Page 1 of 118 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES Date: March 1, 2006 MEMORANDUM SUBJECT: ACETOCHLOR. Revised HED Chapter of the Tolerance Reassessment Eligibility Decision ( TRED) Document. PC Code: 121601, DP Barcode: D292336 Regulatory Action: Tolerance Reassessment Risk Assessment Type: Single Chemical / Aggregate FROM: Alberto Protzel, Ph. D., Branch Senior Scientist, Risk Assessor Toxicology Branch Health Effects Division ( 7509C) AND Linnea Hansen, Ph. D., Toxicologist Toxicology Branch AND Samuel Ary, Chemist Reregistration Branch 2 Health Effects Division ( 7509C) AND Michael R. Barrett, Ph. D, Senior Chemist Ronald Parker, Ph. D., Senior Environmental Scientist Environmental Risk Branch V Environmental Fate and Effects Division ( 7507C)

THROUGH: Louis Scarano, Ph. D., Chief Toxicology Branch Health Effects Division ( 7509C)

TO: Felicia Fort, Chemical Review Manager Reregistration Branch III Special Review and Reregistration Division ( 7508W) Page 2 of 118 Table of Contents 1.0 Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.0 Ingredient Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.1 Summary of Registered/ Proposed Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2 Structure and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.3 Physical and Chemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.0 Metabolism Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.1 Comparative Metabolic Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2 Nature of the Residue in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3.2.1. Description of Primary Crop Metabolism . . . . . . . . . . . . . . . . . . . . . . 13 3.2.2 Description of Livestock Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . 14 3.2.3 Description of Rotational Crop Metabolism, including identification of major metabolites and specific routes of biotransformation . . . . . . . . . 17 3.3 Environmental Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.4 Tabular Summary of Metabolites and Degradates . . . . . . . . . . . . . . . . . . . . . . 19 3.5 Toxicity Profile of Major Metabolites and Degradates . . . . . . . . . . . . . . . . . . 21 3.6 Summary of Residues for Tolerance Expression and Risk Assessment . . . . . . . 25 3.6.1 Tabular Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.6.2 Rationale for Inclusion of Metabolites and Degradates . . . . . . . . . . . . 26 3.6.3 Rationale for Exclusion of Degradates . . . . . . . . . . . . . . . . . . . . . . . . . 26 4.0 Hazard Characterization/ Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.1 Hazard Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.1.1 Database Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.1.2 Mode of Action, Metabolism, Toxicokinetics Data . . . . . . . . . . . . . . . 27 4.1.3 Sufficiency of Studies / Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4.1.4. Toxicological Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4.1.5 Dose Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4.1.6 FQPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.2 FQPA Hazard Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.2.1 Adequacy of the Toxicity Data Base . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.2.2 Evidence of Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.2.3 Developmental Toxicity Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.2.4 Reproductive Toxicity Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 4.2.5 Additional Information from Literature Sources . . . . . . . . . . . . . . . . . 55 Page 3 of 118 4.2.6 Pre and/ or Postnatal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 <IP_ADDRESS> Determination of Susceptibility . . . . . . . . . . . . . . . . . . 55 <IP_ADDRESS> Degree of Concern Analysis and Residual Uncertainties for Pre and/ or Post natal Susceptibility . . . . . . . . . . . . . . . 55 4.3 Recommendation for a Developmental Neurotoxicity Study . . . . . . . . . . . . . . 55 4.3.1 Evidence that supports requiring a Developmental Neurotoxicity study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.3.2 Evidence that supports not requiring for a Developmental Neurotoxicity study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 <IP_ADDRESS> Rationale for the UF DB ( when a DNT is recommended) 56 4.4 Hazard Identification and Toxicity Endpoint Selection . . . . . . . . . . . . . . . . . . 56 4.4.1 Acute Reference Dose ( aRfD)  General Population including Females age 13 49 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.4.2 Chronic Reference Dose ( cRfD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4.3 Incidental Oral Exposure ( Short and Intermediate Term) . . . . . . . . . . . 58 4.4.4 Dermal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4.5 Dermal Exposure ( Short, Intermediate and Long Term) . . . . . . . . . . . 58 4.4.6 Inhalation Exposure ( Short, Intermediate and Long Term) . . . . . . . . . 62 4.4.7 Margins of Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 4.4.8 Recommendation for Aggregate Exposure Risk Assessments . . . . . . . . 64 4.4.9 Classification of Carcinogenic Potential . . . . . . . . . . . . . . . . . . . . . . . . 64 4.5 Special FQPA Safety Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 4.6 Endocrine disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.0 Public Health Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 5.1 Incident Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 5.2 Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 6.0 Exposure Characterization/ Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 6.1 Dietary Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 6.1.1 Residue Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 6.1.2 Acute and Chronic Dietary Exposure and Risk . . . . . . . . . . . . . . . . . . 78 6.2 Water Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 6.3 Residential ( Non Occupational) Exposure/ Risk Pathway . . . . . . . . . . . . . . . . 81 7.0 Aggregate Risk Assessments and Risk Characterization . . . . . . . . . . . . . . . . . . . . 81 7.1 Acute Aggregate Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 7.2 Short Term Aggregate Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 7.3 Intermediate Term Aggregate Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 7.4 Long Term Aggregate Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 7.5 Cancer Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 8.0 Cumulative Risk Characterization/ Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Page 4 of 118 9.0 Occupational Exposure/ Risk Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 10.0 Data Needs and Label Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 10.1 Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 10.2 Residue Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 References: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Appendix 1. Toxicology Data Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Appendix 2. MOE Assessment of Acetochlor Water Degradates . . . . . . . . . . . . . . . . 108 Appendix 3. Tolerance Reassessment Summary for Acetochlor . . . . . . . . . . . . . . . . . 117 Page 5 of 118 1.0 Executive Summary This assessment provides information to support the issuance of a risk management decision document known as a Tolerance Reassessment Eligibility Decision ( TRED) Document for acetochlor. EPA's pesticide reregistration process provides for the review of older pesticides ( those initially registered prior to November 1984) under the Federal Insecticide, Fungicide, and Rodenticide Act ( FIFRA) to ensure that they meet current scientific and regulatory standards. The process considers the human health and ecological effects of pesticides and incorporates a reassessment of tolerances ( pesticide residue limits in food) to ensure that they meet the safety standard established by the Food Quality Protection Act ( FQPA) of 1996.

Acetochlor, 2 chloro N( ethoxymethyl) N( 2 ethyl 6 methylphenyl) acetamide, is a chloroacetanilide herbicide used for preemergence control of weeds in corn. In the United States, acetochlor is conditionally registered for use on field corn to the Acetochlor Registration Partnership ( ARP), which is comprised of Dow AgroSciences, LLC ( Dow) and Monsanto Company ( Monsanto). In addition to the ARP members, Drexel Chemical Company ( Drexel), Syngenta Crop Protection, Inc. ( Syngenta) and Tenkoz, Inc. ( Tenkoz) also have end use products containing acetochlor. Acetochlor is formulated as a variety of emulsifiable concentrate ( EC), soluble concentrate ( SC), microencapsulated ( Mcap), or granular ( G) formulations that can be applied to field corn as a preplant, preemergence, or early postemergence application using only ground equipment.

Acetochlor has low acute toxicity by the oral, dermal and inhalation routes and it is mildly irritating to the eyes. Acetochlor has shown mild skin irritation in one study, however in another study it has been a strong skin irritant. Acetochlor is a strong dermal sensitizer. Acetochlor is well absorbed by the oral route, undergoes extensive biotransformation in the organism leading to chemical reactive species that are responsible for its mode of action in nasal tissue carcinogenesis.

The major target organs affected in rats, dogs and mice exposed to acetochlor appear to be the liver, thyroid ( secondary to liver), nervous system, kidney, testes, and erythrocytes. Speciesspecific target organs include the nasal olfactory epithelium in rats and the lungs in mice. Chronic studies showed that the dog was more sensitive than the rat. Chronic toxicity in the dog appeared in the form of testicular, hepatic, and renal histopathology, and at high doses, brain histopathology. Evidence of neurotoxicity has been observed in several studies. In general, dogs appear to be somewhat more sensitive than rats to effects on the nervous system. There is no evidence that acetochlor is teratogenic or that offspring are more susceptible than adults, but acetochlor does cause developmental toxicity in rats ( but not in rabbits) at maternally toxic doses.

Available developmental toxicity studies in two species and three two generation reproductive toxicity studies in the rat do not show evidence of increased susceptibility of the offspring. It is recommended that the special FQPA SF be reduced to 1X because there are no/ low concerns and no residual uncertainties with regard to pre and/ or postnatal toxicity. Based on neurotoxicity Page 6 of 118 findings in two species, the HED HIARC recommended that a developmental neurotoxicity toxicity ( DNT) study be submitted by the registrant. Pending submission of this study, a 10X database uncertainty factor ( UF DB ) has been proposed for acetochlor to account for the absence of the DNT study.

Endpoints for risk assesment were selected as follows:

! An acute dietary endpoint for females 13 49 years of age was selected from an oral developmental toxicity study in rats, based on decreased fetal weights, increased resorptions and post implantation loss.

! An acute dietary endpoint for the general population was chosen based on acute oral neurotoxicity in female rats.

! The chronic dietary endpoint ( all populations), long term dermal (> 6 months) and inhalation (> 6 months) were selected from a chronic dog feeding study based on increased salivation and histopathology in the testes, liver and kidney.

! Incidental oral short term ( 1 30 days) and intermediate ( 1 6 months) were not selected because there are no registered residential uses for acetochlor.

! The short term dermal ( 1 30 days) endpoint was chosen from a 21 day dermal toxicity study in rabbits based on mortality and clinical signs of toxicity.

! The short term inhalation ( 1 30 days) endpoint was selected from a rabbit oral developmental toxicity study, based on decreased fetal weights, increased resorptions and post implantation loss.

! Intermediate term ( 1 6 months) dermal and inhalation endpoints were selected from a two generation reproductive toxicity study in the rat based on decreased Day 21 mean pup weight and pup weight gain during lactation; focal hyperplasia and polypoid adenomata in nasal epithelium of adult F1 offspring at study termination.

Acetochlor is classified as " likely to be carcinogenic to humans," based on increased incidence of lung tumors in male and female mice, histiocytic sarcoma in female mice and nasal epithelial tumors and thyroid follicular cell adenomas in male and female rats. A non mutagenic ( with threshold) mode of action was established for the nasal and thyroid tumors; no mode of action was established for the other observed tumors.

Acute, chronic, and cancer dietary risk assessments ( food + water) were conducted for all supported acetochlor food uses and were performed to support the tolerance reassessment eligibility decision. The acute, chronic, and cancer dietary exposure assessment incorporated tolerance level residues for all crops and percent crop treated data provided by the Acetochlor Page 7 of 118 Registration Partnership ( ARP). Processing data were available for numerous commodities and incorporated into the assessment. Water values were generated from the ARP acetochlor water monitoring program.

! The acute dietary risk estimates do not exceed HED's level of concern ( less than 100% of the aPAD) at the 99.9th exposure percentile for the U. S. population at 2% of the aPAD and all population subgroups, with the highest exposed population subgroup being infants less than 1 year old at 6% of the aPAD ! The chronic dietary risk estimates do not exceed HED's level of concern ( less than 100% of the cPAD) for the U. S. population and all population subgroups ( all were less than 1% of the cPAD).

! The estimated exposure of the general U. S. population to acetochlor is 0.000026 mg/ kg/ day. Applying the Q 1 * of 3.27 x 10 2 ( mg/ kg/ day) 1 to the exposure value results in a cancer risk estimate of 8.40 x 10 7. Therefore, the cancer dietary risk estimate does not exceed HED's level of concern of 1.0 x 10 6.

There are no residential uses that might contribute to aggregate risks, and occupational exposure is not assessed in this TRED. Acetochlor is part of a Common Mode of Action group with the related chloroacetanilides butachlor and alachlor. A cumulative risk assessment is currently underway for acetochlor and alachlor.

Given that both the ESA and OXA degradates have been detected in water samples ( both groundwater and surface water)  in many cases at concentrations higher than the parent acetochlor  worst case non carcinogenic margin of exposure ( MOE) calculations were done for the two water degradates to better support the MARC conclusion. The values were found to be not of concern to HED. The detailed results are presented in Appendix 2. Page 8 of 118 2.0 Ingredient Profile Acetochlor, 2 chloro N( ethoxymethyl) N( 2 ethyl 6 methylphenyl) acetamide, is a chloroacetanilide herbicide used for preemergence control of annual grasses, yellow nutsedge, and certain broadleaf weeds in corn. In the United States, acetochlor is conditionally registered for use on field corn to the Acetochlor Reregistration Partnership ( ARP). The ARP was originally composed of Monsanto and Zeneca ( ICI); however, Zeneca's interest in the partnership was purchased by Dow in 2000. Acetochlor is formulated as Emulsifiable Concentrate ( EC), SC, Mcap, or G formulations and is marketed by members of the ARP under the trade names Harness ® , TopHand ® , Degree ® , Field Master ® , Surpass   , TopNotch   , Keystone ® , and FulTime   . These formulations can be applied to field corn as a preplant, preemergence, or early postemergence application using only ground equipment.

Under the terms of the conditional registration, the Agency stipulated that the continued registration of acetochlor depended on a significant reduction in the total use of U. S. corn herbicides of concern, including alachlor, metolachlor, atrazine, and 2,4 D. The registration can also be cancelled or suspended if there is evidence of groundwater contamination by acetochlor. In addition, the Agency restricted the use of acetochlor to certified applicators, prohibited aerial application, restricted the types of soils on which acetochlor could be used, and prohibited application to water.

2.1 Summary of Registered/ Proposed Uses Although there are numerous acetochlor EPs registered for use on corn, the use directions are similar among the various labels. Acetochlor is restricted for direct use only on field corn and corn grown only for silage or seed. Labels allowing direct application to pop corn should be amended to prevent such application until the registrant formally petitions for the use. Although several of the labels also allow for a fall application to soybean stubble after crop harvest, replanting the following spring is restricted to field corn. Generally, acetochlor can be applied as either a single broadcast or banded application, preplant, preemergence, or early post emergence. Preplant applications can be made as either a single or split application that is either surface applied or incorporated. The early post emergence application is allowed only on corn up to eleven inches in height. For application, acetochlor may be either diluted with water or liquid fertilizers or impregnated onto dry bulk fertilizers. Only applications using ground equipment are allowed; applications through irrigation systems and using aerial equipment are prohibited. Due to concerns about ground water contamination, use of acetochlor is restricted near water sources and on sands, loamy sands, and sandy loam soils with shallow water tables.

Application rates for acetochlor are dependent on the soil type and the type of weeds to be controlled. However, the maximum single use rate for any soil type is 3.0 lb ai/ A, which is also the maximum seasonal use rate. Formulations containing only acetochlor do not specify pregrazing ( PGI) or preharvest intervals ( PHI); however, the multiple active ingredient formulations contain PGI and PHI restrictions that are based on the other AIs included in the Page 9 of 118 formulation. Following application with acetochlor, the labels only allow for rotation to soybeans, corn ( all types), grain sorghum ( milo), wheat, or tobacco.

The ARP has previously agreed to prohibit use in certain areas with ground water that is highly vulnerable to contamination by pesticides such as acetochlor. Acetochlor product use is restricted on coarse textured, low organic matter soils where groundwater is within 30 feet of the surface. The following language is included with all acetochlor product labels:

Acetochlor products may not by applied to the following soils, if depth to groundwater is 30 feet or less:

° Sands with less than 3% organic matter. ° Loamy sands with less than 2% organic matter. ° Sandy loams with less than 1% organic matter. ° Acetochlor herbicides may be applied to the above soils if depth to groundwater is more than 30 feet.

