kidney weights [ absolute and relative] were observed at the mid and high dose levels at the terminal sacrifice only. In females, increased kidney weights were observed at the mid and high dose levels at both the interim and terminal sacrifices.

Gross pathology findings were comparable among the groups [ both sexes]. Microscopically, there was an increased incidence of lesions in the kidneys of both sexes at the mid and high dose levels. In males at the interim/ terminal sacrifices, renal lesions were characterized as degeneration with regeneration of the descending limb of the proximal tubule in the mid [ 20%/ 50%] and high dose [ 100%/ 96%] males vs none in the low dose or control males; decreased vacuolization of the renal proximal tubule in mid [ 80%/ 78%] and high dose [ 100%/ 96%] males vs 0% in the low dose and control males. Additionally at the terminal sacrifice, there was an increased incidence of mineralization of the tubule( s) in the mid [ 58%] and high [ 72%] dose males compared to the low and control groups [ 38% and 32%, respectively]. In females, the renal lesions were characterized by hypercellularity in the descending part of the proximal tubule at both the interim and terminal sacrifices at the mid [ 80%/ 64%] and high dose [ 100%/ 50%] levels vs 0% in the low and control females. There CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 32 was no treatment related increase in any tumor type in either sex.

The NOAEL is 5 mg/ kg/ day. The LOAEL of 62 mg/ kg/ day [ males]/ 150 mg/ kg/ day [ females] is based on an increased absolute and/ or relative kidney weights and an increased incidence of renal microscopic lesions. There was no treatment related increase in the incidence of any tumor type.

This carcinogenicity study is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 4200b; § 83 2 ( b)] for a carcinogenicity study in the mouse.

NOTE: In the mouse study, there was an increase in hemangiosarcomas of the spleen in male mice at the low and mid doses, which was not sustained at the highest dose; however, the slides from all male mice at the low and mid doses were not evaluated. The HED Carcinogenicity Peer Review Committee [ CPRC; TXR No. 0050017] requested that the slides of the low and mid dose spleens of the males be evaluated. These additional data were submitted [ MRID 44284502]. The spleen of 35 male mice in the low dose [ 5 mg/ kg/ day] group and 33 in the mid dose [ 62.5 mg/ kg/ day] group were examined by light microscopy. No neoplasms were observed. When all of the data on the 50 male mice/ group were combined, there was no dose related increase in hemangiosarcomas or in possible preneoplastic lesions, such as extramedullary hematopoiesis. It was concluded that the additional data do not alter the findings of the study. There was another mouse carcinogenicity study [ MRID 40061801] conducted at dosages of 1, 15, and 45 mg/ kg/ day. No treatment related increase in tumor incidence was found; however, this latter study was classified unacceptable because the high dose was considered inadequate to assess the carcinogenic potential of 2,4 D.

4.7 Mutagenicity Adequacy of data base for Mutagenicity: The data base for mutagenicity is considered adequate based on pre 1991 mutagenicity guidelines. 2,4 Dichlorophenoxyacetic acid has been evaluated extensively [ open literature] in a range of in vivo and in vitro assays that have included tests with human cells. Overall, the pattern of responses observed in both in vivo and in vitro tests indicates that 2,4 dichlorophenoxyacetic acid was not mutagenic, although some cytogenetic effects were observed. The available studies on the amine salts and esters show a similar, nonmutagenic, response.

Ames tests, with and without metabolic activation, were negative consistently. Negative results were also observed in a mouse bone marrow micronucleus assay and in UDS assays in rat hepatocytes. Conflicting results were obtained in Drosophila; positive effects were observed in larvae, and negative results were observed in adults after feeding or injection. Conflicting results were also seen in in vitro mammalian cell cytogenetics assays: 2,4 dichlorophenoxyacetic acid was negative for structural chromosomal damage up to an insoluble level but positive in the presence of metabolic activation at high doses. The positive evidence, however, tends to be weak and generally not supported by the data from in vivo cytogenetic assays. Overall, the pattern of responses observed in both in vivo and in vitro tests indicates that 2,4 dichlorophenoxyacetic acid was not mutagenic, although some cytogenetic effects were observed.

