Halauxifen-methyl l luman Health Risk Assessment DP No. D406145 

UNITED ST ATES ENVIRONMENT AL PROTECTlON AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: March 21, 20 16 

SUBJECT: Halauxifen-methyl - New Active Ingredient Human Health Risk Assessment for 

Proposed Uses on Cereal Grains (Barley, Wheat, and Triticale). 

FROM: 

PC Code: I 1750 I 

Decision No.: 470 169 

Petition No.: 2£8086 

Risk Assessment Type: Single Chemical Aggregate 

TXR No.: NA 

MRI DNo.: A 

DP Barcode: D406 I 45 

Registration No.: 627 19-AAE, AAG, 

AAN, AAR, AAU 

Regulatory Action: Section 3 

Case No.: None 

CAS No.: 94383 1-98-9 

40 Cf'R: New Active Ingredient 

Elizabeth Holman, DrPH, Physical Scientist ~ ~ Linnea Hansen, Ph.D., Biologist 0~'). ~ frv 

Douglas Dotson. Ph.D., Senior Chemist IJ.. t&"'~ \; A 

Zaida Figueroa, DrPH, Industrial Hygienist p~r-J??~/:_ 

Risk Assessment Branch II (RAB II) "r 

Health Effects Division (HED; 7509P) 

THROUGH: Christina Swartz, Branch Chief 

RAB II c~ HED (7509P) 

Charles W. Smith fll, Branch Chief. RAB I Q ~ . El

Ri

i

sk 

zabet

Assessment 

h Mendez, 

Rev

Ph.D

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., 

Comm

Senior 

itt

Toxicolo

ee 

gist ~ 

HED (7509P) 

TO: Dominic Schuler, Risk Manager Revie\.ver 

Reuben Baris, Risk Manager!Product Manager 25 

Dan Kenny, Branch Chief 

Herbicide Branch 

Registration Division (7505P) 

Page 1of1 26 
Halauxifen-methyl Human Health Risk Assessment DP No. 0406145 

1.0 EXECUTIVE SUMMARY ..................................................................... ............. ... .......................... 4 

2.0 HED RECOMME DATIO s ......................................................................................................... 7 

2.1 Data Deficiencies/Conditions of Registration .......................................... .......................... 7 

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

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

2.2.2 International Ha1monization ................................................................................... 8 

2.2.3 Recommended Tolerances ...................................................................................... 8 

2.2.4 Revisions to Petitioned-For Tolerances .................................................................. 9 

2.3 Label Recommendations ............................... .................................................................... 10 

3.0 INTRODUCTION .................................................................................. .......... ............... ............. 10 

3.1 Chemical Identity .............................................................................................................. 10 

3.2 Physical/Chemical Characteristics ............. ....................................................................... 11 

3.3 Pesticide Use Pattern ................................................................. ........................................ 11 

3.4 Anticipated Exposure Pathways ....................................................................................... 12 

3.5 Consideration of Environmental Justice ...... ............. ........................................................ 13 

4.0 H AZARD CHARACTERIZATION A D DOSE-RESPONSE ASSESSMENT ......................................... 13 

4.1 Toxicology Studies Available for Analysis ...................................................................... 14 

4.2 Absorption, Distribution, Metabolism, & Elimination (ADME) ............. .......... ............... 15 

4.2.1 Dermal Absorption ................................................................................................ 16 

4.3 Toxicological Effects ........................................................................................................ 17 

4.3.2 Mode of Action for Liver Toxicity of Halauxifen-Methyl ................................... 20 

4.4 Safety factor for Infants and Children (f.'QPA Safety Factor) .......................................... 23 

4.4.1 Completeness of the Toxicology Database ........................................................... 23 

4.4.2 Evidence of Neurotoxicity .................................... ........ ........................................ 23 

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal ....... 23 

4.4.4 Residual Uncertainty in the Exposure Database ................................................... 24 

4.5 Toxicity Endpoint and Point of Departure Selections ... ................................................... 24 

4.5. 1 Dose-Response Assessment .................................................................................. 24 

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment.. .... 26 

4.5.3 Cancer Classification and Risk Assessment Recommendation ............................ 27 

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

Assessn1ent. ................................................................................................................. 27 

5.0 D IETARY EXPOSURE A D R ISK ASSESSMENT .................. ...... ............... .................................... 30 

5.1 Metabolite/Degradate Residue Profile .............................................................................. 30 

5.1.1 Summary of Plant and Animal Metabolism Studies ............................................. 30 

5.1.2 Summary of Environmental Degradation ....... ............. ......................................... 31 

5.1.3 Comparison of Metabolic Pathways ..................................................................... 32 

5.1.4 Residues of Concern Summary and Rationale ...................................................... 33 

5.2 Food Residue Profile ...... ................................... ....... ..... .................................................... 34 

5.3 Water Residue Profile ....................................................................................................... 35 

5.4 Dietary Risk Assessment .................................................................................................. 35 