2.2 Structure and Nomenclature The PC Code and nomenclature of acetochlor are listed below in Table 2.2 and the physicochemical properties of acetochlor are listed in Table 2.3 The structure of acetochlor and selected classes of metabolites are presented in Table 3.1. Page 10 of 118 N Cl O O CH 3 C H 3 C H 3 Table 2.2. Acetochlor Nomenclature.

Chemical structure Common name Acetochlor Molecular formula C14H20ClNO2 IUPAC name 2 chloro N ethoxymethyl 6' ethylacet o toluidide CAS name 2 chloro N( ethoxymethyl) N( 2 ethyl 6 methylphenyl) acetamide CAS number 34256 82 1 PC Code 121601 Current food/ feed site registration Corn, field, forage; Corn, field, grain; Corn, field, stover 2.3 Physical and Chemical Properties Table 2.3 Physicochemical Properties of Acetochlor.

Parameter Value Reference Molecular weight 269.77 g/ mol Merck Index Online Boiling point/ range 163 ° C at 10 mm Hg; decomposition occurs before the boiling point at atmospheric pressure; ( calculated by extrapolation of vapor pressure at lower temperature) M. Flood, DEB 7474, 2/ 6/ 91 pH 4.41, 1% solution in acetone: water ( 1: 1, v: v) M. Flood, DEB 7474, 2/ 6/ 91 Density at 20 ° C 1.123 g/ mL M. Flood, DEB 7474, 2/ 6/ 91 Water solubility at 25 ° C 223 mg/ L 2001 Farm Chem Handbook Solvent solubility at 25 ° C Infinitely soluble in acetone, benzene, carbon tetrachloride, ethanol, chloroform, and toluene M. Flood, HED Memo, 1/ 21/ 94 Vapor pressure at 25 ° C 0.045 µ Hg ( 4.5 x 10 5 mm Hg) M. Flood, DEB 7474, 2/ 6/ 91 Dissociation constant, pKa Not applicable because acetochlor is neither an acid nor a base. M. Flood, DEB 7474, 2/ 6/ 91 Octanol/ Water partition coefficient 970 or 1082 M. Flood, DEB 7474, 2/ 6/ 91 UV/ Visible absorption spectrum Not available Page 11 of 118 3.0 Metabolism Assessment 3.1 Comparative Metabolic Profile Table 3.1 lists the acetochlor metabolites discussed in this risk assessment.

Studies in rats indicate that acetochlor is extensively absorbed by the oral route ( at least 70% of the oral dose), and its metabolites undergo extensive enterohepatic circulation. After oral administration, acetochlor and its metabolites undergo extensive distribution throughout the organism, with binding to the nasal olfactory epithelium in rats but not in mice and monkeys.

As summarized in Figure 3.1, acetochlor in rats undergoes extensive biotransformation involving enterohepatic recirculation leading to the precursors of a quinone imine ( e. g. the sulfoxide metabolite, Figure 3.1) which can bind to tissue macromolecules. When the mouse was studied it was found that the major in vivo metabolic route was glucuronidation and excretion of the chloramide. Thus, glutathione conjugation, enterohepatic circulation and formation of quinone imine precursors was not a major metabolic pathway in mice and this interspecies difference is consistent with the absence of nasal tumors in the mouse.

Adequate studies are available on the acetochlor metabolism in plants and on the metabolism of various plant metabolites in ruminants and poultry. The major plant metabolites from studies with 14C acetochlor are EMA containing or HEMA containing metabolites and Metabolite 57 ( See Table 3.1 for Structures). Goats given HEMA containing or EMA containing plant metabolites, or Metabolite 57 show that the HEMA moiety, the EMA moiety, or metabolite 57 are essentially not metabolically transformed ( i. e., the major radioactive residues identified are either EMAcontaining HEMA containing, or Metabolite 57, the test substances for the studies). Hens given plant metabolites containing the EMA moiety in the feeding studies show that transformation to the HEMA moiety occurs. When hens are fed Metabolite 57 the major metabolite identified was the test substance Page 12 of 118 N C CH 2 Cl O CH 2 OC 2 H 5 N C O H CH 2 SG N C O H CH 2 SCH 3 N H H N C O H CH 2 SOCH 3 N H H O H N C O H CH 2 SOCH 3 O H N O H N C O CH 2 SOCH 3 O Acetochlor GSH Conjugate S Methyl metabolite Sulfoxide p Hydroxy EMA p Hydroxy sulfoxide Quinone imine Sulfoxide quinone imine Binding to Tissue Macromolecules Reaction with Tissue Nucleophiles ( e. g. GSH) Binding to Tissue Macromolecules Reaction with Tissue Nucleophiles ( e. g. GSH) 2 Ethyl 6 methylaniline ( EMA)

Path A Path B Figure 3.1. Summary of acetochlor biotransformatiom, leading to either methylethylquinone imine ( MEQI) or methylethylquinone imine sulfoxide in the rat Page 13 of 118 3.2 Nature of the Residue in Foods 3.2.1. Description of Primary Crop Metabolism The qualitative nature of acetochlor residues in plants is understood based on the adequate corn metabolism studies. The HED Metabolism Committee concluded that the regulated residues of concern in corn include parent and any metabolites containing the EMA or HEMA moiety ( See Table 3.1 for Structures), expressed in acetochlor equivalents ( M. Flood, 9/ 30/ 1993). With regards to Metabolite 57, which was identified at slightly higher levels in corn forage and fodder than other metabolites in one metabolism study ( ICI study), the Metabolism Committee concluded that this metabolite need not be included in the tolerance expression after further toxicity testing showed that it was not mutagenic ( A. Protzel, TXR No. 0052813, 8/ 31/ 2004).

Adequate plant metabolism studies have been independently submitted by both Monsanto and ICI. Monsanto's corn and soybean metabolism studies were reviewed in 1984 ( R. Cook, PP# 3F2966, 7/ 20/ 1984) and determined to be adequate. In the earlier corn metabolism study from Monsanto, 14C acetochlor was applied preemergence to greenhouse grown corn at 1.5 lb ai/ A and harvested 3.5 months later at maturity. Total radioactive residues were 0.2 ppm in corn grain and 26.7 ppm in corn foliage. Solvent extraction released 81% and 37% of the TRR from foliage and grain, respectively. Parent was not detected in either grain or foliage extracts. Approximately 65 metabolites were observed in corn grain and foliage with individual metabolites each accounting for < 10% of the TRR. However, upon strong acid hydrolysis, the majority of 14C residues were converted to either 2 ethyl 6 methylaniline ( EMA) or 2 hydroxyethyl 6 methylaniline ( HEMA).

In the later corn metabolism study from ICI, which more closely conforms to current guideline requirements, 14C acetochlor ( EC) was applied preplant to greenhouse grown corn at 2.5 lb ai/ A. Samples of forage were collected 55 days after treatment ( DAT) and samples of grain, fodder, and cobs were collected at maturity ( 134 DAT). Initial TRRs were 4.6 4.7ppm in forage and fodder and 0.06 0.08 ppm in grain and cobs. Acetochlor was not detected in forage, fodder or grain. Analyses of fodder ( 5.27 ppm) identified up to eight EMA type metabolites each accounting for # 5.8% TRR and totaling 23.2% of the TRR, along with isomers of two ringhydroxylated metabolites, Metabolite 55 ( 3.2% TRR) and Metabolite 57 ( 12.7% TRR). Results from forage were similar to fodder. Analyses of grain ( 0.069 ppm) identified up to four EMAtype metabolites each accounting for # 3.6% TRR, along with Metabolites 55 ( 3.0% TRR) and Metabolite 57 ( 8.8% TRR). As in the earlier Monsanto study, no HEMA type metabolites were directly identified, but HEMA metabolites were released by strong base hydrolysis. Analysis of corn commodities using the common moiety method with base hydrolysis did not detect quantifiable residues (< 0.02 ppm) of EMA or HEMA metabolites in grain. However, EMA type metabolites accounted for 27.9% of the TRR in forage and 17.1% of the TRR in fodder, and HEMA metabolites accounted for 4.6 4.8% TRR in forage and fodder. Metabolites 57 and 55 were the single major metabolites in forage and fodder and are likely formed following uptake from the soil of Metabolite 17, N ethoxymethyl N( 2N ethyl 6N methylphenyl) oxamic acid. The fact that these isomers were not identified in Monsanto's earlier corn study was attributed by the Page 14 of 118 registrants to the methyl bromide fumigation of the soil in the earlier study, which may have damaged soil microbes active in acetochlor metabolism. Metabolites 57 and 55 are not detected by the current common moiety method.

3.2.2 Description of Livestock Metabolism The qualitative nature of acetochlor residues in livestock is adequately understood, although the available studies dosing goats and hens with 14C acetochlor are not fully acceptable. Adequate studies have been independently submitted by Monsanto and ICI/ Zeneca examining the metabolism of various plant metabolites ( EMA type, HEMA type, and Metabolite 57) in both ruminants and poultry. Based on these studies, the Agency concluded that acetochlor residues in ruminants and poultry include EMA and HEMA type metabolites and Metabolite 57. The HED Metabolism Committee ( M. Flood, 9/ 30/ 1993) concluded that tolerances are not required for livestock commodities to support the use on corn.

Ruminants. The available ruminant metabolism studies summarized below include: two studies from Monsanto and ICI/ Zeneca dosing goats with 14C EMA type metabolites ( MRIDs 00118950 and 41963323); a study from Monsanto dosing goats with an 14C HEMA type metabolite ( MRID 41633601); a study from ICI/ Zeneca dosing a cow with 14C Metabolite 57 ( MRID 42549905); and two studies from ICI/ Zeneca dosing goats with 14C acetochlor ( MRIDs 41565157 and 42549903). Based on the recommended tolerance of 3.0 ppm for field corn forage, the maximum theoretical dietary burden for dairy cattle is 3.77 ppm for the combined residues of EMA type and HEMA type metabolites.

In the earliest Monsanto study, two goats were dosed for five days with a combination of four 14C ring labeled, EMA type metabolites at a level equivalent to approximately 20 ppm in the diet. TRR were 0.007 ppm in milk, 0.025 ppm in kidneys, 0.046 ppm in liver and < 0.001 ppm in muscle and fat. Analysis of urine and feces using a common moiety method indicated that EMAtype metabolites accounted for 83% and 77% of the radioactivity in urine and feces, respectively.

In the similar ICI/ Zeneca study, goats were dosed for seven days with a combination of four 14C ring labeled, EMA type metabolites at a levels equivalent to 10 ppm ( two goats) or 50 ppm ( one goat) in the diet. For the goats dosed at 10 ppm, TRR were # 0.003 ppm in milk, 0.008 ppm in kidneys, 0.136 ppm in liver, 0.006 ppm in muscle, and < 0.005 ppm in fat. For the goat dosed at 50 ppm, TRR were 0.013 0.014 ppm in milk, 0.160 ppm in kidneys, 0.097 ppm in liver, 0.010 ppm in muscle, and < 0.005 ppm in fat. Milk and tissue samples from the 50 ppm goat were extracted for analysis releasing 54 100% of the TRR, and the resulting extracts were analyzed for EMA and HEMA residues using the common moiety method. EMA type metabolites were detected at 0.04 and 0.13 ppm in liver and kidney, respectively, and were < 0.01 ppm in fat, muscle, and milk. HEMA type metabolites were < 0.01 ppm in milk and all tissues. Analysis of urine using a common moiety method indicated that the majority of residues were EMA type metabolites ($ 83% TRR); HEMA type metabolites accounted for # 1% of the radioactivity in urine indicating that oxidation of EMA to HEMA metabolites was limited. Page 15 of 118 In conjunction with a feeding study conducted by Monsanto, four goats were dosed for 28 days with a 14C ring labeled, HEMA type metabolite at a levels equivalent to 0.5, 1.5, or 5 ppm in the diet. At each dosing level, TRR were < 0.001 ppm in milk and tissues. Analysis of milk and tissues using a common moiety method did not detect (< 0.01 ppm) any EMA or HEMA residues. The metabolism of the major oxamic acid plant metabolite was examined in a study from ICI/ Zeneca in which a single cow was dosed with 14C ring labeled Metabolite 57 for seven days at a level equivalent to approximately 25 ppm in the diet. TRR levels peaked on Day 6 in milk at 0.0063 ppm, and TRR in tissues were < 0.004 ppm in muscle, # 0.004 ppm in fat, 0.008 ppm in liver, and 0.015 ppm in kidneys. Metabolite 57 comprised 80% of the TRR in urine and 88% of the TRR in feces, and was the only 14C residue identified in kidneys at 43% of the TRR.

In the first ICI/ Zeneca study examining the direct metabolism of 14C acetochlor in ruminants, goats were dosed for four days with 14C ring labeled acetochlor at levels equivalent to either 1 ppm ( two goats) or 90 ppm ( one goat) in the diet. At both dose levels, the majority of the dose was excreted in the urine ( 57 62% dose) and feces ( 21 25% dose). For the high dose goat, TRR were 0.15 0.19 ppm in milk, 4.55 ppm in liver, 4.16 ppm in kidneys, 0.23 0.25 ppm in muscle, and 0.10 ppm in fat. Characterization was attempted in urine, liver, and kidneys, but milk, muscle, and fat samples were not further analyzed. Acetochlor was not detected (< 0.1% TRR) in any samples. The principal 14C residue identified was the cysteine conjugate of acetochlor ( Metabolite 44) at 37%, 15%, and 18% of the TRR in urine, liver, and kidneys, respectively.

In the subsequent 14C acetochlor metabolism study, two goats were dosed for four days with 14C ring labeled acetochlor at levels equivalent to 10 ppm in the diet. TRR in milk plateaued within two days at 0.016 ppm, and TRR at sacrifice were 0.588 ppm in liver, 0.479 ppm in kidneys, 0.020 0.024 ppm in muscle, and # 0.008 ppm in fat. Characterization was attempted in urine, feces, milk, muscle, liver, and kidneys. Acetochlor was detected at low levels ( 0.8% TRR) in feces, but was not detected in urine, milk, or tissues. Metabolite 44 was identified as the principal 14C residue in urine ( 23.6% TRR) and milk ( 18.6% TRR). The remaining components in urine and milk each accounted for < 5% of the TRR. No metabolites were identified in muscle, liver or kidney. Analysis of muscle, kidney, and liver using the common moiety method detected residues of EMA and HEMA type metabolites at combined levels of 0.007 ppm in muscle, 0.04 ppm in kidney and 0.08 ppm in liver.

The Agency noted that both 14C acetochlor goat metabolism studies are inadequate due to insufficient characterization of 14C residues in liver and kidneys.