2,4 dICHLOROPHENOXYACETIC ACID [ 2,4 D aCID] CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 33 Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41409801 classification: Acceptable 2,4 D 100 1000

g/ plate w/ S9; 66.7 6670

g/ plate w/ out S9. No evidence of bacterial mutation in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, w/ and w/ out S9 Guideline 870.5300, hamster V79 fibroblasts, HPRT locus Pavlica, et al., 1991 classification: N/ A 2,4 D 10 100

g/ mL. Positive Cytogenetics Guideline 870.5385, in vitro chromosome aberration [ human lymphocytes]; Mustonen, et al. 1986 classification: N/ A 2,4 D 0.125 0.35 mM; 0.125 1.250 mM. No evidence of increased chromosome aberrations in human lymphocytes.

Guideline 870.5385, in vivo chromosome aberration [ Wistar rat bone marrow] Adhikari & Grover, 1988 classification: N/ A 2,4 D 0, 17.5, 35, 70 mg/ kg/ day ip 2X. Equivocal [+ at top 2 doses, but results were similar to DMSO control].

Guideline 870.5385, in vivo chromosome aberration [ Charles River rat bone marrow] Turkula & Jalal, 1987 classification: N/ A 2,4 D 0 350 mg/ kg ip for 4 or 24 hours/ 3 replicates. Equivocal [+  75 mg/ kg but only in one replicate].

Guideline 870.5395, micronucleus assay; MRID 41409804, 41870101 classification: Acceptable 2,4 D ( 96.1%) 40 400

g/ kg. ICR mouse ( bone marrow). Negative Other Genotoxicity Guideline 870.5450, Unscheduled DNA synthesis assay; MRID 41409807; classification: Acceptable 2,4 D ( 96.1%) 0.969 2890

g/ mL Fisher 344 primary rat hepatocytes. No evidence of induction of unscheduled DNA synthesis.

The following tables provide summaries of the mutagenicity studies on the 2,4 D amine salts and esters for comparison.

BUTOXYETHYL ESTER [ BEE]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41388204, 41797903, 42015701 classification: Acceptable BEE 5 5000

g/ plate w/ S9; 1.6 1667

g/ plate w/ out S9. No evidence of bacterial mutation in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, w/ and w/ out S9 CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 34 Guideline 870.5300, HPRT locus ( CHO) cells [ in vitro] MRID 43394201 classification: Acceptable BEE Doses: 200 700 and 600 1000 µ g/ mL without S9; 150 1400 µ g/ mL with S9 activation [ Aroclor induced rat liver]; cytotoxic at  800 µ g/ mL without S9 activation; non cytotoxic with S9. There was no evidence of mutagenic effect at any dose level with or without activation.

Cytogenetics Guideline 870.5375, in vitro chromosome aberration [ rat lymphocytes]; MRID 43327305 classification: Unacceptable need new study BEE Doses: 87.5, 175, 350, 700, and 1400 µ g/ mL with or without metabolic activation. Negative.

Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 41478301, 42015704, 42015701, 42015707 classification: Unacceptable BEE Doses: 37.5, 125, or 375 mg/ kg; did not cause a significant increase in frequency of micronucleated polychromatic erythrocytes ( MPEs); however, no evidence of compound toxicity in the test animals or cytotoxicity in the target organ.

Other Genotoxicity Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 41498101. classification: Unacceptable BEE 5.0 to 500 µ g/ mL; did not induce an appreciable increase in the net nuclear grain counts in two independent unscheduled DNA ( UDS) assays, but technical problems preclude accurate assessment of results; excessively high background cytoplasmic and nuclear grain counts in all groups make it difficult to distinguish between a positive and negative genotoxic response.