5.4.1 Description of Residue Data Used in Dietary Assessment.. ..... ... ......................... 36 

5.4.2 Dietary Exposure Risk Estimates .......................................................................... 36 

5.4.3 Dietary Risk Assessment Summary Table ............................................................ 36 

6.0 R ESIDENTIAL (NON-0CCUPATl01 AL) EXPOSURE/RISK CllARACTERIZJ\TION .......................... 37 

6.1 Residential Bystander Post-Application Inhalation Exposure .. ........................................ 37 

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Halauxifen-methyl Human Health Risk Assessment DP No. D406145

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6.2 Spray Drift ........................................................................................................................ 37

7.0 AGGREGATE EXPOSURE/RISK CHARACTERIZATION................................................................. 38

8.0 CUMULATIVE EXPOSURE/RISK CHARACTERIZATION ............................................................... 38

9.0 OCCUPATIONAL EXPOSURE/RISK CHARACTERIZATION ........................................................... 38

9.1 Short- and Intermediate-Term Handler Risk .................................................................... 38

9.2 Short- and Intermediate-Term Post-Application Risk ...................................................... 40

10.0 REFERENCES ...................................................................................................................... 41

APPENDIX A. TOXICOLOGY PROFILE AND EXECUTIVE SUMMARIES............................................ 42

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

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

A.3 Hazard Identification and Endpoint Selection ............................................................ 63

A.4 Executive Summaries.................................................................................................. 66

APPENDIX B. METABOLISM SUMMARY TABLES.......................................................................... 117

APPENDIX C. PHYSICAL/CHEMICAL PROPERTIES ........................................................................ 125

APPENDIX D. REVIEW OF HUMAN RESEARCH ............................................................................. 126
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

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1.0 Executive Summary

Dow AgroSciences LLC has petitioned the Agency to register the herbicide halauxifen-methyl

for use on wheat, barley, and triticale and to establish permanent tolerances for residues resulting 

from those uses. This document presents the HED aggregate human health risk assessment for 

halauxifen-methyl.

Halauxifen-methyl (methyl (4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)-2-

pyridinecarboxylate) is a new arylpicolinate post-emergence herbicide, and was reviewed as part 

of a global joint review project. The review partners were the United States Environmental 

Protection Agency (U.S. EPA), the Pest Management Regulatory Agency (PMRA) of Canada 

and the Australian Pesticides and Veterinary Medicines Authority (APVMA). The submitted 

studies make up a complete database for the purpose of conducting an aggregate human health 

risk assessment. No risks of concern were identified in this assessment, from either occupational 

exposures or from dietary exposures, for the general U.S. population or any population subgroup. 

Under the proposed use pattern, halauxifen-methyl may be applied as a post-emergence 

broadcast spray through a variety of application methods including ground and aerial equipment 

at a maximum single application rate of 0.0091 lb ai/A. There is a potential for occupational 

exposure associated with handler activities (i.e., mixing, loading, and applying) and for workers 

re-entering treated areas. Based on the use sites, there is the potential for dietary exposure to 

halauxifen-methyl and/or its breakdown products via consumption of treated crops and from 

drinking water. There are no proposed uses that would result in residential exposures. 

The toxicology database for halauxifen-methyl is considered adequate and no additional data are 

required at this time. Following oral exposure, halauxifen-methyl is rapidly hydrolyzed in the 

liver to its major metabolite, XDE-729 acid, which then enters systemic circulation. The liver is 

therefore exposed pre-systemically to parent halauxifen-methyl prior to its being metabolized to 

XDE-729 acid, but other organs are exposed to XDE-729 acid. The guideline studies in support 

of registration of halauxifen-methyl were conducted on XDE-729 acid and identified the kidney 

as the main target organ. Although halauxifen-methyl is rapidly converted to XDE-729 acid, 

kidney effects were not observed after halauxifen-methyl exposure because the blood levels of 

the acid from exposure to the parent compound were lower than those seen with comparable 

exposure to the acid per se. Bridging studies designed to compare the toxicity profile of 

halauxifen-methyl and XDE-729 acid identified liver as the target organ for halauxifen-methyl. 

However, chronic toxicity data from the XDE-729 acid could not be used for bridging because 

liver toxicity from subchronic exposure to halauxifen-methyl occurred at lower doses. In lieu of 

conducting long-term studies on halauxifen-methyl, mechanistic studies were conducted. These 

studies identified activation of the liver aryl hydrocarbon receptor (AhR) as the mode of action 

(MOA), and the molecular initiating event (MIE), for liver toxicity, for which increased liver 

Cyp1a1 gene expression serves as a biomarker. Due to the distinct toxicities of the halauxifenmethyl and XDE-729 acid and the unique MOA for liver toxicity of halauxifen-methyl, risk from 

the two compounds was assessed separately. Therefore, bridging and mechanistic studies were 

considered, along with the guideline studies, in selection of the dose and endpoint for halauxifenmethyl. Based on the abundance of guideline and mechanistic data available, a MOA approach 

was used for the identification and characterization of hazard. A point of departure (POD) for 
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increased Cyp1a1 expression was identified in the rat 90-day dietary study and was selected for 

risk assessment of halauxifen-methyl, since it protects for the initial step in liver toxicity. 