Poultry. The available poultry metabolism studies summarized below include: two studies from Monsanto and ICI/ Zeneca dosing hens with 14C EMA type metabolites ( MRIDs 40365602 and 41963324); a study from ICI/ Zeneca dosing hens with 14C Metabolite 57 ( MRID 42549904); and a study from ICI/ Zeneca dosing hens with 14C acetochlor ( MRIDs 41565158 and 41565159). Based on reassessed tolerances, the maximum theoretical dietary burden for poultry is 0.044 ppm for the combined residues of EMA and HEMA type metabolites. Page 16 of 118 In the first Monsanto study, groups of hens were dosed for six days with a combination of four 14C ring labeled, EMA type metabolites at a levels equivalent to either 15 or 100 ppm in the diet. At both dosing levels, 95 98% of the administered dose was recovered in the excreta and 0.02% of the dose was recovered in eggs. For the 15 ppm dose groups, maximum TRR levels were 0.009 ppm in egg whites, 0.027 ppm in yolks, 0.010 ppm in muscle, 0.045 ppm in liver and 0.005 ppm in fat. For the 100 ppm dose groups, TRR levels were 0.107 ppm in egg whites, 0.173 ppm in yolks, 0.032 ppm in muscle, 0.266 ppm in liver and 0.007 ppm in fat. Eggs and tissues were analyzed using the common moiety method. EMA type metabolites accounted for 63% of the TRR in liver, 20 48% of the TRR in muscle, and 86% of the TRR in fat. Analyses of muscle also detected HEMA type metabolites at 10 1% of the TRR and 2( 1 hydroxyethyl 6 hydroxymethylaniline ( HEHMA) type metabolites at 3 6% of the TRR. Analyses of egg whites detected both EMA and HEMA type metabolites together accounting for 45 52% of the TRR, with the HEMA type metabolites being 2 4x more abundant than the EMA type metabolites. Analyses of egg yolks detected both EMA ( 65% TRR) and HEMA ( 27% TRR) type metabolites together accounting for 92% of the TRR.

In the ICI/ Zeneca study, a group of hens was dosed for seven days with a combination of four 14C ring labeled, EMA type metabolites at a level equivalent to 10 ppm in the diet. Excreta and cage wash together accounted for 92% of the administered dose. The mean TRR in egg whites was 0.006 ppm ( Days 3, 5 and 7), with a single highest value of 0.009 ppm from one sample on Day 7. The mean TRR in egg yolks on Day 7 was 0.015 ppm, with a maximum of 0.017 ppm. Maximum TRR levels were 0.013 ppm in kidney, 0.031 ppm in liver, 0.002 ppm in muscle, and 0.010 ppm in fat. Samples of liver, egg yolks, and excreta were analyzed using the common moiety method. In excreta, EMA and HEMA type metabolites accounted for 84% and 8% of the TRR, respectively. EMA type metabolites were also detected at 0.015 ppm in yolks and 0.01 ppm in liver, but HEMA type metabolites were not detected.

In the ICI/ Zeneca study examining metabolism of Metabolite 57, a group of hens were dosed for 10 days with 14C ring labeled Metabolite 57 at a level equivalent to 10 ppm in the diet. Radioactivity in excreta accounted for > 98% of the administered dose. Maximum residues in eggs yolks were attained on Day 8 at 0.016 ppm; TRR in egg whites were generally < 0.003 ppm although a value of 0.007 ppm was obtained from one sample. Maximum TRR in tissues were 0.025 ppm in fat, 0.005 ppm in muscle, and 0.006 ppm in liver. Samples of excreta, fat and egg yolks were used for analysis. Metabolite 57 comprised 77% of the TRR in excreta, 41% of the TRR in fat, and 36% of the TRR in egg yolks. No other components were identified.

In the ICI/ Zeneca study examining the direct metabolism of 14C acetochlor, two group of hens were dosed for four days with 14C ring labeled acetochlor at levels equivalent to either 1 or 90 ppm in the diet. At both dose levels, the majority of the dose was recovered in the excreta and cage wash ( 76 89% dose). For hens dosed at 1 ppm, TRR were < 0.004 ppm in eggs, 0.041 0.055 ppm in liver, 0.053 0.062 ppm in kidneys, 0.004 0.006 ppm in muscle, and < 0.010 ppm in fat. For hens dosed at 90 ppm, TRR were 0.16 ppm in eggs ( Day 4), 2.48 5.13 ppm in liver, 3.48 7.48 ppm in kidneys, 0.30 0.63 ppm in muscle, and 0.16 0.46 ppm in fat. Characterization Page 17 of 118 was attempted only on liver and excreta. Acetochlor was detected at 5.6% of the TRR in liver, and analysis using the common moiety method indicated that EMA type metabolites accounted for 12% of the TRR. In excreta, the principal 14C residue identified was N( 2 ethyl 6 methylphenyl) oxamic acid ( Metabolite 27) at 10% of the TRR. Analysis of excreta using the common moiety method detected EMA type metabolites at 19% of the TRR. This study was deemed inadequate due to insufficient characterization of 14C residues.

3.2.3 Description of Rotational Crop Metabolism, including identification of major metabolites and specific routes of biotransformation The requirement for confined accumulation in rotational crops is satisfied. The requirements for field accumulation in rotational crops are satisfied.

Confined Accumulation in Rotational Crops. The requirement for confined accumulation in rotational crops is satisfied ( Guideline 860.1850). Following an application of 14C acetochlor to a sandy loam soil at 3.0 lb ai/ A ( 1x rate), TRR were > 0.01 ppm in all commodities of lettuce, radish, and wheat that were planted 30, 120, and 365 DAT, with 14C residues generally being the highest at the 120 day plant back interval ( PBI). Extraction and analysis of the rotational crop commodities identified or characterized $ 63% of the TRR in each commodity from each interval. Acetochlor was detected only in radish foliage from one interval ( 120 DAT) accounting for 4% of the TRR ( 0.03 ppm). A total of 17 metabolites were identified in rotational crops, including 12 EMA producing metabolites, 3 HEMA producing metabolites, and 2 HMEA producing metabolites. In each commodity, the EMA producing metabolites were present at the highest concentrations, followed by the HEMA and HMEA type metabolites, respectively. Metabolism in the rotational crops was similar to corn except that HMEA type metabolites were not observed in corn, and Metabolite 57 was not observed in rotational crops, possibly of the same reason indicated for one of the earlier corn studies.

Based on the above results and the data from the extensive rotational crop field trials discussed below, the HED MARC concluded that tolerances for rotational crops should be expressed as acetochlor and its EMA and HEMA producing metabolites ( M. Flood, MARC Memorandum, 9/ 30/ 93). Residues of HMEA containing metabolites, expressed in acetochlor equivalents, should not be included in the tolerance expression, but will be used for purposes of risk assessment.

Field Accumulation in Rotational Crops. The requirements for field accumulation in rotational crops are satisfied ( Guideline 860.1900). The available extensive rotational crop field trials are adequate and support the current tolerances on sorghum, soybean, and wheat commodities. Labels for EPs registered to members of the ARP currently include the following rotational crop restriction: do not rotate to crops other than soybeans, corn ( all types), milo ( sorghum), wheat or tobacco.

Extensive rotational field crop trials were conducted on sorghum, soybean, and wheat rotated Page 18 of 118 with treated corn. Acetochlor ( EC) was applied as a single broadcast preemergence application to a primary crop of corn at 3 lb ai/ A ( 1x maximum rate) at 19 test locations throughout the corn growing regions of the U. S. After corn harvest, rotational crops of winter wheat, soybeans, and sorghum were planted on the test plots at PBIs of 3.0 5.9 months for wheat and 10.4 14.2 months for soybeans and sorghum. Duplicate samples of sorghum, soybean, and wheat commodities were collected from each test at the appropriate harvest intervals.

Samples were analyzed for residues of acetochlor and its EMA, HEMA, and HMEA containing metabolites using an adequate HPLC/ OCED method. The LOQ for each analyte is 0.01 ppm, expressed in acetochlor equivalents. Residue values were corrected for average concurrent recoveries. The field trials are supported by the available storage stability data. For all three rotational crops, concentrations of EMA type metabolites were highest, followed by HEMA and HMEA type metabolites, respectively.

For rotated wheat, the combined residues of EMA, HEMA, and HMEA yielding compounds, expressed in acetochlor equivalents, were < 0.03 ppm (< LOQ) in 38 samples of grain, < 0.03 0.531 ppm in 44 forage samples, and < 0.03 0.124 ppm in 38 straw samples. Considering only the currently regulated EMA and HEMA containing compounds, maximum residues were < 0.02 ppm in grain, 0.457 ppm in forage, and 0.104 ppm in straw. No data were provided on hay, which is a RAC of wheat.

For rotated sorghum, the combined residues of EMA, HEMA, and HMEA yielding compounds were < 0.03 ppm (< LOQ) in 34 samples of grain, < 0.03 0.103 ppm in 36 forage samples, < 0.03 0.082 ppm in 34 fodder ( stover) samples, < 0.03 0.206 ppm in 34 hay samples, and < 0.03 0.068 ppm in 36 silage samples. Considering only the currently regulated EMA and HEMA containing compounds, maximum residues were < 0.02 ppm in grain, 0.093 ppm in forage, 0.068 ppm in fodder, 0.186 ppm in hay, and 0.057 ppm in silage.

For rotated soybeans, the combined residues of EMA, HEMA, and HMEA yielding compounds were < 0.03 0.128 ppm in 32 samples of seed, < 0.03 0.769 ppm in 36 forage samples, and < 0.034 1.217 ppm in 32 hay samples. Considering only the currently regulated EMA and HEMA containing compounds, maximum residues were 0.101 ppm in seed, 0.648 ppm in forage, and 1.064 ppm in hay.

3.3 Environmental Degradation The major routes of dissipation for acetochlor appear to be microbially mediated degradation, runoff, and leaching. Although acetochlor generally degrades rapidly when applied to soil, in some field situations it can be relatively persistent ( e. g. field dissipation half lives were up to 36 days) and it has been found in groundwater at numerous locations. There is variable evidence as to the persistence of acetochlor in subsoil horizons with a published study by the registrant reporting only a modest increase in persistence from surface soils at two sites using in situ Page 19 of 118 N Cl O O CH 3 C H 3 C H 3 N R2 O R1 CH 3 C H 3 methods ( Mills et al. 2001). Lavy et al. ( 1996) have reported a much more substantial increase in persistence at two sites for alachlor, a herbicide that is chemically related to acetochlor and tends to have a very similar environmental fate profile.

Laboratory degradation data indicate that acetochlor does not degrade by abiotic processes ( hydrolysis and photolysis). While acetochlor has relatively short half lives in fine textured aerobic soil, it may be moderately persistent in coarser soils ( this may be related to the lower rate of microbial activity in sandy, low organic matter soils).

The aerobic soil metabolism degradates oxanilic acid ( oxamic acid), and sulfonic acid are the major environmental degradates of acetochlor. Another metabolite is thioacetic acid sulfoxide.

3.4 Tabular Summary of Metabolites and Degradates Table 3.1. presents presents the structures of major residues and environmental degradates of acetochlor. Acetochlor ESA and acetochlor OXA are environmental degradates. All other compounds in Table 3.1 were reported in plants or animals.

Table 3.1 Chemical Names and Structures of Acetochlor and Major Metabolites and Degradates.

Company Name ( Codes) Chemical Name Structure 1 Acetochlor MON 097 2 chloro N( ethoxymethyl) N( 2 ethyl 6 methylphenyl) acetamide EMA type metabolites N/ A = not applicable Table 3.1 Chemical Names and Structures of Acetochlor and Major Metabolites and Degradates.

Company Name ( Codes) Chemical Name Structure 1 Page 20 of 118 N R2 O R1 CH 3 C H 3 OR 3 N R2 O R1 CH 2 OH C H 3 N H O COOH C H 3 CH 3 OH N CH 3 SO 3 H O O CH 3 C H 3 HEMA type metabolites N/ A HMEA type metabolites N/ A Metabolite 57 N( 6 ethyl 3 hydroxy 2 methylphenyl) oxamic acid Table 3.1 Chemical Names and Structures of Acetochlor and Major Metabolites and Degradates ( Contd).

Company Name ( Codes) Chemical Name Structure 1 Acetochlor ESA MON 52754 R290131 Acetochlor ethane sulfonic acid ( ESA) Table 3.1 Chemical Names and Structures of Acetochlor and Major Metabolites and Degradates ( Contd).

Company Name ( Codes) Chemical Name Structure 1 Page 21 of 118 N CH 3 O CH 3 C H 3 O O H O Acetochlor OXA MON 52755 R290130 Acetochlor oxanilic acid ( OXA)

1. For the EMA, HEMA, and HMEA type metabolites the R1 functional group can consist of  H or  CH2OCH2CH3 and the R2 functional group can consist of many different moieties. The R3 functional for HEMA type metabolites can consist of  H or a variety of components such as sugars.

3.5 Toxicity Profile of Major Metabolites and Degradates Based on their structures, the Metabolism Assessment Review Committee ( MARC) was unable to conclude that the metabolites in raw agricultural commodities ( RACs) containing the EMA, HEMA or HMEA moieties ( See Table 3.1) will be significantly less toxic than the parent and therefore recommended that for risk assessment purposes the residues of concern in plants are the parent and the metabolites containing the EMA or HEMA moiety. In rotational crops, for risk assessment purposes, the residues of concern are the parent and the metabolites containing the EMA, HEMA, or HMEA moiety.

In the case of water, the MARC concluded, based on toxicity data, that the water degradates acetochlor ESA and acetochlor OXA are significantly less toxic than the parent and should not be included in the risk assessment. The ESA and OXA degradates have been monitored in water samples ( both groundwater and surface water)  and in many cases were found at concentrations higher than the parent acetochlor. Thus, to support the MARC's conclusion on the water degradates, Margin of Exposure ( MOE) calculations were done to estimate acute and chronic risk of acetochlor ESA and acetochlor OXA ( See Appendix 2).