DIETHANOLAMINE [ DEA]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41920910 classification: Acceptable DEA 500, 1000, 2500, 5000, 10000, 14000

g/ plate [ equivalent to 369, 738, 1845, 3690, 7380, 10332

g/ plate 2,4 D DEA]. Not mutagenic in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, w/ and w/ out S9 Cytogenetics Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 41920908 classification: Acceptable DEA Doses ( oral): 60, 200, 600 mg/ kg; equivalent to 44.28, 147.6, 442.8 mg/ kg. Not clastogenic; no significant increase in frequency of micronucleated polychromatic erythrocytes ( MPEs); toxic signs in males at HDT included ataxia, languidness, and squinted eyes.

Other Genotoxicity CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 35 Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 41920909 classification: Acceptable DEA Doses [ 10 to 500 µ g/ mL; equivalent to 7.38 to 369 µ g/ mL]. Negative; no appreciable increase in net nuclear grain counts of treated rat hepatocytes.

DIMETHYLAMINE [ DMA]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41409802 classification: Acceptable DMA 333 10000

g/ plate. No evidence of bacterial mutation in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, w/ and w/ out S9 Cytogenetics Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 41409805, 41870102 [ micronucleus] classification: Acceptable DMA Doses: 60, 120, or 600 mg/ kg. Not clastogenic; no significant increase in frequency of MPE's. Toxic signs [ languidness, prostration, rough hair coat or death] at 600 mg/ kg.

Other Genotoxicity Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 41409808 classification: Acceptable DMA Doses [ 2.5 to 100 µ g/ mL]. Negative. No appreciable increase in net nuclear grain counts of treated rat hepatocytes.

ETHYLHEXYL ESTER [ EHE]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41409803 classification: Acceptable EHE 333 10000

g/ plate. No evidence of bacterial mutation in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, w/ and w/ out S9 Cytogenetics Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 41409806, 41870103 classification: Acceptable EHE Doses: 60, 120, or 600 mg/ kg. Not clastogenic; no significant increase in frequency of MPE's. Toxic signs [ languidness, prostration, rough hair coat or death] at 600 mg/ kg.

Other Genotoxicity CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 36 Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 41409809 classification: Acceptable EHE Doses 2.5 to 100 µ g/ mL. Negative. No appreciable increase in net nuclear grain counts of treated rat hepatocytes.

ISOPROPYLAMINE [ IPA]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41388203, 41797902, 42015701 classification: Acceptable IPA 333 10000

g/ plate. Not mutagenic w/ and w/ out S9 in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100,

Guideline 870.5300, HPRT locus ( CHO) cells [ in vitro] MRID 43327304 classification: Acceptable IPA Doses: 500, 1,000, 1500, 2000, and 3000 µ g/ mL with and without S9 activation [ Aroclor induced rat liver]; cytotoxic at dose of 2500 µ g/ mL with and without S9 activation. There was no evidence of mutagenic effect at any dose level with or without activation.

Cytogenetics Guideline 870.5375, in vitro chromosome aberration [ rat lymphocytes]; MRID 43327303 classification: Acceptable IPA Doses: 96, 192, 384, 767, 1534, 3068, and 61370 µ g/ mL with or without metabolic activation. Negative; not clastogenic at dose levels upto 3000

g/ mL Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 41478303, 42015701, 42015703, 42015706 classification: Acceptable IPA Doses ( oral): 75, 150, or 750 mg/ kg; Not clastogenic; no significant increase in frequency of micronucleated polychromatic erythrocytes ( MPEs); toxic signs were languidness with ataxia and/ or rough hair coat.

Other Genotoxicity Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 41498103.

classification: Unacceptable IPA 5 doses [ 5.0 to 500 µ g/ mL] did not induce an appreciable increase in the net nuclear grain counts in two independent unscheduled DNA ( UDS) assays, but technical problems preclude accurate assessment of results; excessively high background cytoplasmic and nuclear grain counts in all groups make it difficult to distinguish between a positive and negative genotoxic response.