There was no evidence of dermal toxicity, neurotoxicity or immunotoxicity. Inhalation studies 

were waived because test atmospheres could not be generated for either compound, and the 

margin of exposure (MOEs) for inhalation exposure using the POD of 3 mg/kg/day were 

adequately protective (>1000); therefore, an additional database uncertainty factor (UFDB) is not 

required in the absence of the inhalation study. Neither compound was acutely toxic (Category 

IV). Both are mild eye irritants (Category III) but not dermal irritants or sensitizers. XDE-729 

acid is classified as “not likely to be carcinogenic to humans.” Halauxifen-methyl is classified as 

“not likely to be carcinogenic to humans at doses that do not induce Cyp1a1 expression,” based 

on the premise that AhR activation and subsequent promotion of hepatocellular tumors due to a 

sustained increase in hepatocellular proliferation depend upon this MIE. Neither show evidence 

of genotoxicity.

There was no evidence of increased prenatal susceptibility to either compound and no evidence 

of postnatal susceptibility to XDE-729 acid. Residual concerns for postnatal susceptibility to 

halauxifen-methyl in the absence of reproductive toxicity data are low, due to selection of a 

highly conservative endpoint and assumptions for dietary exposure, and the low level of 

exposure expected from proposed use patterns. The FQPA safety factor (SF) is therefore 

reduced to 1x.

Acute dietary assessments were not conducted due to lack of single-dose effects. For chronic 

dietary exposure to XDE-729 acid, the NOAEL of 20.3 mg/kg/day from the rat chronic toxicity/

carcinogenicity study was selected, based on kidney effects. For chronic dietary exposure to 

halauxifen-methyl, the POD of 3.0 mg/kg/day for mildly increased liver Cyp1a1 expression in 

the rat 90-day study was selected. Although this endpoint was not adverse - no liver effects were 

observed at the POD - in the absence of long-term data, it is considered protective because liver 

toxicity will not result without the MIE. For non-dietary exposure, only occupational endpoints 

for halauxifen-methyl were selected because there are no proposed residential uses. 

Occupational handlers/applicators will be exposed only to the parent in product formulations. 

Dermal exposure was not assessed due to lack of toxic effects via the dermal route. For 

inhalation exposure, the MIE (POD of 3 mg/kg/day), selected in the absence of inhalation data, 

was considered conservative and protective of potential portal of entry and systemic effects. 

Toxicity via the inhalation route was assumed by default to be equivalent to toxicity via the oral 

route.

The plant and livestock metabolism studies are adequate for determination of the residues of 

concern in plants and livestock for both tolerance enforcement and risk assessment for the 

purposes of the current tolerance petition. HED has determined that the residue of concern in 

primary crops and rotational crops for both tolerance enforcement and risk assessment is parent 

halauxifen-methyl only. The residue of concern in ruminants and poultry for both tolerance 

enforcement and risk assessment is the metabolite X11449757 (4-amino-3-chloro-6-(4-chloro-2-

fluoro-3-hydroxyphenyl)-pyridine-2-carboxylic acid). Based on environmental fate studies, the 

breakdown of halauxifen-methyl in drinking water is more complex. In drinking water, the 

residues may include parent halauxifen-methyl, as well as transformation products XDE-729 
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acid, X11449757, X11406790, degradates 1, 2, 4, 10, 11, and 14. The distribution of residues 

between these compounds will be dependent on the source of the drinking water (surface or 

groundwater) as well as the amount of elapsed time since application of the product. Regulatory 

enforcement in primary crops is based on parent halauxifen-methyl only. Enforcement of 

tolerances can be accomplished by a validated LC-MS/MS analytical method with a limit of 

quantitation of 0.01 ppm. No livestock commodity tolerances are required for the proposed uses 

because there is no reasonable expectation of finite residues in these commodities. 

Following application of halauxifen-methyl according to the directions for use on the proposed 

labels, residues of halauxifen-methyl are expected to be below the limit of quantitation of 0.01 

ppm in wheat and barley grain and most of the associated livestock commodities; the only 

detectable residues were found in wheat straw, hay, and forage. 

As discussed above, the toxicological effects caused by parent halauxifen-methyl and the XDE729 acid metabolite are different. The parent targets the liver, while the metabolite causes 

kidney effects. In the field trials, no residues of parent or acid were found in wheat or barley 

grain. The acid metabolite is a major degradate in drinking water. As the parent and metabolite 

have different toxicological effects, separate chronic toxicity endpoints were selected for them

and separate chronic dietary exposure assessments were performed for the two compounds. For 

parent halauxifen-methyl, an assessment was performed for food and drinking water. This

assessment was based on tolerance level residues in food and the assumption that 100% of all 

barley, wheat, and triticale consumed will be treated with halauxifen-methyl. Drinking water 

residues for halauxifen-methyl were negligible and did not contribute to the dietary exposure. 