3.5.1 Summary of Available Toxicity Studies on Acetochlor Degradates The toxicity data used to evaluate the relative toxicities of the ESA degradate relative to acetochlor is presented below in Table 3.2. Data for the OXA degradate is presented in Table 3.3. Mutagenicity data for the ESA and OXA degradates ( and a mixture of compounds 57 and 55) are presented below in Table 3.4:

Table 3.2. Comparison of Toxicity of Acetochlor and Acetochlor ESA Page 22 of 118 Test Acetochlor Acetochlor ESA Acute oral LD50 LD50( M& F) = 4124 ( 3557 4691) mg/ kg ( MRID 41565104) LD50( M& F) > 2000 mg/ kg ( no mortality at limit dose) ( MRID 44632704)

Metabolism > 80% oral absorption, extensive biotransformation, precursors of quinonone imine, binding to nasal turbinates ( MRIDs 41565125,  26,  27; 41592007,  08; 44496203,  10,  12) Poorly absorbed by the oral route ( about 10 12% of the dose). Limited biotransformation ( 75 79%) excreted untransformed. Does not bind to nasal turbinates. ( MRID 45300504)

4 Week feeding range finding ( ESA)/ thyroid mechanistic ( acetochlor) ( rats) Males administered acetochlor in diet at 100.6 or 280.9 mg/ kg/ day for 14, 28 or 56 days: increased liver and thyroid weights, hepatic UDPGT activity, circulating serum TSH, T4; decreased T3, some or all time points. Males administered acetochlor at 10.4, 91.9 or 270.3 mg/ kg/ day for 90 or 160 days: increased liver weight ( slight increase in thyroid wt) at 91.9 mg/ kg/ day, increased hepatic UDPGT at 270.3 mg/ kg/ day. ( MRID 44496208) NOAEL = 370.3 / 374.6 mg/ kg/ day [ M/ F] LOAEL = 766.6/ 762.3 mg/ kg/ day [ M/ F], based on decreased body weights and body weight gain and increased TSH and free T3 in males. At 1578.7 mg/ kg/ day ( HDT) there was a statistically significant increase in T4 UDPGT ( microsomal enzyme) ( MRID 45300503)

90 Day feeding ( rats) Study 1: NOAEL = 16.1 / 19.1 mg/ kg/ day [ M/ F] LOAEL = 161 / 191 mg/ kg/ day [ M/ F] based on hematology, small but significant increases relative liver, kidney & brain weights. ( MRID 41565115) Study 2: NOAEL = 40 mg/ kg/ day LOAEL = 100 mg/ kg/ day, based on body weight loss & food consumption. ( MRID 00050933) NOAEL = 225.4 / 259.1 mg/ kg/ day [ M/ F] LOAEL = 919.4 / 1073.2 mg/ kg/ day [ M/ F], based on reduced body weights, body weight gains and food utilization in both sexes. Decreased cell proliferation in nasal passages was seen at the LOAEL, but not statistically significantly diff. with controls because of variability. ( MRID 45313801)

Developmental toxicity Study 1( rats): NOAEL( maternal & developmental) = 150 mg/ kg/ day, LOAEL ( mat & dev) = 600 mg/ kg/ day; Maternal: based on clinical signs & decr. b. wt. gain. Developmental: based on incr. resorptions & decr. fetal body weights ( MRID 41592005) Study 2 ( rats): NOAEL( maternal & developmental) = 200 mg/ kg/ day, LOAEL ( mat & dev) = 400 mg/ kg/ day; Maternal: based on clinical signs & decr. b. wt. gain. Developmental: based on decr. fetal body weights. ( MRID 00050929) No data for Acetochlor ESA. Data are for Alachlor ESA. Rats: NOAEL ( maternal & developmental): greater than or equal to 900 ( HDT) mg/ kg/ day LOAEL ( maternal & developmental): greater than 900 mg/ kg/ day ( MRID 43908101)

Mutagenicity No Concern ( See Table 3.4) No Concern ( See Table 3.4)

Table 3.3. Comparison of toxicity of Acetochlor and Acetochlor OXA Test Acetochlor Acetochlor OXA Acute oral LD50 LD50( M& F) = 4124 ( 3557 4691) mg/ kg ( MRID 41565104) LD50( M& F) > 2000 mg/ kg ( MRID 44632703) Page 23 of 118 Metabolism Rats: > 80% oral absorption, extensive biotransformation, precursors of quinone imine, binding to nasal turbinates ( MRIDs 41565125,  26,  27 ; 41592007,  08; 44496203,  10,  12) Rats: About 33.9 38.6% of the dose absorbed by the oral route ( as seen in urine) Limited biotransformation: ( 81.4 84.9% excreted untransformed. Two unidentified metabolites ( 5 & 2% of dose, resp. ). Does not bind to nasal turbinates. No data for mouse. ( MRID 45300507)

4 Week feeding range finding ( OXA)/ mechani stic thyroid toxicity ( acetochlor) ( rats) Males administered acetochlor in diet at 100.6 or 280.9 mg/ kg/ day for 14, 28 or 56 days: increased liver and thyroid weights, hepatic UDPGT activity, circulating serum TSH, T4; decreased T3, some or all time points. Males administered acetochlor at 10.4, 91.9 or 270.3 mg/ kg/ day for 90 or 160 days: increased liver weight ( slight increase in thyroid wt) at 91.9 mg/ kg/ day, increased hepatic UDPGT at 270.3 mg/ kg/ day. ( MRID 44496208) NOAEL = 372.6 / 367.2 mg/ kg/ day [ M/ F] LOAEL = 768.5 / 737.3 mg/ kg/ day [ M/ F], based on decr. TSH & T3 and incr. absolute & relative thyroid weights in males and decr. TSH & T3 in females. ( MRID 45300506)

90 Day feeding ( rats) Study 1: NOAEL = 16.1 / 19.1 mg/ kg/ day [ M/ F] LOAEL = 161 / 191 mg/ kg/ day [ M/ F] based on hematology, small but significant increases relative liver, kidney & brain weights. ( MRID 41565115) Study 2: NOAEL = 40 mg/ kg/ day LOAEL = 100 mg/ kg/ day, based on body weight loss & food consumption. ( MRID 00050933) NOAEL = 230.2 / 268.0 mg/ kg/ day [ M/ F] LOAEL = 955.2/ 1082.7 mg/ kg/ day [ M/ F], based on reduced body weights, body weight gains and food utilization in both sexes. Thyroid weight increases were not seen in this study. T4 UDPGT activity was slightly, but not significantly, increased in high dose males and was statistically significantly decreased in high dose females ( MRID 45313805)

Developmental toxicity Study 1( rats): NOAEL( maternal & developmental) = 150 mg/ kg/ day, LOAEL ( mat & dev) = 600 mg/ kg/ day; Maternal: based on clinical signs & decr. b. wt. gain. Developmental: based on incr. resorptions & decr. fetal body weights. ( MRID 41592005) Study 2 ( rats): NOAEL( maternal & developmental) = 200 mg/ kg/ day, LOAEL ( mat & dev) = 400 mg/ kg/ day; Maternal: based on clinical signs & decr. b. wt. gain. Developmental: based on decr. fetal body weights ( MRID 00050929) Rats: NOAEL ( maternal) = 500 mg/ kg/ day, LOAEL ( maternal ) = 1000 mg/ kg/ day, based on maternal mortality. NOAEL ( developmental) is equal or greater than 1000 mg/ kg/ day ( limit dose). LOAEL ( developmental) greater than 1000 mg/ kg/ day ( MRID 45313807)

Mutagenicity No Concern ( See Table 3.4) No Concern ( See Table 3.4) Page 24 of 118 Table 3.4. MUTAGENICITY STUDIES WITH ACETOCHLOR WATER DEGRADATES Test System MRID ( year) Purity Batch No. Result Acetochlor ethane sulfonic acid ( ESA)

870.5100 Bacterial Gene Mutation Assay( plate or preincubation) Salmonella typhimurium, Escherichia coli 44632706 ( 1997) 97.6% R290131 Acceptable/ guideline 100 5000 µ g/ plate  /+ S9 plate or preincubation Negative up to the limit dose ( 5000 µ g/ plate /+ S9) plate or preincubation 870.5300 In vitro mammalian cell gene mutation L5178Y mouse lymphoma cells 45313803 ( 2000) 97.6% R290131 Acceptable/ guideline 250 3010 µ g/ mL ( equiv to 10mM) +/ S9; two trials Negative up cytotoxic levels ( 3010 µ g/ mL /+ S9).

870.5375 Cytogenetics In vitro mammalian cell chromosomal aberration assay human lymphocytes 45313804 ( 2000) 97.6% R290131 Acceptable/ guideline 250 3010 µ g/ mL ( equiv to 10mM) +/ S9; two trials Negative up to the limit dose, 3010 µ g/ mL ( equiv to 10mM) +/ S9 870.5395 Mammalian Erythrocyte Micronucleus Test CD 1 Mice 45313802 ( 2000) 97.6% R290131 Acceptable/ guideline 0, 500, 1000, 2000 mg/ kg oral gavage 1X Negative up to the limit dose ( 2000 mg/ kg).

Acetochlor oxanilic acid ( OXA)

870.5100 Bacterial Gene Mutation Assay( plate or preincubation) Salmonella typhimurium, Escherichia coli 44632705 ( 1998) 97% R290130 Acceptable/ guideline 100 5000 µ g/ plate  /+ S9 plate or preincubation Negative up to the limit dose ( 5000 µ g/ plate /+ S9) plate or preincubation 870.5300 In vitro mammalian cell gene mutation L5178Y mouse lymphoma cells 45313809 ( 2000) 93.15% R290130 Acceptable/ guideline 250 2650 µ g/ mL ( equiv to 10mM) +/ S9; two trials Negative up cytotoxic levels or the limit dose ( 2650 µ g/ mL). Positive: S 8in MF at 2000 & 2650 µ g/ mL + S9 both trials  outside of historical control range. Predominantly small colony mutants MUTAGENICITY STUDIES WITH ACETOCHLOR WATER DEGRADATES Test System MRID ( year) Purity Batch No. Result Acetochlor oxanilic acid ( OXA) Table 3.4. MUTAGENICITY STUDIES WITH ACETOCHLOR WATER DEGRADATES Test System MRID ( year) Purity Batch No. Result Page 25 of 118 870.5375 Cytogenetics In vitro mammalian cell chromosomal aberration assay human lymphocytes 45313810 ( 2000) 93.15% R290130 Acceptable/ guideline 250 2650 µ g/ mL ( equiv to 10mM) +/ S9; two trials Negative up to the limit dose ( 2650 µ g/ mL /+ S9).

870.5395 Mammalian Erythrocyte Micronucleus Test CD 1 Mice 45313808 ( 2000) 93.15% R290130 Acceptable/ guideline 0, 500, 1000, 2000 mg/ kg oral gavage 1X Negative up to a the limit dose ( 2000 mg/ kg).

Compound 57 870.5375 Cytogenetics In vitro mammalian cell chromosomal aberration assay human lymphocytes 43785702 ( 1995) 99% P3 Acceptable/ guideline 500 2500 µ g/ mL ( Donor 1) 200 2500 µ g/ mL ( Donor 2) +/ S9 Negative up to cytotoxicity ( 2500 µ g/ mL S9) or the highest tested level w S9 ( 2500 µ g/ mL) .

In vivo/ in vitro unscheduled DNA synthesis Alderley Park ( Alpk: APfSD) rats 43785701 ( 1995) 99% P3 Acceptable/ nonguideline 1250 or 2000 mg/ kg oral gavage 1X Negative up to a the limit dose ( 2000 mg/ kg).

PJ 2 ( mixture of compound 57 and its 3 OH isomer, compound 55)

870.5100 Bacterial Gene Mutation Assay( plate or preincubation) Salmonella typhimurium, Escherichia coli 42713118 ( 1991) 96% ASW 1351 R Acceptable/ guideline 100 5000 µ g/ plate  /+ S9 plate or preincubation Negative up to the limit dose ( 5000 µ g/ plate /+ S9) plate or preincubation S = Significant ( p< 0.01) NS= Nonsignificant MF = Mutation frequency 3.6 Summary of Residues for Tolerance Expression and Risk Assessment Summarizing, MARC has determined that the tolerance expression for residues in/ on corn and rotational crop commodities should include only acetochlor and its metabolites containing the EMA or HEMA moiety, expressed in acetochlor equivalents ( M. Flood, MARC Memorandum, 9/ 30/ 93). For purposes of the dietary risk assessment, residues in/ on rotational crops should also include metabolites containing the HMEA moiety, expressed in acetochlor equivalents. The HED MARC also determined that parent only in drinking water should be considered in the dietary risk assessment ( A. Protzel, MARC Memorandum, 8/ 31/ 2004). Table 3.5 is a summary of the HED MARC decisions concerning the residues of concern in plants, rotational crops, and drinking water. Page 26 of 118 3.6.1 Tabular Summary for Residues for Tolerance Expression and Risk Assessment Table 3.5. Residues for Tolerance Expression and Risk Assessment.

Matrix Residues included in Risk Assessment Residues included in Tolerance Expression Plants  field corn parent and metabolites containing the EMA or HEMA moiety parent and metabolites containing the EMA or HEMA moiety Rotational Crops  sorghum, soybean, and wheat parent and metabolites containing the EMA, HEMA, or HMEA moiety parent and metabolites containing the EMA or HEMA moiety Drinking Water parent only parent only 3.6.2 Rationale for Inclusion of Metabolites. Based on their structures, the MARC was unable to conclude that the metabolites in raw agricultural commodities ( RACS) containing the EMA, HEMA or HMEA moieties ( See Table 3.1) will not be significantly less toxic than the parent and therefore recommended that these residues be included in the risk assessment, as summarized in Table 3.5.

3.6.3 Rationale for Exclusion of Degradates The HED MARC met on May 13, 2004 to consider whether the ESA and OXA degradates of acetochlor should be included in the dietary drinking water assessment, based on the available toxicity data ( TXR No. 0052813). Based on consideration of the data ( summarized in Tables 3.2 3.4) and structure activity relationships, the MARC concluded that: ( 1) the ESA and OXA degradates of acetochlor should not be included in the water risk assessment with the parent. The weight of the evidence determination was based on the following rationales for carcinogenic potential and non carcinogenic toxicity:

Carcinogenicity: In contrast to acetochlor, both the OXA and ESA degradates show the following characteristics that would make them unlikely to be carcinogenic:

( 1) are highly polar compounds showing poor to limited absorption; the majority of the administered dose is excreted without biotransformation; ( 2) are nonmutagenic; ( 3) lack reactive chlorine; ( 4) lack the capacity to form a quinoneimine species leading to nasal tumor formation; ( 5) do not show thyroid and liver effects indicating potential to disrupt thyroid pituitary homeostasis Non carcinogenicity toxicity assessment: The toxicity data on the OXA and ESA degradates indicated that they are of lower anticipated toxicity than the parent acetochlor based on the following data: Page 27 of 118 ( 1) are highly polar compounds showing poor to limited absorption, with the majority excreted untransformed; ( 2) subchronic toxicity in the rat is lower than acetochlor ( 3) developmental toxicity is not observed in the rat.

Given that both the ESA and OXA degradates have been detected in water samples ( both groundwater and surface water)  in many cases at concentrations higher than the parent acetochlor  worst case non carcinogenic margin of exposure ( MOE) calculations were done for the two water degradates to better support the MARC conclusion. The values were found to be not of HED concern. The detailed results are presented in Appendix 2.

4.0 Hazard Characterization/ Assessment 4.1 Hazard and Dose Response Characterization 4.1.1 Database Summary <IP_ADDRESS> Studies available and considered Acute oral neurotoxicity, rat; LD50 ( oral and dermal), rat; LC50 ( inhalation), rat; primary dermal/ eye irritation, rabbit; dermal sensitization, guinea pig. Subchronic oral toxicity, rat ( 2); oral, dog ( 2); 21 day dermal toxicity, rabbit and rat; oral neurotoxicity, rat. Chronic and/ or carcinogenicity oral toxicity, rat ( 3 combined chronic toxicity/ carcinogenicity); oral toxicity, dog ( 2); oral carcinogenicity, mouse ( 2). Reproductive/ developmental oral developmental toxicity, rat ( 2), oral developmental toxicity, rabbit ( 2); oral multigeneration reproductive toxicity, rat ( 3). Other general metabolism, rat ( 2); genotoxicity screening studies, all categories; dermal penetration, rat; special nonguideline mechanistic studies provided on the mechanism of nasal, thyroid and liver tumor formation in the rat; pathology working group ( PWG) reevaluations of tumors.

4.1.2 Mode of Action, Metabolism, Toxicokinetic Data Acetochlor is a chloroacetanilide herbicide related to alachlor, butachlor, propachlor and metolachlor. These structurally similar compounds show an overlapping, but not identical, tumor profile. On March 19, 1997, this group of compounds was presented as a case study for evaluation of a common mechanism of action to the OPP Science Advisory Panel ( SAP) as part of the presentation on methodology for evaluation of cumulative toxicity ( EPA, 1997, 2001). The Panel determined that there was sufficient evidence to support a common mode of action of acetochlor, alachlor and butachlor for nasal olfactory epitheilial and thyroid follicular cell tumorigenesis. The SAP also recommended that only the nasal tumors and not thyroid tumors Page 28 of 118 should be included in the cumulative assessment. Numerous mechanistic studies on acetochlor have been submitted in support of the proposed mechanisms. The HED MTARC/ CARC reviewed the mechanistic data on these two tumors for acetochlor on April 21 22, 2004 and concurred that it supported a non genotoxic mode of action for formation of the nasal olfactory epithelial and thyroid follicular cell tumors ( HED TXR# 0052727).