ISOPROPYLESTER [ IPE]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 43935101 classification: Acceptable IPE 100 5000

g/ plate + S9/ 10 2500

g/ plate  S9. Not mutagenic w/ and w/ out S9 in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100,

Cytogenetics CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 37 Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 43930801 classification: Acceptable IPE oral [ gavage] doses of 100, 200, and 400 mg/ kg to CD 1 mice; did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes.

Other Genotoxicity Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 43930501 classification: Acceptable IPE 5.01 200 µ g/ mL. No significant increases in net nuclear grain counts at any concentration.

2,4 D TRIISOPROPANOLAMINE [ TIPA]

Gene Mutation Guideline 870.5265, Ames, reverse mutation; MRID 41388202 ,41797901, 42015701 classification: Acceptable TIPA 1000 10000

g/ plate. No evidence of bacterial mutation in S. Typhimurium strains TA1535, TA1537, TA1538, TA98, TA100, w/ and w/ out S9 Guideline 870.5300, HPRT locus ( CHO) cells [ in vitro] MRID 43327302 classification: Acceptable TIPA Doses: 800, 1,000, 1,250, 2,500, and 5,000 µ g/ mL with and without S9 activation [ Aroclor induced rat liver]; cytotoxic at the limit dose of 5,000 µ g/ mL without S9 activation. There was no evidence of mutagenic effect at any dose level with or without activation.

Cytogenetics Guideline 870.5375, in vitro chromosome aberration [ rat lymphocytes]; MRID 43327301 classification: Acceptable TIPA Doses: 78, 156, 313, 625, 1,250, 2,500, and 5,000 µ g/ mL with or without metabolic activation. Negative.

Guideline 870., in vivo chromosome aberration [ mouse bone marrow] MRID 41478302., 42015701, 42015702, 42015705 classification: acceptable TIPA Doses: 75, 250, or 750 mg/ kg; did not cause a significant increase in frequency of micronucleated polychromatic erythrocytes ( MPEs); 2 deaths occurred at 750 mg/ kg Other Genotoxicity Guideline 870.5450, Unscheduled DNA synthesis assay [ rat hepatocytes]; MRID 41498102.

classification: Unacceptable TIPA 5 doses [ 5.0 to 500 µ g/ mL] did not induce an appreciable increase in the net nuclear grain counts in two independent unscheduled DNA ( UDS) assays, but technical problems preclude accurate assessment of results; excessively high background cytoplasmic and nuclear grain counts in all groups make it difficult to distinguish between a positive and negative genotoxic response.

4.8 Neurotoxicity Adequacy of data base for Neurotoxicity: The data base for neurotoxicity is not considered complete. A developmental neurotoxicity study is required and is considered a datagap. Evidence of neurotoxicity was CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 38 observed following both acute and repeated dose exposure at dose levels above saturation of renal clearance. Following acute exposure, there was an increased incidence of incoordination, slight gait abnormalities and decreased motor activity. There was evidence of neuropathology [ increased incidence of bilateral retinal degeneration] following repeated dosing and increased fore/ hindlimb grip strength.

870.6100 Delayed Neurotoxicity Study  Hen This study is not required for this chemical.

870.6200a Acute Neurotoxicity Screening Battery EXECUTIVE SUMMARY: In an acute neurotoxicity study [ MRID 43115201], Fischer 344 rats ( 10/ sex/ dose) were orally gavaged once with 2,4 D [ 96.6% a. i.] at doses of 0 ( corn oil), 15, 75, or 250 mg/ kg ( actual: 0, 13, 67 or 227 mg/ kg). Neurobehavioral evaluations, consisting of Functional Observational Battery ( FOB) and motor activity, were conducted at Day  1 ( prestudy), Day 1 ( approximately 5 6 hrs postdosing, peak time of effect) and Days 8 and 15. At terminal sacrifice ( Day 15), animals were euthanized and neuropathological examination performed on control and treated animals ( 5/ sex/ dose).