For the acid metabolite, the assessment was based on drinking water alone because the acid 

metabolite is not a residue of concern in food, but it is a residue of concern in water. 

Dietary risk estimates for halauxifen-methyl and XDE-729 acid are well below HED’s level of 

concern. In both of the chronic dietary exposure assessments that were performed, the general 

U.S. population and all population subgroups used <1% of the chronic population adjusted dose 

(cPAD). For the chronic dietary exposure assessments with parent and XDE-729 acid, the 

maximum exposure estimates were for the “all infants” subpopulation. 

As there are no residential uses for halauxifen-methyl, there are no residential exposure 

contributions to the aggregate risk estimates. Therefore, aggregate risk estimates are equivalent 

to the dietary risk estimates and are also below HED’s level of concern.

For occupational scenarios, long-term exposures (> 6 months) are not expected to occur; 

however, short- (1-30 days) and intermediate-term (1-6 months) exposures may occur. 

Occupational assessments for halauxifen-methyl are based on inhalation exposures only. All 

occupational handler scenarios for the proposed uses resulted in MOEs greater than 100 at 

baseline personal protective equipment (PPE; i.e., baseline attire with no respirator; engineering 

control for aerial applications (enclosed cockpits)) and, therefore, are not of concern. Although a 

POD from an oral study was used to assess handler inhalation risk, the calculated MOEs are all 

≥2,500, thus providing an ample margin to account for any uncertainties in the route-to-route 

extrapolation. Based on the Agency's current practices, a quantitative non-cancer occupational 

post-application inhalation exposure assessment is not being performed for halauxifen-methyl at 
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this time. If new policies or procedures are put into place, the Agency may revisit the need for a 

quantitative occupational post-application inhalation exposure assessment for halauxifen-methyl.

There are no human health risk concerns that would preclude granting the requested registrations 

for halauxifen-methyl and establishing tolerances for residues at the recommended levels. 

Potential areas of environmental justice concerns were considered in this human health risk 

assessment to the extent possible. For more information, see Section 3.5.

This risk assessment relies in part on data from studies in which adult human subjects were 

intentionally exposed to a pesticide or other chemical. Those studies are subject to ethics review, 

have received that review, and are compliant with applicable ethics requirements.

2.0 HED Recommendations

Provided the recommended changes are made to the halauxifen-methyl end-use product labels

(see Section 2.3), HED recommends registering halauxifen-methyl for use on wheat, barley, and 

triticale. Furthermore, HED recommends establishing permanent tolerances for residues related 

to halauxifen-methyl as discussed in Section 2.2.

2.1 Data Deficiencies/Conditions of Registration

None.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

Suitable methods for tolerance enforcement have been developed and independently validated. 

For all matrices and analytes, the limit of quantitation (LOQ), defined as the lowest spiking level 

where acceptable precision and accuracy data were obtained, was determined to be 0.01 mg/kg. 

The limit of detection (LOD) was defined to be 30% of the LOQ (i.e., 0.003 mg/kg). 

The FDA multi-residue methods are not suitable for detection and enforcement of halauxifenmethyl and XDE-729 acid in non-fatty matrices. The QuEChERS multiresidue method was 

evaluated for the quantitative determination of residues of halauxifen-methyl and XDE-729 acid 

in agricultural commodities representative of the four OECD crop types: wet crop, dry crop, oily 

crop, and acidic crop. This QuEChERS multiresidue method was found to be adequate for the 

determination of residues of halauxifen-methyl and XDE-729 acid in the four types of crops

tested. 

Plant Commodity Enforcement Method

Residues of halauxifen-methyl and XDE-729 acid are extracted with acetonitrile/water (80:20, 

v/v) and the sample is centrifuged. An aliquot of supernatant is filtered/centrifuged and cleaned 

up by solid-phase extraction. After preparation, the samples are analyzed by liquid 

chromatography using a C8 column coupled with positive-ion electrospray LC-MS/MS, 
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monitoring two MS/MS transitions characteristic of each analyte and one MS/MS transition 

characteristic of each internal standard. For halauxifen-methyl, the ion transition m/z 345

[M+H]+ → 250 is used for quantification, and the transition m/z 345 → 235 is used for 

confirmation. For XDE-729 acid, the ion transition m/z 331 [M+H]+ → 250 is used for 

quantification, and the transition m/z 331 → 285 is used for confirmation. 

Livestock Commodity Enforcement Method

Dow AgroSciences proposed the method described in Report 110505 as the analytical 

enforcement method for halauxifen-methyl and XDE-729 acid in livestock matrices. However, 

HED has determined that the residue of concern in livestock commodities is the metabolite 

X11449757. Based on the available data for residues in livestock feedstuffs associated with this 

petition, quantifiable residues in livestock matrices are not expected, and a validated livestock 

enforcement method is not necessary at this time. In the cattle feeding study, samples of cattle 

commodities were analyzed using a modification of the proposed enforcement method. In the 

event that tolerances are needed for livestock commodities in the future, the petitioner will need 

to submit an adequate enforcement method for residues of X11449757 in livestock commodities 

and an independent laboratory validation (ILV) for the method.