In the rat, acetochlor is highly metabolized and rapidly excreted, primarily in the urine. Increased levels of radioactivity are excreted in the feces with increased dose. Radioactivity is not retained in tissues to a significant extent, but does show binding to red blood cells and to nasal olfactory epithelium and is found in at higher levels in highly perfused organs. The major urinary metabolite of acetochlor is a mercapturic acid conjugate of N deethylated acetochlor. Biliary excretion is also significant and the major biliary metabolite is a glucuronide conjugate.

Mode of action for nasal olfactory epithelial cell tumorigenesis: The formation of nasal tumors in the rat is considered a non genotoxic event that occurs secondary to binding of a cytotoxic benzoquinoneimine metabolite. This metabolite is formed in situ by the metabolizing enzymes of the nasal olfactory epithelial cells from circulating metabolites of acetochlor that are delivered to the nasal olfactory epithelial cells. The benzoquinoneimine metabolite, a highly reactive compound that binds to cellular proteins, may also cause oxidative stress and cell death, consistent with findings of lipofucsin pigment accumulation in the nasal olfactory epithelial cells. Cytotoxicity to the nasal olfactory epithelial cells results in replacement of the cells by respiratory metaplasia of undifferentiated cells; stimulation of cellular proliferation eventually leads to tumor formation. Mechanistic studies submitted to characterize nasal tumorigenesis in the rat indicate that the reactive agent ( the benzoquinoneimine) has higher rates of formation in the nasal tissue of rats than in mice, primates or humans. Although rats are considered to be much more sensitive to formation of these nasal tumors, the potential for acetochlor to cause nasal tumors in humans cannot be ruled out at this time, based on apparent potential for the human liver to produce the EMA metabolite, which could then be delivered to other organs.

Mode of action for thyroid follicular cell tumorigenesis: Acetochlor has been shown to cause disruption of thyroid pituitary homeostasis by increased activity of hepatic UDPGT, leading to increased clearance of thyroid hormones and compensatory increases in pituitary release of thyroid stimulating hormone ( TSH). Although some of the experimental parameters that are used to support this mechanism ( EPA, 1998) were not evaluated in the study ( e. g., evaluation of thyroid follicular cell proliferation), the data were considered sufficient by the HED CARC/ MTARC when considered together with the data on the structural analog alachlor, to classify the thyroid tumors under this non genotoxic mechanism. These tumors were not considered as part of the quantitative assessment for the carcinogenicity of acetochlor.

Effects of acetochlor on liver: At high doses, acetochlor causes acute toxicity to the liver. Studies evaluating the acute effects of acetochlor on rat liver showed that hepatic toxicity was associated with depletion of hepatocellular glutathione reserves at doses at which a slight increase in UDS are observed. The data provide some evidence that the UDS is secondary to depleted glutathione, Page 29 of 118 rather than direct genotoxicity. Hepatocellular proliferation was also evaluated in mice administered acetochlor in the diet for 90 days. At higher doses, BrdU incorporation was shown to increase during this time, providing some evidence that a non genotoxic, proliferative mechanism may be involved in formation of hepatocellular tumors. Significantly increased hepatocellular tumors were only observed at excessively toxic doses in both the mouse and the rat.

Modes of action for other types of toxicity observed with acetochlor have not been characterized.

4.1.3 Sufficiency of Studies/ Data The toxicological database for acetochlor is incomplete at this time. The following studies are required: ( 1) A developmental neurotoxicity ( DNT) study in the rat, due to observations in several oral studies indicating effects on the nervous system and uncertainties regarding the sensitivity of fetal and neonatal animals to neurotoxic effects. The effects observed in the available studies included clinical observations indicating neurotoxicity in several studies in the rat and the dog, and in the dog, frank microscopic pathology of the brain in a chronic oral study. A small but statistically significant decrease in brain acetylcholinesterase of males was also observed in one subchronic oral study in the rat at the highest dose tested. An alternative test to the rat DNT which addresses the sensitivity observed in the dog may also be considered. ( 2) In addition, submission of the positive control ( validation) studies for the rat acute and subchronic neurobehavioral screening studies ( MRIDs 45357501, 45357502), as cited in the neurobehavioral study reports, are requested as confirmatory data to upgrade the studies to Acceptable/ guideline.

The requirement of a 28 day inhalation toxicity study is deferred until the next assessment of acetochlor where occupational exposures are evaluated.

Although the above information is required for acetochlor, the database is nonetheless considered sufficient to provide endpoints and a dose response evaluation for risk assessment, based on the available studies in the rat, rabbit, dog and mouse.

4.1.4. Toxicological Effects The database for acute toxicity for acetochlor is complete and is summarized below in Table 4.1a. Acetochlor is classified as Toxicity Category III for acute exposure via the oral, dermal and inhalation routes and in ocular irritation studies. Although one primary dermal irritation study reported low levels of dermal irritation, severe irritation was observed in a second study, including microscopic changes ( hair follicle hyperplasia, and in some animals, acanthosis and inflammatory cell infiltration). Acetochlor has been shown to be a potent dermal sensitizer in two studies.

The subchronic, chronic and other guideline studies submitted to support acetochlor registration are summarized below in Table 4.1b. Page 30 of 118 Table 4.1a Acute Toxicity Profile  Test Substance Guideline No. Study Type MRID( s) Results Toxicity Category 870.1100 Acute oral, rat ( 1) 41565104 ( 1986) Acceptable/ guideline ( 2) 00118944 ( 1982) Acceptable/ guideline ( 1) LD50 = 4238 mg/ kg ( males); 4025 mg/ kg ( females); 4124 mg/ kg ( combined) ( 2) LD50 = 2389 mg/ kg ( males; 1929 mg/ kg ( females); 2148 mg/ kg ( combined) III ( both studies)

870.1200 Acute dermal, rabbit ( 1) 41565105 ( 1986) Acceptable/ guideline ( 2) 00118945 ( 1982) Unacceptable/ guidelin e ( upgradable with submission of dosing procedure ( surface area treated) ( 1) LD50 > 2000 mg/ kg ( combined males and females)

( 2) LD50 = 4166 ( combined males and females). Estimated between 3536 5000 mg/ kg for males and females. III ( both studies)

870.1300 Acute inhalation, rat ( 1) 41565106 ( 1989) Acceptable/ guideline ( 2) 40994401( 1988) Acceptable/ guideline LC50 > 4.46 mg/ L ( males) LC50 = 3.99 mg/ L ( females) ( 2) LC50 > 3.0 mg/ L ( both males and females) III ( both studies)

870.2400 Acute eye irritation, rabbit ( 1) 41592003 ( 1989) Acceptable/ guideline ( 2) 00118947 ( 1982) Acceptable/ guideline slightly irritating ( both studies) III ( both studies)

870.2500 Acute dermal irritation, rabbit ( 1) 41565107 ( 1989) Acceptable/ guideline ( 2) 00118946 ( 1982) Acceptable/ guideline ( 1) severe ( including microscopic changes) ( 2) mild irritation at 72 hrs ( 1) II ( 2) IV 870.2600 Skin sensitization, guinea pig ( 1) 00131396 ( 1983) Acceptable/ guideline ( 2) 41565108 ( 1989) Acceptable/ guideline Both studies very strong dermal sensitizer N/ A The major target organs of acetochlor appear to be the liver, thyroid ( secondary to liver), nervous system, kidney, testes, erythrocyte and in the rat, nasal olfactory epithelium. Liver enlargement was sometimes observed to be accompanied by increased plasma levels of GGT or other liver enzymes and microscopic alterations. Acute liver toxicity studies in the rat demonstrated depletion of Page 31 of 118 hepatocellular glutathione ( GSH) reserves and cytotoxicity at higher dose levels. Increases in thyroid weight and thyroid follicular cell tumors in chronic/ carcinogenicity studies in the rat are considered secondary to induction of hepatic UDPGT and subsequent perturbations to thyroid hormone levels, although acetochlor is not a potent inducer of this enzyme. Testicular atrophy was observed in dogs with chronic exposure. Interstitial nephritis was observed in some studies. The proposed mechanisms for nasal epithelial hyperplasia and subsequent tumor formation are discussed above in Section <IP_ADDRESS>. Decreased body weight/ weight gain were observed in most studies but not always at the LOAEL.

Evidence of neurotoxicity has been observed in several studies. In general, dogs appear to be somewhat more sensitive than rats to effects on the nervous system. Clinical signs such as excessive salivation or perianal staining were observed in dog subchronic and chronic studies and in the rat developmental toxicity studies. Females in the acute rat neurotoxicity study showed decreased motor activity; at higher dose levels but clinical signs were observed in both sexes. In the rat subchronic neurotoxicity study, a transient decrease in hindlimb grip strength in males was observed at week 2 but not at later times. In one chronic dog study, frank neuropathology ( degeneration of neurons in the cerebellum) accompanied by clinical effects such as ataxia, abnormal head movements and other findings, were observed. A small number of females in one rat chronic toxicity/ carcinogenicity study had peripheral neuropathy, but this was also at an excessively toxic dose. A slight but statistically significant decrease in brain acetylcholinesterase was observed at the high dose in a subchronic rat study and small but statistically significant effects on brain weights have been observed in some studies, although the direction of the weight change ( increase or decrease) was not consistent and in some instances was associated primarily with body weight changes.

Measurement of tissue distribution of radioactivity in rat metabolism studies shows that significant amounts are found in whole blood bound to erythrocytes. This finding may explain the slight anemia observed in some subchronic and chronic dietary studies at higher dose levels. It is not known whether the toxicity to the erythrocyte is related to glutathione depletion, as observed in hepatocytes, or is due to some other mode of toxicity.

There is no evidence that acetochlor is teratogenic or that offspring are more susceptible than adults, but acetochlor does cause developmental toxicity at maternally toxic doses. In both of the rat developmental toxicity studies, decreased fetal weights are observed. Offspring in rat multigeneration reproductive studies show reduced pup weight on lactation day 21 at doses causing maternal toxicity. One rat developmental study also showed increased early resorptions and postimplantation loss; the second study tested at lower doses. One rat reproductive toxicity study also showed slightly reduced live litter size and another showed decreased implantations. A low incidence of nasal epithelial hyperplasia was also observed in the adult F1 offspring. The rabbit is less sensitive than the rat and no developmental effects have been seen at maternally toxic levels.

Mutagenicity studies on acetochlor do not indicate that it has significant genotoxic potential in vivo. Bacterial gene mutation studies are negative or show equivocal increases, but the positive responses were only observed in the presence of significant cytotoxicity. A weak increase in sister chromatic Page 32 of 118 exchange ( SCE) induction has been reported but SCE is not known to be linked to cancer risk at this time. Although acetochlor is clastogenic in in vitro clastogenicity assays, in vivo clastogenicity assays ( rat or mouse bone marrow assays, dominant lethal studies) are negative. An unscheduled DNA synthesis study in rat hepatocytes also showed a weak positive response, but this appeared to be related to liver cytotoxicity ( depletion of glutathione). A comet assay specifically evaluating nasal olfactory and respiratory tissues of rats exposed to acetochlor at doses causing nasal tumors was negative.

As discussed above ( Section <IP_ADDRESS>), acetochlor causes an increased incidence of nasal cell tumors in the olfactory region of the nasal cavity and thyroid follicular cell tumors in the rat. These tumors are considered to show a threshold for induction dependent on their tumorigenic mode of action. A dose related increase in the incidence of lung tumors in male and female mice and histiocytic sarcomas in female mice was also observed. Although the genotoxicity data do not indicate a strong potential for acetochlor to act as a genotoxic carcinogen, there are no mechanistic data available at this time to characterize the mechanism of formation of these two tumor types. A statistically significant increase in hepatocellular tumors in mice and rats was also reported, but only at a dose level that resulted in excessive toxicity in both species.

Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results 870.3100 90 Day oral toxicity ( rat) 00050933 ( 1980) Acceptable/ guideline 0, 800, 2000, 6000 ppm ( diet) 0, 40, 100, 300 mg/ kg/ day NOAEL = 40 mg/ kg/ day LOAEL = 100 mg/ kg/ day based on decreased mean body weight/ weight gain in males and females.

870.3100 90 Day oral toxicity ( rat) 41565115 ( 1986) Acceptable/ guideline 0, 20, 200, 2000 ppm ( diet) males 0, 1.6, 16.1, 161.1 mg/ kg/ day; females 0, 1.9, 18.7, 191.9 mg/ kg/ day NOAEL = 16.1 mg/ kg/ day males ( 18.7 mg/ kg/ day females) LOAEL = 161.1 mg/ kg/ day males ( 18.7 mg/ kg/ day females) based on decreased body weight/ weight gain in males and females.

870.3150 119 Day oral toxicity ( dog) 00050928 ( 1980) Acceptable/ guideline 0, 25, 75, 200 ( capsule) NOAEL = 25 mg/ kg/ day LOAEL = 75 mg/ kg/ day based on death of 1 male ( diarrhea, emaciation also in this animal) and in both sexes, decr. body weights and decr. food consumption, liver effects. ( It is noted that in this study, mid and high dose animals were acclimated by increasing dose weekly: mid dose started at 25 mg/ kg/ day week 1, 50 mg/ kg/ day week 2, then 75 mg/ kg/ day. High dose started at 50 mg/ kg/ day, incr. weekly by 50 mg/ kg/ day then 200 mg/ kg/ day) Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 33 of 118 870.3150 90 Day oral toxicity ( dog) 41565116 ( 1986) Acceptable/ guideline 0, 2, 10, 60 mg/ kg/ day ( capsule) NOAEL = 10 mg/ kg/ day LOAEL = 60 mg/ kg/ day based on mucous diarrhea, decr. body wt, slight anemia, incr. alanine aminotransferase, incr. relative liver weight, decr. blood glucose.

870.3200 21 Day dermal toxicity ( rabbit) 00116637 ( 1981) Acceptable/ guideline 0, 100, 400, 1200 mg/ kg/ day ( applied 5 days/ week for 3 weeks) systemic NOAEL = 400 mg/ kg/ day systemic LOAEL = 1200 mg/ kg/ day based on high mortality and agonal clinical signs of toxicity. Local dermal irritation was observed at all dose levels.

870.3200 21 Day dermal toxicity ( rat) 41565117 ( 1989) Acceptable/ guideline 0, 0.1 1.0, 10, 100 mg/ kg/ day ( applied 5 days/ week for 3 weeks) systemic NOAEL = 100 mg/ kg/ day ( HDT) systemic LOAEL = > 100 mg/ kg/ day ( not tested at higher dose levels due to reported excessive dermal irritation in preliminary testing). Local dermal irritation reported in this study in all groups including controls but epithelial hyperplasia was observed at 100 mg/ kg/ day.

870.3465 90 Day inhalation toxicity none submitted ( not required) N/ A 870.3700a Prenatal developmental in rats 00050929 ( 1980) Acceptable/ guideline 0, 50, 200, 400 mg/ kg/ day ( gavage) Maternal NOAEL = 200 mg/ kg/ day LOAEL = 400 mg/ kg/ day based on clinical signs of toxicity and decreased maternal body weight/ weight gain. Developmental NOAEL = 200 mg/ kg/ day LOAEL = 400 mg/ kg/ day based on slightly decreased mean fetal weight.