There were no treatment related mortalities, and no significant differences were noted in the mean body weights or mean body weight gains. Treatment related, clinical signs [ uncoordinated movement/ behavior] were observed in the high dose rats only. During the Day 2 and 3 clinical examinations, incoordination was noted in high dose animals of both sexes [ Day 2, 2/ 10 males and 5/ 10 females; Day 3, 2/ 10 males, 1/ 10 females; Day 4, 0/ 10 males, 1/ 10 females]. The incidence of incoordination decreased to control levels by Day 4 in males and Day 5 in females. Neurobehavioral evaluation revealed treatment related changes. During the Day 1 FOB evaluations, increased incidences of incoordination ( 6/ 10, males; 4/ 10, females) and slight gait abnormalities, described as forepaw flexing or knuckling, were observed in high dose animals ( 8/ 10, males; 8/ 10 females). Slight gait abnormalities, observed in a single mid dose female, were not judged to be treatment related since no other signs of toxicity were evident. Minimal [ equivocal] gait abnormalities, not judged to be treatment related, were observed in one low dose female and one each mid and high dose male. In high dose animals, total motor activity was significantly lower at Day 1 only. No treatment related gross or neuropathological findings were present at any dose level in either sex.

The NOAEL for neurotoxicity is 67 mg/ kg, based on an increased incidence of incoordination and slight gait abnormalities [ described as forepaw flexing or knuckling] and decreased total motor activity at the LOAEL of 227 mg/ kg. The NOAEL for systemic toxicity was 227 mg/ kg [ the highest dose tested] in males and females.

This acute neurotoxicity study in the rat is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 6200a; § 81 8] for an acute neurotoxicity study in the rat.

870.6200b Subchronic Neurotoxicity Screening Battery In lieu of the 90 day subchronic neurotoxicity study, a one year chronic neurotoxicity study was performed. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 39 EXECUTIVE SUMMARY: In this chronic toxicity/ carcinogenicity study with a chronic neurotoxicity screening battery substudy [ MRID 43293901], 50 Fischer 344 rats/ sex/ group [ main study]; 15/ sex/ group [ substudy] were administered 2,4 dichlorophenoxyacetic acid [ 96.4%] via the diet for up to 24 months at concentrations of 0 [ basal diet], 5 mg/ kg/ day, 75 mg/ kg/ day, and 150 mg/ kg/ day [ achieved doses were 4.6, 71.2, and 141.1 mg/ kg/ day [ males] and 4.6, 68.0, and 138.9 mg/ kg/ day [ females], respectively. Additionally, 10 rats/ sex/ group were sacrificed at 12 months [ interim sacrifice]. NOTE: This DER presents the results of the interim [ one year] sacrifice and neurotoxicity substudy.

NEUROTOXICITY STUDY No treatment related, hand held, FOB observations were noted at any of the evaluation periods. Relative forelimb grip strength was significantly increased in both sexes at the high dose level, but there was no treatment related change in absolute grip strength. There was no treatment related effect on motor activity. In agreement with the chronic toxicity portion of the study, an increased incidence of bilateral retinal degeneration was observed in the high dose females.

The NOAEL for neurotoxicity is 75 mg/ kg/ day, based on increased relative forelimb grip strength and increased incidence of bilateral retinal degeneration at the LOAEL of 150 mg/ kg/ day.

CHRONIC TOXICITY STUDY There were no treatment related deaths or clinical signs of toxicity. Decreased body weight was observed throughout the first year of the study at the high dose level in both sexes [ males 91%/ females 88% of control at the interim sacrifice] and at the mid dose level in females [ 94% of control at the interim sacrifice]. At 90 days, the decrease in body weight was very slight in the high dose males [ 96% of control] and somewhat greater in the mid [ 95% of control] and high dose [ 90% of control] females. Body weight gains were decreased throughout the first year at the mid and high dose levels in both sexes, although statistical significance was not always attained in males at the mid dose level [ 2 week interval ( males: mid dose 90%/ high dose 84%; females: mid dose 81%/ high dose 63% of control); 3 month interval ( males: mid dose 98%/ high dose 88%; females: mid dose 87%/ high dose 75% of control); and 1 year interval ( males: mid dose 95%/ high dose 82%; females: mid dose 89%/ high dose 73% of control). Consistent with the decreased body weight gains was a slight decrease in food consumption, which was observed in both sexes at the high dose level.