2.2.2 International Harmonization

Halauxifen-methyl is a new active ingredient, and no MRLs have been established by Codex, 

Canada, or Mexico. The U.S., Canada, and Australia, all agreed to use the OECD MRL 

calculation procedures for this tolerance petition. In addition, the three countries are in 

agreement with respect to the residues of concern for tolerance enforcement. The residue of 

concern in plant commodities is parent halauxifen-methyl only. The residue of concern in 

livestock commodities is the metabolite X11449757.

The tolerances/MRLs that are being recommended by the U.S. and Canada are harmonized to the 

extent possible. In the cases where tolerances/MRLs are not being harmonized, it is because of 

policy differences in the way the two countries establish tolerances/MRLs. The only MRLs that 

are not being harmonized are those for livestock feed items and livestock commodities. Canada 

does not establish MRLs for livestock feed items, whereas the U.S. does establish tolerances. 

The U.S. is establishing tolerances for residues in wheat forage, hay, and straw, as well as for 

barley hay and straw. On the other hand, the U.S. generally does not establish tolerances for 

livestock commodities when there is no reasonable expectation of finite residues. Canada 

establishes LOQ MRLs in these situations. As a result, Canada is establishing LOQ MRLs for 

ruminant, poultry, and swine commodities, whereas the U.S. is not establishing tolerances.

2.2.3 Recommended Tolerances

HED recommends that 40 CFR be amended by establishing tolerances for the plant commodities 

listed in the table below. The residue definitions for plant and livestock commodities are 

different, as a result, HED has prepared two separate tolerance summary tables for them. For 

plant commodities, the tolerances should be established as follows: 

Tolerances are established for residues of halauxifen-methyl, including its metabolites
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and degradates, in or on the commodities in the table below. Compliance with the

tolerance levels specified below is to be determined by measuring only halauxifen-methyl

(methyl (4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxyphenyl)-2

-pyridinecarboxylate).

Table 2.2.3.1. Tolerance Summary for Halauxifen-methyl.

Commodity as Proposed by 

Petitioner

Proposed 

Tolerance 

(ppm)

Recommended 

Tolerance 

(ppm)

Comments

(correct commodity definition)

Plant Commodity Tolerances

barley, grain 0.01 0.01 Barley, grain

barley, hay 0.01 0.01 Barley, hay

barley, straw 0.01 0.01 Barley, straw

wheat, forage 0.5 0.50 Wheat, forage

wheat, grain 0.01 0.01 Wheat, grain

wheat, hay 0.04 0.03 Wheat, hay

wheat, straw 0.015 0.015 Wheat, straw

Dow AgroSciences did not propose a tolerance for triticale, and does not need to because a 

tolerance for wheat automatically applies to triticale (40 CFR §180.1(g)).

Tolerances are not needed for livestock commodities due to the lack of quantifiable residues in 

livestock commodities associated with the proposed uses. The tolerances that the petitioner 

proposed are listed in the following table.

Table 2.2.3.2. Petition Proposed Livestock Commodity Tolerance Summary for Halauxifen-methyl.

Commodity

Proposed 

Tolerance 

(ppm)

Recommended 

Tolerance (ppm) Comments

(correct commodity 

definition)

cattle, fat 0.01 None

cattle, meat 0.01 None

cattle, meat byproducts 0.01 None

goat, fat 0.01 None

goat, meat 0.01 None

goat, meat byproducts 0.01 None

horse, fat 0.01 None

horse, meat 0.01 None

horse, meat byproducts 0.01 None

milk 0.01 None

sheep, fat 0.01 None

sheep, meat 0.01 None

sheep, meat byproducts 0.01 None

2.2.4 Revisions to Petitioned-For Tolerances
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As can be seen in Table 2.2.3.1, above, the only recommended tolerance for treated crops that is 

different from the proposed tolerance is that for wheat hay. The petitioner proposed a tolerance 

of 0.04 ppm, whereas HED determined that a tolerance of 0.03 ppm is appropriate. When the 

petitioner determined the proposed tolerances, it included the metabolite XDE-729 acid as a 

residue of concern. HED subsequently determined that this metabolite is not a residue of 

concern for tolerance enforcement. The petitioner should submit a revised Section F in which a 

tolerance of 0.03 ppm is proposed for wheat, hay. In addition, the petitioner proposed livestock 

commodity tolerances, but HED has determined that they are not required because quantifiable 

residues are not expected in livestock commodities. 

2.3 Label Recommendations

The proposed labels specify the single application rates and the maximum seasonal application 

rates. HEDs risk assessment is based on these maximum application rates. It is noted that the 

labels do not specify the maximum number of applications that may be made per season. For 

this assessment, HED has inferred the number of applications per season by comparing the single 

application rate to the maximum seasonal application rate. On each of the labels, the petitioner 

needs to specify the maximum number of applications that may be made per growing season. 

Some of the end-use products proposed for registration contain multiple active ingredients. For 

those products, RD should ensure that the use restrictions on the proposed labels comply with 

any restrictions associated with the co-formulated active ingredients.