870.3700b Prenatal developmental in rats 41592005 ( 1989), 42054903 Acceptable/ guideline 0, 40, 150, 600 mg/ kg/ day ( gavage) Maternal NOAEL = 150 mg/ kg/ day LOAEL = 600 mg/ kg/ day based on mortality, clinical signs of toxicity and decreased maternal body weight gain. Developmental NOAEL = 150 mg/ kg/ day LOAEL = 600 mg/ kg/ day based on increased early resorptions, postimplantation loss and decreased fetal weight.

870.3700b Prenatal developmental in rabbits 40134101( 1986) Acceptable/ guideline 0, 30, 100, 300 mg/ kg/ day ( gavage) Maternal NOAEL = 300 mg/ kg/ day LOAEL > 300 mg/ kg/ day ( HDT; study acceptable based on results of a range finding study). Developmental NOAEL = 300 mg/ kg/ day LOAEL > 300 mg/ kg/ day .

870.3700b Prenatal developmental in rabbits 41592006 ( 1989) Acceptable/ guideline 0, 15, 50, 190 mg/ kg/ day ( gavage) Maternal NOAEL = 50 mg/ kg/ day LOAEL = 190 mg/ kg/ day based on body weight loss. Developmental NOAEL = 190 mg/ kg/ day LOAEL > 190 mg/ kg/ day ( HDT). Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 34 of 118 870.3800 Reproduction and fertility effects rats 00131391( 1982) Acceptable/ guideline 0, 500, 1500, 5000 ppm ( diet) F0 premating 0, 30.8, 60.4 or 316 mg/ kg/ day, males; 0, 46.2, 130.4 or 442 mg/ kg/ day, females; F1 premating 0, 29.9, 87.8 or 333 mg/ kg/ day, males; 0, 43.6, 129.8 or 441 mg/ kg/ day, females. Parental/ Systemic NOAEL = 30.8/ 46.2 mg/ kg/ day males/ females LOAEL = 60.4/ 130.4 mg/ kg/ day males/ females, based on decreased maternal gestation body weight gain and slightly reduced male and female premating body weight gain. Offspring NOAEL = 30.8/ 46.2 mg/ kg/ day males/ females LOAEL = 60.4/ 130.4 mg/ kg/ day males/ females, based on slightly decreased mean pup weights in F2b pups at lactation day 21. Reproductive NOAEL = 316/ 442 mg/ kg/ day males/ females. LOAEL = mg/ kg/ day ( HDT).

870.3800 Reproduction and fertility effects rats 41565120 ( 1989) Acceptable/ guideline 0, 18, 175, 1750 ppm ( diet) Premating F0 males 0, 1.27, 12.6 or 123.8 mg/ kg/ day; F0 females 0, 1.63, 15.5 or 157.4 mg/ kg/ day Premating F1 males 0, 1.53, 15.2 or 152.1 mg/ kg/ day; F1 females 0, 1.83, 18.3 or 192.4 mg/ kg/ day Parental/ Systemic NOAEL = 12.6 mg/ kg/ day males/ 15.2 mg/ kg/ day females LOAEL = 123.8 mg/ kg/ day males/ 157.4 mg/ kg/ day females, based on decreased body weight gain during premating. Offspring NOAEL = 12.6 mg/ kg/ day males/ 15.2 mg/ kg/ day females. LOAEL = 123.8/ 157.4 mg/ kg/ day males/ females, based on reduced pup weight during lactation. Reproductive NOAEL = 123.8/ 157.4 mg/ kg/ day ( HDT). LOAEL > 123.8/ 157.4 mg/ kg/ day males/ females.

870.3800 Reproduction and fertility effects rats 45357503 ( 2001) Acceptable/ guideline 0, 200, 600, 1750 ppm ( diet) Premating F1 males 0, 21.2, 65.6, 196.4 mg/ kg/ day; Premating F1 females 0, 22.4, 70.9, 215.9 mg/ kg/ day Parental/ Systemic NOAEL = males 21.2 mg/ kg/ day; females 22.4 mg/ kg/ day LOAEL = males 65.6 mg/ kg/ day; females 70.9 mg/ kg/ day, based on focal hyperplasia and polypoid adenomata in nasal epithelium of adult F1 offspring at study termination. Offspring NOAEL = males 65.6 mg/ kg/ day; females 70.9 mg/ kg/ day LOAEL = males 196.4 mg/ kg/ day; females 215.9 mg/ kg/ day based on decreased pup weights during lactation ( F1 and F2), decreased F2 litter size at birth, focal hyperplasia and polypoid adenomata in nasal epithelium of adult F1 offspring at study termination. Reproductive NOAEL = males 65.6 mg/ kg/ day; females 70.9 mg/ kg/ day LOAEL = males 196.4 mg/ kg/ day; females 215.9 mg/ kg/ day based on decreased implantations. Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 35 of 118 870.4100a Chronic toxicity ( rat) See 870.4200, below 870.4100b Chronic toxicity ( dog) 00116631, 00164944 ( 1981) Acceptable/ guideline 0, 4, 12, 40 mg/ kg/ day ( capsule) NOAEL = 12 mg/ kg/ day LOAEL = 40 mg/ kg/ day based on decr. body wt. ( females) and decr. wt. gain ( males), incr. adrenal wt. ( females), incr. liver weight ( males and females), decr. testes wt., testicular atrophy.

870.4100b Chronic toxicity ( dog) 41565118 ( 1988), Acceptable/ guideline 0, 2, 10, 50 mg/ kg/ day ( capsule)

46100901 ( 2003) Unacceptable/ nonguidelin e ( reexamination of testicular / epididymal tissue) NOAEL = 2 mg/ kg/ day LOAEL = 10 mg/ kg/ day based on incr. salivation, incr. ornithine carbamyltransferase and triglycerides, decr. blood glucose; incr. incidence of interstitial nephritis, testicular degeneration/ hypospermia and liver glycogen depletion. [ Neurotoxic effects seen at 50 mg/ kg/ d  salivation, ataxia, histopathological changes in the brain]

Reexamination of testicular/ epididymal tissue ( MRID 46100901) was not performed according to Agency pathology working group/ peer review policy and the findings do not change the original dog study ( MRID 41565118) conclusions.

870.4200 Carcinogenicity ( rat) 00131088, 40484801 ( 1983) 0, 500, 1500 or 5000 ppm in diet males: 0, 22, 69 or 250 mg/ kg/ day, 115 weeks females: 0, 30, 93 or 343 mg/ kg/ day for 103 weeks ( females; discontinued earlier due to high mortality). NOAEL < 22 mg/ kg/ day LOAEL = 22 mg/ kg/ day based on decreased body weight/ weight gain ( males) and increased abs/ rel thyroid weight in females. HDT ( 250 males, 343 females) considered excessive based on high mortality and markedly reduced body weight/ weight gain, both sexes. evidence of carcinogenicity) at 69/ 93 mg/ kg/ day and higher increased incidence of nasal epithelial adenomas ( males). At 250/ 343 mg/ kg/ day ( excessively toxic dose based on high mortality and markedly decreased body weight/ weight gain ( both sexes) increased incidence of nasal epithelial carcinomas and thyroid follicular cell adenomas ( males) and hepatocellular tumors ( both sexes). Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 36 of 118 870.4200 Carcinogenicity ( rat) 40077601 ( 1986) Acceptable/ guideline 0, 40, 200 or 1000 ppm in diet 0, 2, 10 or 50 mg/ kg/ day in diet ( estimated for both sexes using standard conversion factor of 0.05) NOAEL = 10 mg/ kg/ day LOAEL = 50 mg/ kg/ day based on decreased body weight/ weight gain ( both sexes, slight in females), increased bilirubin in females and in males, increased GGT, cholesterol, thyroid c cell hyperplasia, papillary hyperplasia of the nasal epithelium, hepatocellular alterations and necrosis. evidence of carcinogenicity at 50 mg/ kg/ day, increased incidence of nasal epithelial papillary adenoma in males and females and thyroid follicular cell adenoma/ cystadenoma in females. PWG reevaluation of liver did not show increase with treatment.

870.4200 Carcinogenicity ( rat) 41592004 ( 1988) Acceptable/ guideline 0, 18, 175 or 1750 ppm in diet males 0, 0.67, 6.37 or 66.9 mg/ kg/ day; females 0, 0.88, 8.53 or 92.1 mg/ kg/ day NOAEL = 6.37 mg/ kg/ day LOAEL = 66.9 mg/ kg/ day based on decreased body weight/ weight gain, increased GGT and cholesterol ( both sexes; marginal in females), nasal epithelial hyperplasia, degeneration of retinal outer layer, renal pelvic epithelial hyperplasia, stromal fatty infiltration of the pancreas. evidence of carcinogenicity increased incidence of nasal epithelial adenoma/ carcinoma and thyroid follicular cell adenomas in males and females. Benign chondroma of femur and basal cell tumor of the stomach were reevaluated and reclassified by the PWG and are not considered treatment related tumors.

870.4300 Carcinogenicity ( mouse) 00131089 ( 1983) Acceptable/ guideline 0, 500, 1500 or 5000 ppm in diet 0, 75, 225, 750 mg/ kg/ day ( estimated for both sexes using standard conversion factor of 0.15) NOAEL = 75 mg/ kg/ day LOAEL = 225 mg/ kg/ day based on reduced survival ( females), slightly decreased body weight during part of study ( males), anemia ( females), increased abs/ re/ thyroid weight ( females) and renal interstitial nephritis ( males). evidence of carcinogenicity incidence of lung tumors showed a dose related increase. A PWG reevaluation of liver showed increases only at the HDT ( excessive dose); ovarian and kidney tumors were not considered treatment related.

870.4300 Carcinogenicity ( mouse) 41565119 ( 1989) Acceptable/ guideline 0, 10, 100 or 1000 ppm in diet Males 0, 1.1, 11, 116 mg/ kg/ day; females 0, 1.4, 13, 135 mg/ kg/ day NOAEL = 1.1 mg/ kg/ day males; 135 mg/ kg/ day females LOAEL = 11 mg/ kg/ day in males, based on increased incidence of brochiolar hyperplasia and possibly renal tubular hyperplasia; > 135 mg/ kg/ day in females. evidence of carcinogenicity increased incidence of lung adenomas ( females) and adenoma/ carcinoma ( males) at 116/ 135 mg/ kg/ day. Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 37 of 118 Gene Mutation 870.5100 Bacterial reverse mutation Salmonella typhimurium 00050930 ( 1978) Acceptable/ guideline 0.001 1 µ l/ plate  /+ S9 Negative up to the highest dose tested ( 1 µ l/ plate  /+ S9); higher concentrations ($ 10 µ l/ plate  /+ S9) were cytotoxic Gene Mutation 870.5100 Bacterial reverse mutation Salmonella typhimurium 41565121 ( 1989) Acceptable/ guideline 1.6 5000 µ g/ plate  /+ S9 Equivocal positive in TA 1538 at 2500 and 1000 µ g/ plate + S9; reproducible at 1000 µ g/ plate but < 2 fold, not doserelated and not seen in TA98 Gene Mutation 870.5100 Bacterial reverse mutation Salmonella typhimurium ( strain T1538 only) 44863202 ( 1989) Acceptable/ nonguideline 100 5000 µ g/ plate  /+ S9 ( Arochlor 1254 or Phenobarbital /$ naphthoflavone induced rat livers) Negative in TA1538 using 3 different batches of acetochlor ( 89.8 99.6%) in two separate tests Gene Mutation 870.5300 Mammalian cell gene mutation Chinese hamster ovary ( CHO) cells 00131395 ( 1983) Acceptable/ guideline 25 150 µ g/ mL  S9 25 125 µ g/ mL + 10% S9 Positive $ 2 fold in mutation frequency ( MF) at 125 or 150 µ g/ mL  S9 & 125 µ g/ mL + S9 accompanied by cytotoxicity ( 61% or 93% decrease in cell survival /+ S9)

Gene Mutation 870.5300 Mammalian cell gene mutation CHO cells 42713106 ( 1989) Acceptable/ guideline 50 200 µ g/ mL  S9 50 300 µ g/ mL + 1, 2, 5 or 10% S9 Negative up to cytotoxic levels ($ 200 µ g/ mL /+ 10% S9)

Cytogenetics 870.5300 Mammalian cell gene mutation Mouse lymphoma L5178Y cells 00131394 ( 1982) Acceptable/ guideline 20 400 µ L/ mL  S9 5 250 µ L/ mL + S9 Positive 30 50 µ L/ mL + S9 2.2 5.2 fold increase accompanied by cytotoxicity (< 10% survival at $ 50 µ L/ mL + S9) Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 38 of 118 870.5375 Cytogenetics In vitro mammalian cell chromosomal aberration assay human lymphocytes ( whole blood vs separated blood) 44863204 ( 1998) Acceptable/ guideline ( 1) 0, 10, 75 150 µ g/ mL  /+ S9 ( 2) 0, 100 µ g/ mL  S9 ( whole blood) 0, 75 µ g/ mL  S9 ( separated blood) ( 1) Whole Blood: Positive at 75 and 150 µ g/ mL  S9 and 150 µ g/ mL + S9 accompanied by slight reduction in mitotic indices at 150 µ g/ mL ( 31%  S9; 13 % + S9decrease). Types of aberrations: breaks, fragments and minutes.

( 2) Whole Blood: Positive 9 fold increase in aberrations at 100 µ g/ mL Separated Blood: Positive 26 fold increase in aberrations at 75 µ g/ mL 870.5375 Cytogenetics In vitro mammalian cell chromosomal aberration assay human lymphocytes 41565122 ( 1989) Acceptabe/ guideline 0, 10, 50, 100 µ g/ mL +/ S9 Positive at 50 and 100 µ g/ mL + S9 accompanied by marked reduction in mitotic indices at 100 µ g/ mL ($ 59% decrease). Types of aberrations: breaks, fragment and minutes.

870.5385 Mammalian Bone Marrow Chromosomal Aberration Test Rat 00131392 ( 1983) Acceptable/ guideline 0, 40, 150, 500 mg/ kg IP injection Negative up to overt toxicity ( significant decrease in body weight gain)

870.5395 Mammalian Erythrocyte Micronucleus Test CD 1 Mice 00164941 ( 1986) Acceptable/ guideline 0, 200, 660, 2000 mg/ kg oral gavage Negative up to overt toxicity ( mortality) & cytotoxicity ( significant decrease in PCE: NCE ratio at 2000 mg/ kg, both sexes combined)

870.5395 Mammalian Erythrocyte Micronucleus Test CD 1 Mice 41565123 ( 1989) Acceptable/ guideline 0, 898 or 1436 mg/ kg %

0, 1075 or 1719 mg/ kg& Negative up to a cytotoxic dose ( significant decrease in PCE: NCE ratio) seen at both doses in %&

870.5450 Cytogenetics Dominant Lethal Rat 44069502 ( 1996) Unacceptable/ guideline 0, 200, 1000, 1500 ppm for 10 weeks Negative for dominant lethal mutations but dosage was insufficient 870.5450 Cytogenetics Dominant Lethal Rat 41963309/ 44093703 ( 1991/ 1996) Acceptable/ guideline 0, 200, 1000, 2000 mg/ kg oral gavage Negative; earlier report of positive results now considered to be due to reproductive ( infertility) toxicity Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 39 of 118 870.5450 Cytogenetics Dominant Lethal Mouse 44093701( 1996) Unacceptable/ guideline 0, 200, 1000, 3500 ppm for 8 weeks Negative for dominant lethal mutations but dosage was insufficient 870.5900 Other Genotoxicity In vitro sister chromatid exchange assay Human Lymphocytes Hill et al. ( 1997) Acceptable/ guideline 10 µ M ( 2.7 µ g/ mL) Weak evidence of positive response ( 1.5 fold increase) in one of two donor cells 870.5550 Other Genotoxicity In vitro UDS in Primary Rat Hepatocytes 00131393 ( 1983) Acceptable/ guideline 0.032 320 µ g/ well Negative up to cytotoxic concentrations ($ 10.6 µ g/ well)

870.5550 Other Genotoxicity In vitro UDS in Primary Rat Hepatocytes 41565124 ( 1989) Acceptable/ guideline 0, 500, 1000, 2000 mg/ kg oral gavage Weak positive response accompanied by major hepatic pathology ( necrosis, 70% decreased GSH, 60 fold increase in aspartate transaminase )

870.6200 Other Genotoxicity In vivo Comet Assay in Rat Olfactory and respiratory cells 44863208 ( 1999) Acceptable/ nonguideline 1750 ppm ( 175 mg/ kg/ day) 7 days Negative at a tumorigenic dose in vivo 870.6200a Acute neurotoxicity screening battery MRID 45357501 ( 2001) Unacceptable ( guideline) upgradable 0, 150, 500, 1500 mg/ kg NOAEL = 150 mg/ kg ( females); 500 mg/ kg ( males) LOAEL = 500 mg/ kg based on decreased motor activity in females ( 1500 mg/ kg in males based on clinical signs in the FOB and decreased body weight/ weight gain).