Ophthalmology findings were comparable among the groups at the interim sacrifice. Decreased RBC [ mid and high dose females ( 6 & 12 months) and high dose males ( 12 months)], HCT [ mid and high dose females and high dose males ( 6 & 12 months)], HGB [ high dose females ( 6 months)], WBC high dose females ( 6 months)], and platelet counts [ mid and high dose females and high dose males ( 6 & 12 months) were observed. A doserelated increase in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase was observed in the mid and high dose males at 6 months but not at 12 months. The mid and high dose females displayed a dose related increase in alkaline phosphatase values at both the 6 and 12 month intervals. Cholesterol levels were decreased in the mid ( males at 12 months only) and high dose rats of both sexes at both time intervals. T4 values were decreased at both time points in both sexes at the mid and high dose levels, although the middose males at 6 months did not attain statistical significance. The only urinalysis finding was a decrease in specific gravity , which was observed in both sexes and time points at the mid and high dose levels. CHEMICAL NAME/ MONTH/ YEAR of update RED ( or Registration) Toxicology Chapter 40 Thyroid weights [ absolute and relative] were statistically significantly increased in the mid dose females [

20% greater than control] and in both sexes at the high dose level [

20% greater than control] at the 12 month interim sacrifice. Decreased testes weights [ absolute and relative to brain] were observed at the high dose level at 12 months [

15% lower than control]. Kidney weights [ absolute and relative] were increased at the 12 months sacrifice in males at the mid [ 7% 12% greater than control] and high dose [ 8% 16% greater than control] levels [ dose related].

Gross pathology findings included decreased fat in high dose females [ 4/ 10] and multifocal pale foci in the lungs [ 1/ 10 mid dose, 10/ 10 high dose females]. Microscopically, there were increased incidences of lesions in the bone marrow [ decreased hematopoiesis in high dose females], eyes [ bilateral retina degeneration in 1 male and 10 females at high dose], kidney [ proximal tubule degeneration in mid and high dose males and females], liver [ altered tinctorial properties in mid dose females and both sexes at high dose], lungs [ multifocal , subacute to chronic inflammation ( mid dose females, both sexes at high dose), alveolar histiocytosis in females at high dose], adipose tissue [ atrophy in mid and high dose females], testes [ atrophy at high dose], and thyroid [ decreased secretory material, epithelial cells high dose females].

The NOAEL is 5 mg/ kg/ day. The LOAEL of 75 mg/ kg/ day is based on decreased body weight ( females)/ body weight gain ( both sexes), alterations in hematology [ decreased RBC, HCT, and platelets ( females)], clinical chemistry parameters [ increased alanine and aspartate aminotransferases ( males), alkaline phosphatase ( both sexes), decreased cholesterol ( both sexes), and decreased T4 ( both sexes)], and urinalysis [ decreased specific gravity ( both sexes)], increased kidney weights ( males), increased incidence of degeneration of the descending proximal tubules ( both sexes), hepatocellular hypertrophy with altered tinctorial properties ( females), lung inflammation ( females), adipose tissue atrophy ( females). At the high dose level, there also were microscopic lesions in the eyes ( both sexes), liver ( males), testes ( males), thyroid ( females), and lungs ( males).

This chronic toxicity/ neurotoxicity study in the rat is classified ACCEPTABLE/ Guideline, and it satisfies the guideline requirement [ OPPTS 870. 6200b; § 82 7] for a subchronic neurotoxicity study in the rat.

870.6300 Developmental Neurotoxicity Study This is a datagap.