3.0 Introduction

3.1 Chemical Identity

Table 3.1. Nomenclature and Identification Information for Halauxifen-Methyl.

Active Ingredient

Common Name Halauxifen-methyl

Company Experimental Name XDE-729 methyl or XDE-729 methyl ester 

Company Experimental Number X11393728

IUPAC Name Methyl 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-

methoxyphenyl)pyridine-2-carboxylate

Molecular Formula C14 H11O3N2Cl2F 

Molecular Weight 345.16

CAS Name Methyl 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-

methoxyphenyl)-2-pyridinecarboxylate 

CAS Number 943831-98-9
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3.2 Physical/Chemical Characteristics

Technical-grade halauxifen-methyl is a solid at room temperature. The compound has a low 

vapor pressure (1.1 x 10-10 mm Hg at 25°C), and a low proposed use rate (0.0091 lb ai/A); 

therefore, there is a relatively low likelihood of exposure to halauxifen-methyl in the vapor 

phase. The technical-grade material has a log KOW of 3.8 indicating a somewhat-fat-soluble 

compound, but not to the degree that bioaccumulation is of particular concern. A listing of the 

physical/chemical properties of halauxifen-methyl and the XDE-729 acid metabolite are 

provided in Appendix C.

3.3 Pesticide Use Pattern

Halauxifen-methyl has been proposed for use on wheat, barley, and triticale. Halauxifen-methyl 

is a post-emergent herbicide developed for control of a broad spectrum of broadleaf and sedge 

weeds. Application is intended prior to harvesting crops in the field. 

The proposed use directions are summarized in Table 3.3. Four agricultural products are 

proposed for use on wheat, barley, and triticale: GF-2685, GF-2686, GF-2687, and GF-2688. 

These end use products may be applied through a variety of application methods including: aerial 

and ground equipment. These products are not to be applied using any type of chemigation 

equipment. The proposed Restricted Entry Interval (REI) is 12 hours for all crops.

The labels do not specify the maximum number of applications that may be made. The 

recommended single application rate is approximately 0.005 lb ai/A. For GF-2685, the 

maximum seasonal application rate is 0.0091 lb ai/A. These application rates indicate that the 

total number of applications of GF-2685 should not exceed two.

The label for GF-2685 states that livestock may be grazed 1 day following application. The only 

active ingredient in this product is halauxifen-methyl. The labels for GF-2686, GF-2687, and 

GF-2688 state that livestock may be grazed 7 days following application. These products 

contain either florasulam or fluroxypyr in addition to halauxifen-methyl. 

The PPE statement on the proposed labels requires applicators and other handlers to wear: longsleeved shirt and long pants, chemical-resistant gloves, and shoes plus socks. 
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Table 3.3. Proposed Uses of Halauxifen-Methyl on Wheat, Barley, and Triticale.

Applic. 

Timing, 

Type, and 

Equip.a

Formulation (EPA 

Reg. No.)b

Application Rate

(lb ai/A)c

Max No. 

Applic. Per 

Seasond

Max. Seasonal 

Applic. Rate

(lb ai/A) PHIe

Use 

Directions 

and 

Limitationsf

Ground 

and Aerial 

Equipment

GF-2685 WG

(62719-AAN)

0.0045-0.0091

1-2

(depending 

on rate)

0.0091

60 

Days

See 

Footnotes

GF-2686 WG

(62719-AAR)

0.0049 1 0.0049

GF-2687 WG

(62719-AAE)

0.0049 1 0.0049

GF-2688 EC

(62719-AAG) 0.0058 1 0.0058

a: Do not apply using any type of chemigation equipment. Do not apply halauxifen-methyl to more than two 

growing seasons per year (i.e., Spring and/or Fall). 

b: WG: Water dispersible granule.

 EC: Emulsifiable concentrate.

c: For the proposed label GF-2685, the maximum single application rate of 0.0091 lb ai/A is for heavy infestations 

or larger stages of weeds.

d: The maximum number of applications per season per year is not specified on the labels. Those given in the table 

are inferred based on the single application rates and the maximum seasonal application rate.

e: PHI: Do not apply within 60 days of crop harvest. Do not apply closer than 21 days before cutting of hay.

 Livestock may be grazed on treated crops 1 day following application.

f: To minimize spray drift, apply in a total spray volume of 3 gallons or more per acre.

3.4 Anticipated Exposure Pathways

Based on the proposed use pattern on grain crops, humans may be exposed to halauxifen-methylrelated residues in food from treated crops and from migration of residues to surface and/or 

groundwater sources of drinking water. From crops, the exposure will be primarily to parent 

halauxifen-methyl. In drinking water, the residues may include parent halauxifen-methyl, as 

well as transformation products XDE-729 acid, X11449757, X11406790, and degradates 1, 2, 4, 

10, 11, and 14. The distribution of residues between these compounds will be dependent on the 

source of the drinking water (surface or groundwater) as well as the amount of elapsed time since 

application of the product.