Positive control ( validation) studies cited in study required to confirm endpoints.

870.6200b Subchronic neurotoxicity screening battery MRID 45357502 ( 2001) Unacceptable ( guideline) upgradable. 0, 200, 600, 1750 ppm (% 0, 15.4, 47.6, 139.0 mg/ kg/ day; & 0, 18.3, 55.9, 166.5 mg/ kg/ day) NOAEL = 47.6 mg/ kg/ day LOAEL = 139.0 mg/ kg/ day based on decreased body weight/ weight gain in males and females, possible transiently decreased hindlimb grip strength in males at week 2.

Positive control ( validation) studies cited in study required to confirm endpoints. Table 4.1b Subchronic, Chronic and Other Toxicity Profile Guideline No./ Study Type MRID No. ( year)/ Classification / Doses Results Page 40 of 118 870.6300 Developmental neurotoxicity Not available N/ A 870.7485 Metabolism and pharmacokinetics ( rat) 00130839 ( 1983) Acceptable/ guideline Oral 10 mg/ kg, 400 mg/ kg ( 14C acetochlor) Rapidly eliminated (> 70% of administered dose by day 2), elimination biphasic ( rapid phase ½  life 5.4 10.4 hrs, slow phase 128.6 286.4 hrs for 10 mg/ kg dose). Tissues did not retain high levels of radioactivity except for RBC, which retained about 2.5% of dose at study termination. Acetochlor was completely metabolized, via N dealkylation and glucuronide conjugation, primarily to mercapturic acid derivatives, with other sulfur containing derivatives identified.

870.7485 Metabolism and pharmacokinetics ( rat) 41565125,  26,  27, 41592007,  08 ( 1987, 1989, 1990) Acceptable/ guideline Oral 10 mg/ kg, 200 mg/ kg and 10 mg/ kg/ day x 14 days ( 14C acetochlor) Well absorbed and rapidly eliminated ( 92 96% of dose by day 5, ½ life of elimination 20 30 hrs), primarily excreted in urine ( about 60% by 24 hrs) but significant fecal excretion observed, especially males and at high dose, with biliary excretion observed. Elimination biphasic. Retention in tissue/ carcass negligible, primarily in blood ( binding to RBC) and well perfused organs ( heart, spleen, kidney, lungs, liver). Acetochlor was completely metabolized ( 15 compounds separated in urine, 4 in bile, 5 in feces), with glutathione, mercapturic acid or glucuronide conjugation of n dealkylated acetochlor a major route of metabolism; sulphoxymethyl and cysteine conjugates also identified in feces. In urine, major metabolite was mercapturic acid conjugate of N deethylated acetochlor; in bile, major metabolite was the glucuronide conjugate. Major fecal metabolite not characterized.

870.7600 Dermal in vivo penetration ( rat) 41778303 ( 1990) Acceptable/ guideline Acetochlor was dermally absorbed in rats in a dose and timerelated manner. Maximum % of dose absorbed during a 10 hr exposure duration ( used for unintentional occupational exposure) was 19 23% using an application of 1/ 70 or 1/ 1000 dilution of formula concentrate ( equivalent to 11.772 or 0.763 mg/ g, respectively). Page 41 of 118 In addition to the above studies that were submitted to fulfill guideline requirements for acetochlor, numerous special and mechanistic studies have been submitted to address the mechanism of nasal and thyroid tumorigenesis ( see Section <IP_ADDRESS>, above). These studies have been reviewed and a toxicology profile is presented below in Table 4.1c:

Table 4.1c Special ( Nonguideline) Mechanistic Study Toxicity Profile Study Type MRID No. ( year)/ Classification / Doses Results Studies on Nasal Tumorigenesis Comparative metabolism ( rat/ mouse) 44496203 ( 1998) Acceptable/ nonguideline ( 1) 200 mg/ kg single gavage dose or ( 2) 1750 ppm in diet for 6 months, then 200 mg/ kg single dose or ( 30 0, 10, 200, 1000 or 2000 mg/ kg single gavage dose ( 14C acetochlor) Differences in metabolism between rats and mice were seen. Initial reactions both species were oxidative O deethylation of the N ethoxymethyl sidechain and glucuronidation of the methylol group. In rats glucuronide conjugate excreted in the bile, followed by hepatic removal of methylol group and glutathione conjugation, yielding mercapturic acid derivative of the glutathione conjugate ( major urinary metabolite in rats). Sulfoxides and sulfone derivatives also identified. In mice major urinary metabolite was a chloramideenterohepatic circulation not observed and glutathione conjugation not a major route of metabolism.

Nasal cell proliferation ( rat) 44496207 ( 1996) Acceptable/ nonguideline 0, 200, 1750 or 5000 ppm in diet for 160 days Cell proliferation in nasal turbinate olfactory respiratory epithelium, but not respiratory epithelium, was significantly increased at 1750 and 5000 ppm as measured by tritiated thymidine incorporation into DNA at each dose level at 60 days ( 5000 ppm only), 90 or 160 days of treatment. Bromodeoxyuridine incorporation also showed significant increases after 160 days at 1750 and 5000 ppm, but not at 200 ppm. No increase was seen in respiratory epithelium. Cell proliferation increased 1.5 2.0 fold at 5000 ppm and 1.3 1.5 fold at 1750 ppm.

Nasal cell proliferation ( mouse) 44496209 ( 1996) Acceptable/ nonguideline 0, 1000 or 5000 ppm in diet for 60 and 90 days. Acetochlor did not cause increased nasal olfactory or respiratory epithelial cell proliferation in mice as evaluated by bromodeoxyuridine nuclear incorporation. Table 4.1c Special ( Nonguideline) Mechanistic Study Toxicity Profile Study Type MRID No. ( year)/ Classification / Doses Results Page 42 of 118 Quinoneimine protein binding, autoradiography ( rat) 44496210 ( 1998) Unacceptable/ nonguideline ( upgradable) 1710 or 5170 ppm acetochlor in diet containing 14C acetochlor for 14 days. In rat nasal turbinate tissue, a dose dependent formation of 3 ethyl, 5 methyl benzoquinoneimine cysteine ( EMIQ cysteine) adducts was observed ( 119 and 206 pmole/ mg protein at 1710 and 5170 ppm, respectively) ( determined by acid hydrolysis and HPLC). Whole body autoradiography showed localization of radioactivity in gut, stomach contents, urinary bladder, highly perfused organs and in the nasal turbinates, adrenal and preputial glands. Microautoradiography of decalcified noses showed localization in Bowman's glands at 1720 and 5170 ppm and RBC at 5170 ppm, with equivocal localization in the neuron layer of the olfactory surface epithelium.

Quinoneimine protein binding autoradiography ( mouse) 44496211 ( 1998) Unacceptable/ nonguideline ( upgradable) 1800 or 4750 ppm acetochlor in diet containing 14C acetochlor) for 14 days. EMIQ cysteine adduct formation not observed in mice as assessed by acid hydrolysis and HPLC.

Quinoneimine protein binding autoradiography acetochlor secondary sulfide ( rat) 44496212 ( 1998) Unacceptable/ nonguideline ( upgradable) 7 mg/ kg/ day 14C acetochlor for either 5 consecutive days or single dose, sacrificed either one or 5 days after final dose. EMIQ cysteine adducts were observed in nasal turbinate tissue as assessed by acid hydrolysis and HPLC. Autoradiography showed localization in nasal turbinates and microautoradiography of decalcified noses showed binding in the Bowman's glands.

Quinoneimine protein binding autoradiography ( Rhesus monkey) 44496213 ( 1998) Acceptable/ nonguideline 126 mg/ kg 14C acetochlor for 14 days EMIQ cysteine adducts were not detected in nasal turbinate tissues as assessed by acid hydrolysis and HPLC.

Nasal tumor mapping ( rat) 44496214 ( 1997) Acceptable/ nonguideline nasal passages examined from rats in chronic/ carcinogenicity dietary studies on acetochlor ( 1750 ppm) and butachlor ( 3000 ppm) and a one year gastric initiation promotion study on alachlor ( 126 mg/ kg). Hyperplastic and preneoplastic/ neoplastic lesions for all compounds were located primarily in the ethmoid turbinates, regions normally lined by olfactory mucosa, with many near the olfactory respiratory junctions. Olfactory to respiratory metaplasia was a significant feature of neoplastic progression. Females given acetochlor also showed basal cell hyperplasia in the region underlying Bowman's glands in dorsal and medial airways. Table 4.1c Special ( Nonguideline) Mechanistic Study Toxicity Profile Study Type MRID No. ( year)/ Classification / Doses Results Page 43 of 118 In vitro metabolism ( rat/ mouse/ one human sample) 44530001 ( 1998) Acceptable/ nonguideline 14C acetochlor sulfoxide ( 0.025 mM, 15.5 kBq) incubated w/ microsomes from rat liver, nasal olfactory and nasal respiratory epithelia; mouse nasal olfactory and liver cells; and human nasal epithelia ( mixed olfactory/ respiratory) Acetochlor sulfoxide was rapidly hydroxylated in rat and mouse olfactory microsomal fractions, but not respiratory or liver fractions. Major metabolites were ( 1) side chain oxidation product of acetochlor sulphone and ( 2) parahydroxy metabolite of acetochlor sulfoxide. Hydroxylation of acetochlor sulfoxide was not detected in the sample of human nasal tissue.

In vitro metabolism ( rat/ mouse/ squirrel monkey) 44530002 ( 1998) 46081803 ( 2003) Unacceptable/ nonguideline ( upgradable) 14C acetochlor ( 30 mM, 0.05 mBq) Study evaluated rates of steps in metabolism of acetochlor to p hydroxy 2 ethyl 6 methylaniline ( pOH EMA), a precursor to quinoneimine formation, in cellular fractions from rat and mouse liver, nasal olfactory epithelia and nasal respiratory epithelia; and from monkey combined nasal olfactory/ respiratory epithelia. In mice and rat tissues, the rate of acetochlor GSH conjugation of acetochlor was comparable, but slightly higher in rat olfactory tissue than mouse olfactory tissue. Rate of secondary sulfide hydrolysis to EMA was significantly lower in olfactory and respiratory tissues of mice vs. rats; p hydroxylation of EMA was comparable in nasal tissues of rats and mice but lower in rat liver than mouse liver. Overall conversion of acetochlor to pOH EMA was slower in mice than rats, lowering potential to form reactive intermediates. Rates of all reactions were much lower in monkey nasal tissue than rat nasal or liver tissue, suggesting lower potential to form reactive intermediates.

Protein adduct formation ( rat) 46009402 ( 2001) in vivo protein binding, 10 mg/ kg 14C acetochlor sulfoxide; in vitro binding, 0.4 mM 14C acetochlor sulfoxide, 407 458 Kbq to cellular fractions of nasal and liver tissue Unacceptable/ nonguideline ( upgradable) ( 1) HPLC analysis comparing radioactivity of acid hydrolysates from olfactory vs respiratory mucosa showed significantly higher levels of radioactivity in the olfactory mucosa; ( 2) SDS PAGE of bound proteins from incubation of olfactory epithelial microsomal fractions showed similar patterns for carbonyl and phenyl labeled acetochlor sulfoxide, indicating that the sulfoxide moiety was retained in much of the bound radioactivity; ( 3) Histoautoradiography of the olfactory and respiratory regions of the rat nasal cavity at 8 and 24 hrs postdosing showed the highest levels of bound radioactivity over Bowman's glands in the olfactory mucosa, with none in the respiratory region. The areas of binding coincide with the cellular location of xenobiotic metabolizing enzymes in the nasal passages. Table 4.1c Special ( Nonguideline) Mechanistic Study Toxicity Profile Study Type MRID No. ( year)/ Classification / Doses Results Page 44 of 118 In vitro metabolism ( rat/ mouse/ squirrel monkey/ human) 46009401 ( 2000) 46081802 ( 2003) Unacceptable/ nonguideline ( upgradable) Rate of hydroxylation of acetochlor sulfoxide to p OHacetochlor sulfoxide evaluated. Highest levels of activity observed in the rat and mouse nasal olfactory tissues, no detectable activity seen in monkey or human samples. Enzyme characterization studies indicated that the reaction is catalyzed by a cytochrome similar to the CYP2A family, but not coumarin hydroxylase itself.

Studies on thyroid tumorigenesis Characterization of thyroid toxicity and liver effects time course ( rats) 44496208 ( 1996) Acceptable/ nonguideline 0, 1750 or 5000 ppm ( 0, 100.6 or 280.9 mg/ kg/ day) in diet for 14, 28 or 56 days; 0, 200, 1750 or 5000 ppm ( 0, 10.4, 91.9 or 270.3 mg/ kg/ day) in diet for 160 days. Effects on liver and thyroid weights, thyroid hormones and liver UDPGT activity were observed at 1750 and 5000 ppm, consistent w/ perturbation of thyroid pituitary homeostasis via UDPGT mediated clearance of T4. Increased hepatic UDPGT activity ( by day 14), increased TSH ( by day 14 at 5000 ppm and day 56 at 1750 ppm) and T4 ( day 14 only) and decreased T3 ( day 14 only) were observed. Liver and thyroid weights were increased ( days 14 90; liver also at day 160).

Studies on acute liver toxicity ( supplemental data for UDS studies) and liver cell proliferation Acute liver toxicity ( rats) 44863205 ( 1993) Unacceptable/ nonguideline ( upgradable with test material purity information) ( 1) 2000 mg/ kg via gavage in corn oil ( evaluation of UDS); ( 2) 0, 500, 1000 or 2000 mg/ kg via gavage in corn oil ( evaluation of liver tissue non protein sulphydryl groups) Dose dependent depletion of hepatocellular glutathione leading to mild to marked necrosis at $ 500 mg/ kg was observed, with slight stimulation of UDS at 2000 mg/ kg. Increased serum AST and ALT were observed at 2000 mg/ kg. UDS therefore observed at conditions of excessive hepatocellular toxicity and reduced hepatocellular glutathione levels.