At this time, there are no proposed residential uses of halauxifen-methyl, and exposure via 

residential pathways is not expected.

In an occupational setting, there is a potential for occupational exposure associated with handler 

activities (i.e., mixing, loading, and applying). Post-application exposure is expected for workers 

re-entering treated areas.

 
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3.5 Consideration of Environmental Justice

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

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

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

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

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

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

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

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

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

Health and Nutrition Survey/What We Eat in America (NHANES/WWEIA) and are used in 

pesticide risk assessments for all registered food uses of a pesticide. These data are analyzed and 

categorized by subgroups based on age and ethnic group. Additionally, OPP is able to assess 

dietary exposure to smaller, specialized subgroups and exposure assessments are performed 

when conditions or circumstances warrant. Whenever appropriate, non-dietary exposures based 

on home use of pesticide products and associated risks for adult applicators and for toddlers, 

youths, and adults entering or playing on treated areas post-application are evaluated. Further 

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

development of specialized software and models that consider exposure to bystanders and farm 

workers as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0 Hazard Characterization and Dose-Response Assessment

The hazard characterization for halauxifen-methyl takes into consideration the distinct 

toxicological profiles of both halauxifen-methyl and its major metabolite, XDE-729 acid. 

Following oral exposure, halauxifen-methyl is absorbed in the gut and transported via the 

enterohepatic circulation to the liver, where it is rapidly and completely metabolized via 

hydrolysis at the methyl ester to form XDE-729 acid, before entering systemic circulation. 

Therefore, the liver is exposed pre-systemically to halauxifen-methyl, but exposure to other 

tissues and organs through systemic circulation is to XDE-729 acid. Based on the rapid 

hydrolysis of the parent to acid and the similarity of their structures, it was anticipated that they 

would show comparable toxicity. The guideline toxicology studies for halauxifen-methyl were 

conducted on XDE-729 acid, with additional bridging studies conducted on halauxifen-methyl

(with the exception that the immunotoxicity study was conducted on the parent compound). 

In fact, very different toxicological profiles were observed. Comparison of the subchronic rat 

studies showed that the kidney is the target organ for XDE-729 acid, whereas for halauxifenmethyl, the target organ is the liver. Furthermore, the liver effects of halauxifen-methyl occurred

at significantly lower doses than those at which XDE-729 acid caused kidney toxicity. 

Therefore, the data on halauxifen-methyl cannot be used to bridge to the acid.

In lieu of conducting a complete set of guideline toxicity studies on halauxifen-methyl to 

evaluate long-term toxicity, mechanistic studies were performed to characterize the mode of 

action (MOA) for liver toxicity, with the anticipation that this would provide adequate 

information, when considered together with the bridging data and studies on XDE-279 acid, to 
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characterize toxicity from halauxifen-methyl. The mechanistic data support a MOA of activation 

of liver aryl hydrocarbon receptor (AhR) by halauxifen-methyl (but not XDE-729 acid). AhR 

activation is a well-known MOA that has been characterized for a number of compounds, most 

notably dioxin, and that may affect different tissue types (reviewed in Nebert et al., 2004; Cohen, 

2010; Safe et al., 2013). In the liver, this MOA causes liver hypertrophy, histopathology and 

cellular proliferation, and with sustained AhR activation, over time may promote the formation 

of hepatocellular tumors due to increased liver cell proliferation. AhR activation through 

binding by the ligand, halauxifen-methyl, is considered the molecular initiating event (MIE), an 

initial prerequisite event upon which the subsequent effects are dependent. An early indicator of 

AhR activation is increased expression of the liver Cyp1a1 gene, which serves as a readily 

measurable biomarker for the MIE. Therefore, in addition to the liver toxicity reported in the rat 

subchronic toxicity studies, increases in Cyp1a1 gene expression may be used to identify dose 

levels at which there is the potential for liver effects from AhR activation with long-term 

exposure, in the absence of long-term animal toxicity studies. The MOA and supporting data are 

summarized below (Section 4.3.2) and in greater detail in a separate document (TXR # 

0051197). The extensive guideline and mechanistic study database for halauxifen-methyl and 

XDE-729 acid allowed the Agency to use a MOA approach for characterization of hazard and 

identification of a MIE for halauxifen-methyl and endpoints for risk assessment.

4.1 Toxicology Studies Available for Analysis

The toxicology database is considered adequate for assessment of human health risk from 

exposure to halauxifen-methyl. No additional studies are required at this time. This 

determination is in accordance with the global review partners, Australia OCS and Canada 

PMRA, as well as the European Union. In mammals, halauxifen-methyl is rapidly hydrolyzed 

(demethylated) in vivo to its major metabolite, XDE-729 acid, and therefore the majority of the 

guideline toxicity studies were conducted on the acid, with bridging studies conducted on 

halauxifen-methyl. 