Acute liver toxicity ( rats) 44863207 ( 1994) Unacceptable/ nonguideline ( upgradable with submission of test material purity) 0, 500, 1000 or 2000 mg/ kg via gavage in corn oil Dose dependent depletion of hepatocellular glutathione observed at $ 500 mg/ kg, peaking 6 12 hrs post dosing ( 17 to 63% of control levels between 3 12 hrs). Necrosis and serum liver enzymes returned to normal levels thereafter and normal levels of glutathione were observed by 48 hr.

Hepatocellular proliferation ( mice) 44863601 ( 1999) Acceptable/ nonguideline 0, 1000 or 5000 ppm in diet for 90 days ( males only). Equivalent to 0, 166.6 or 887.9 mg/ kg/ day). Incorporation of BrdU in mice treated with acetochlor was approximately doubled ( 0.15, 0.35, 0.38 at 0, 1000 and 5000 ppm, respectively). Page 45 of 118 4.1.5 Dose Response Appropriate endpoints were identified for all potential exposure scenarios for acetochlor. Studies considered in the development of this risk assessment showed developmental effects on , weight gain decrements in offspring, evidence of neurotoxicity and microscopic effects in several tissues. Although weight gain decrements in young adult/ adult animals were consistently observed in studies on acetochlor, they tended to occur at higher dose levels than these other, more sensitive findings.

The acute oral RfD for the general population including females age 13 49 was derived from the acute neurotoxicity study in the rat, based on decreased motor activity in females at the time of peak effect. The endpoint selected is considered to be protective of pregnant or nursing females because the NOAEL selected ( 150 mg/ kg) is same as the developmental toxicity NOAEL in the rat, which was based on an increase in fetal resorptions/ postimplantation loss, an effect that is potentially caused by a single exposure. The chronic RfD was derived from a chronic oral toxicity study in the dog, which provided the most sensitive endpoint available and was based on clinical signs ( excessive salivation) and microscopic findings in the liver, testes and kidney. Incidental oral endpoints were not selected because there are presently no residential uses for acetochlor.

For dermal risk assessment, a 21 day dermal exposure study in the rabbit provided the endpoint for short term exposure. The route of exposure and study duration were appropriate: although significant mortality was observed in this study, it occurred at a dose exceeding the limit dose and significantly higher than the NOAEL. The endpoint is considered protective of pregnant or nursing females who may be occupationally exposed ( the dermal endpoint is lower than the developmental endpoint from the oral developmental toxicity study when the latter is adjusted for 20% dermal absorption).

For intermediate term dermal exposure, the findings of two subchronic toxicity studies in the dog were considered together to provide the highest NOAEL and lowest LOAEL on which to base the quantitative hazard estimate. It is noted that the offspring NOAELs/ LOAELs from reproductive toxicity studies on acetochlor provide additional support for the endpoint selected. Long term exposure was assessed using the NOAEL from the dog chronic study as discussed above. These oral endpoints were adjusted for anticipated dermal absorption of 20%, as determined in a rat dermal penetration study.

Since there are no inhalation studies for acetochlor, oral studies were selected for inhalation risk assessment. The developmental NOAEL from the developmental toxicity study in the rat was selected to provide an appropriate endpoint that would be protective for pregnant females who may be occupationally exposed. The intermediate and long term inhalation exposure scenarios used the same studies as the dermal exposure scenarios of the same length ( see above).

For all exposure scenarios, uncertainty factors of 10x for interspecies and 10x for intraspecies variation ( total UF of 100x) were used. In addition, a database uncertainty factor of 10x ( total UF = 1000) was used for the acute dietary assessment to account for the lack of a developmental Page 46 of 118 neurotoxicity study in the rat.

Acetochlor is classified as " likely to be carcinogenic to humans," showing dose related increases in lung tumors in male and female mice, histiocytic sarcoma in female mice, nasal epithelial tumors in male and female rats and thyroid tumors in male and female rats. A Q 1 * of 3.27 x 10 2 was calculated, based on increased lung tumor incidence in male mice ( HED TXR# 0052743). Nasal epithelial cell tumors in the rat were considered to have a non linear dose response, based on the proposed mode of action for induction of these tumors ( see Section <IP_ADDRESS>).

4.1.6 FQPA The database is considered adequate for selection of study endpoints and determination of a doseresponse to characterize the potential prenatal or postnatal toxicity of acetochlor to infants and children. No increase in susceptibility was seen in developmental toxicity studies in rat and rabbit or in three multigeneration reproductive toxicity studies in the rat. Toxicity to offspring was observed at dose levels the same or greater than those causing maternal or parental toxicity. A developmental neurotoxicity study is required because evidence of neurotoxicity, was observed in studies in the dog and the rat including frank neuropathology in a chronic study in the dog. The uncertainty associated with the lack of this data is accounted for by use of a database uncertainty factor ( UF DB ) of 10, where appropriate. Since there are no residual uncertainties that indicate the need for a special safety factor, the factor has been reduced to 1X.

4.2 FQPA Hazard Considerations 4.2.1 Adequacy of the Toxicity Data Base The database for acetochlor is considered incomplete at this time. A developmental neurotoxicity study is required for acetochlor based on neurological observations, primarily in the dog ( see below), or an alternative test which addresses the sensitivity of the dog to neurological effects. In addition, submission of the positive control studies for validation of the laboratory methodology used in the acute and subchronic rat oral neurotoxicity screening studies is required as confirmatory data and to upgrade these studies to Acceptable/ guideline. However, the endpoints identified in the neurotoxicity studies are considered adequate for use in this risk assessment. The need for a 28 day inhalation study has not been evaluated at this time.

4.2.2 Evidence of Neurotoxicity Evidence of neurotoxicity from exposure to acetochlor was observed in several studies. Salivation and other clinical signs ( anogenital staining, diarrhea) were reported in some studies in both the rat ( two developmental toxicity studies) and the dog ( subchronic and chronic oral). A marginal decrease in brain cholinesterase was observed at the high dose in one subchronic rat study. The dog appears to be more sensitive than the rat or mouse to effects on the nervous system, in that salivation occurred at lower dose levels and frank neuropathology of the brain was observed in one study. In a Page 47 of 118 one year oral ( capsule) toxicity study in the dog, pronounced neurological signs ( ataxia, abnormal head movements, tremor, depressed righting, hopping and flexor reflexes, exaggerated tonic neck reflex and stiffness and rigidity of the hindlimbs) were observed at the high dose and were associated with degenerative lesions of the cerebellum. An acute and a subchronic oral neurotoxicity screening study in the rat were submitted for acetochlor. In the acute study at the time of peak effect, decreased total motor activity at mid and high dose was observed in females, but not males; at the highest dose, both males and females showed clinical signs of toxicity in the FOB ( perioral staining, piloerection, hunched posture). Single animals showed signs such as chromodacryorrhea, upward curvature of the spine and hypothermia. In the subchronic study, decreased body weight/ weight gain in both sexes at the high dose was observed. A decrease in hindlimb grip strength in males at 2 weeks, but not at later times, was observed and considered a possible effect of treatment. The neurotoxicity studies did not evaluate cholinesterase levels.

Acute Neurotoxicity Screening Study in Rats In an acute neurotoxicity study ( MRID 45357501), groups of fasted, 42 day old Alpk: AP f SD ( Wistar derived) rats ( 10/ sex/ dose) were given a single oral dose of acetochlor ( 94.7% a. i., batch/ lot # P11) at doses of 0, 150, 500 or 1500 mg/ kg bw and observed for 14 days. Neurobehavioral assessment ( functional observational battery and motor activity testing) was performed at pre test and study Days 1 ( time of peak effect), 8 and 15. Cholinesterase activity was not determined. At study termination, 5 animals/ sex/ group were euthanized and perfused in situ for neuropathological examination. Of the perfused animals, 5 rats/ sex from the control and high dose groups were subjected to histopathological evaluation of brain and peripheral nervous system tissues.

At 1500 mg/ kg bw, body weights adjusted for initial weight were significantly lower than the control group on Day 8 for males and on Days 1 ( peak effect), 8 and 15 for females. Body weight gains were significantly lower for the Day   7 to 8 time period for males ( 77% of controls) and during throughout the study for females ( 65 76% of controls). Food consumption by the high dose males and females was significantly reduced during the first week of the study compared with the controls. During the FOB, findings were limited to the time of peak effect at the high dose level. These consisted of hunched posture observed in 5 6 animals/ sex, piloerection on 7 10/ sex, and staining around the mouth seen in 3 4/ sex. The severity was considered slight in the males and from slight to moderate in females. Other findings at 1500 mg/ kg bw were decreased activity in one female, chromodacryorrhea in one female, hypothermia in one female, labored breathing in one male, sides pinched in in one male, and upward curvature of the spine in one female. No effects of treatment were noted on landing foot splay measurement, time to tail flick, or grip strengths. No effects of treatment were noted for motor activity in males. Total activity counts for high dose females on Day 1 were significantly decreased compared to controls at 500 and 1500 mg/ kg ( 401.7 and 251.7, respectively, vs. 571.4 for controls; equivalent to reductions of 30% and 56%) and also were decreased by 43.8% of the pre test value. A statistically significant increase in motor activity of 28% was also observed in high dose females on Day 8. There were no treatment related effects on brain weights or gross and histologic pathology or neuropathology. The LOAEL in females was 500 mg/ kg bw, based on dose related decreases in motor activity on Day 1 and the NOAEL was Page 48 of 118 150 mg/ kg bw. In males, the LOAEL was 1500 mg/ kg bw, based on decreased body weights and body weight gain ( males and females), reduced food consumption ( males and females), increased incidence of clinical signs during the FOB ( males and females) at the time of peak effect, with a NOAEL of 500 mg/ kg bw.

This neurotoxicity study is classified as Unacceptable/ Guideline ( upgradable) pending submission of information on positive control studies conducted at the performing laboratory and does not satisfy the guideline requirement for an acute neurotoxicity study in rats ( 870.6200; OECD 424). If such data have already been submitted to the agency, the registrant or performing laboratory should provide reference to the data and when the study( ies) were conducted. In addition, methods and equipment used for the quantitative measures during the FOB were not described. This information should also be provided.

4.2.3 Developmental Toxicity Studies Developmental Toxicity in Rats ( Two Studies Available)

( 1) In an oral developmental toxicity study ( MRID 00050929), 25 mated female Crl: CD ( SD) BR VAF/ Plus rats were administered acetochlor ( tech. 91.4% a. i., lot XHK 119) by gavage in corn oil vehicle ( dosing volume of 10 mL/ kg bw) as single daily doses of 0 ( corn oil only), 50, 200 or 400 mg/ kg/ day from Gestation Days 6 through 19, inclusive, with sacrifice and cesarean evaluations on Day 20.

Maternal toxicity: At 400 mg/ kg/ day, excessive salivation ( post dosing in 3 females on one occasion) and increased frequency of urogenital staining ( 13 rats, vs. 5, 9 and 6 in control, low and mid dose groups, respectively) were observed. Mean body weight gain during treatment ( Days 6 20) was decreased ( 30% less than controls) and gestational weight gain ( Days 0 20) adjusted for gravid uterine weight was approximately  50% less than controls ( adjusted gains from controls to high dose, respectively, were 43, 56, 46 and 20 g). There were no treatment related effects on survival or cesarean parameters. Food consumption was not evaluated in this study. The maternal toxicity LOAEL is 400 mg/ kg/ day, based on clinical signs of toxicity and reduced body weight gain during treatment. The NOAEL is 200 mg/ kg/ day.

Developmental toxicity: At 400 mg/ kg/ day, a slight but not statistically significant reduction in mean fetal weight was observed. No other cesarean parameters were affected and there were no treatment related increases in fetal variations or malformations. The developmental toxicity LOAEL is 400 mg/ kg/ day, based on slightly ( not statistically significant) reduced mean fetal weight. The developmental toxicity NOAEL is 200 mg/ kg/ day.

This oral developmental toxicity study in the rat is classified Acceptable ( guideline) and satisfies the guideline requirement for a developmental toxicity study ( OPPTS 870.3700; OECD 414) in the rat. [ Note: the original DER did not present data from the study and the only copy available of the original study report had cesarean tables that were illegible; the percent decrease in fetal weight at Page 49 of 118 400 mg/ kg/ day is therefore not available for this supplemental review. However, the reported findings are consistent with the findings in a later acceptable developmental toxicity study ( MRID 41592005; see supplemental DER in this TXR) and therefore a new copy of this study report is not required at this time.]

( 2) In an oral developmental toxicity study ( MRIDs 41592005, 42054902 and 42054903), 25 mated female Crl: CD ( SD) BR VAF/ Plus rats were administered acetochlor ( tech. 90.5% a. i., batch/ lot # 2) by gavage in corn oil vehicle ( dosing volume of 10 mL/ kg bw) as single daily doses of 0, 40, 150 or 600 mg/ kg/ day from Gestation Days 6 through 15, inclusive.

Maternal toxicity: At 600 mg/ kg/ day, two females died ( on GD 13 and 15), apparently due to treatment. Clinical signs of increased salivation post dosing and urogenital staining were reported. Mean body weight gain was reduced during the dosing period ( 47% below controls; mean corrected body weight gain from Days 6 to 20 was  38% below controls). A significant reduction in food consumption on Days 6 7 and 8 9 ( 20% and  16% less than controls, respectively) was observed only between Days 6 9 of dosing; thereafter, consumption was comparable among all groups. The maternal toxicity LOAEL is 600 mg/ kg/ day, based on mortality, clinical signs of toxicity and reduced body weight gain during treatment. The NOAEL is 150 mg/ kg/ day.

Developmental toxicity: At 600 mg/ kg/ day, the following effects showed statistically significant differences from controls and were considered treatment related: increased early resorptions/ dam ( 0.8 vs. 0.2, controls) and total resorptions/ dam ( 1.0 vs. 0.3, controls); increased postimplantation loss ( 7.6% vs. 3.5%, controls) and reduced mean fetal weight ( 7.1% less than controls). There were no treatment related increases in fetal variations or malformations. The developmental toxicity LOAEL is 600 mg/ kg/ day, based on increased fetal early resorptions and postimplantation loss and reduced mean fetal weight. The developmental toxicity NOAEL is 150 mg/ kg/ day..

This oral developmental toxicity study in the rat is classified Acceptable ( guideline) and satisfies the guideline requirement for a developmental toxicity study ( OPPTS 870.3700; OECD 414) in the rat.

Developmental Toxicity in the Rabbit ( Two Studies Available)

( 1) In a developmental toxicity study ( MRID 40134101), MON 097 ( acetochlor tech., 94.2% a. i., lot # XLF 349) was administered to 20 artificially inseminated female New Zealand White rabbits/ dose by gavage ( in a dosing volume of 0.5 mL/ kg bw corn oil) at dose levels of 0, 15, 50 or 190 mg/ kg bw/ day from days 7 through 19 of gestation, inclusive.

Maternal toxicity: At 190 mg/ kg/ day, maternal body weight loss during the treatment period was observed ( mean  146 g, compared to gain of + 81 g, + 112g and + 93 g for control, low and mid dose groups, respectively, from Days 7 19 of gestation). Loss of weight during treatment was reported in 14/ 19 high dose females compared to 4 5/ 18 19 in the other groups. A rebound in body weight gain was observed postdosing ( control to high dose mean gain from days 19 through 29 was 129, 30, 134 Page 50 of 118 and 231 g, respectively). There were no treatment related effects on survival, clinical signs of toxicity or cesarean parameters. The maternal toxicity LOAEL is 190 mg/ kg bw/ day, based on weight loss during treatment. The maternal toxicity NOAEL is 50 mg/ kg bw/ day.