The following guideline studies were submitted:

Halauxifen-methyl XDE-729 acid

Acute toxicity battery (except for acute 

inhalation)

Acute toxicity battery (except for acute 

inhalation)

28- and 90-day subchronic (rat) 28- and 90-day subchronic (rat, mouse, dog)

- 28-day dermal (rat)

Prenatal developmental (rat, rabbit) Prenatal developmental (rat, rabbit)

- Two-generation reproductive toxicity (rat)

- Chronic toxicity (dog)

- Carcinogenicity (mouse)

- Chronic toxicity/carcinogenicity (rat)

Genotoxicity battery Genotoxicity battery

- Neurotoxicity (rat, acute and subchronic)

Metabolism (rat) Metabolism (rat)

Dermal penetration, rat (in vivo and in vitro) -

Immunotoxicity (rat) -

A 28- or 90-day inhalation study was not conducted for either compound because of difficulties 

in generating a test atmosphere containing respirable particles (1 to 4 μm diameter) and clogging 

of the aerosol delivery apparatus. However, the available oral endpoint was adequately 
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protective for inhalation exposure, based on high MOEs (Level of Concern or LOC=1000; 

MOEs all 2500 or greater), and both compounds show low volatility. Based on these 

considerations, the HED Hazard Science Policy Council (HASPOC) concluded that the 

inhalation study is not needed (TXR No. 0056954). 

The bridging studies on halauxifen-methyl indicated that its toxicological profile is very different 

from that of XDE-729 acid. Liver toxicity, an effect not observed with the acid form, was 

observed at lower dose levels of halauxifen-methyl than those of XDE-729 acid at which kidney 

effects were observed. To elucidate the basis for these differences, mechanistic studies on 

halauxifen-methyl were also submitted to characterize the mode of action (MOA) for liver 

toxicity, and to evaluate the comparative metabolism of XDE-729 acid in the dog, rat and mouse. 

Genotoxicity studies were submitted for the minor metabolite X11449757 [4-Amino-3-chloro-6-

(4-chloro-2-fluoro-3-hydroxyphenyl)-pyridine-2-carboxylic acid], which is derived from XDE729 acid by demethylation at the methoxy group of the phenyl ring. No additional data are 

required for this metabolite, as it is a more polar compound and is not expected to be more toxic 

than either halauxifen-methyl or XDE-729 acid.

There is no published literature information specific to halauxifen-methyl available for 

consideration, as this is a new proposed active ingredient.

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

Rat metabolism studies were conducted on both XDE-729 acid and halauxifen-methyl. XDE-729 

acid was rapidly and well-absorbed following a single 10 mg/kg gavage dose, with a time to 

peak plasma concentration (Tmax) of ~0.2 hr. Elimination was primarily via the urine (68-92% 

administered dose (AD)), with 11-29% excreted in feces and most of the AD excreted within 24 

hr of dosing. Fecal excretion was mostly due to biliary excretion, based on fecal excretion in the 

IV dose group. Females excreted somewhat higher radioactivity levels in the urine than males. 

Elimination was biphasic, with an initial phase of 0.5-0.9 hr and a slower terminal phase of 6-16 

h at 10 mg/kg. At 750 mg/kg, the initial elimination phase was slower (2.7-4.3 hr), but the 

terminal phase was faster (5.6-8.7 hr), and the plasma area under the curve (AUC) was 2.0-2.6-

fold higher than the low dose. Taken together, the data suggest saturation of excretion at higher 

doses. Radioactivity was not retained in tissues. XDE-729 acid was mostly excreted 

unmetabolized. Minor metabolites (<5% AD) included the acyl-glucuronide conjugate, hydroxy 

XDE and O-demethylated XDE-729 acid, and corresponding sulfate and glucuronide conjugates. 

Halauxifen-methyl showed similar kinetics following a 10 mg/kg gavage dose. Tmax was slightly 

longer (0.50-0.44 h, M/F) than XDE-729 acid (0.19-0.15 hr, M/F), and Cmax was lower (1.6 to 

3.6-fold lower), but maximum absorption and biphasic elimination t1/2s, and the AUCs, were 

comparable. Radioactivity was not retained in tissues. At or near the peak plasma radioactivity 

concentration, the primary analyte was XDE-729 acid, indicating rapid hydrolysis of the ester in 

vivo. The major radioactive component in urine and feces was XDE-729 acid (80% AD), with 

minor metabolites (<5% AD) similar to those of XDE-729 acid. 
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Additional metabolism studies were conducted to evaluate metabolism of XDE-729 acid in the 

Beagle dog and comparative metabolism in the rat and mouse. Dogs also showed rapid 

absorption and excretion, primarily in the urine, with most radioactivity excreted unmetabolized. 

Clearance was somewhat slower than in the rat. In the comparative study, absorption, 

metabolism and elimination were similar in rats and mice at the doses tested. 

Toxicokinetic evaluations were conducted in many of the oral toxicity studies on halauxifenmethyl and XDE-729 acid, and included assessment of parent and/or metabolite in the blood and, 

in some studies, the urine and liver. In rat 28- and 90-day dietary studies on halauxifen-methyl, 

XDE-729 acid was found in the blood at all dose levels; low levels of halauxifen-methyl were 

observed only at the highest doses. Urinary excretion was primarily as XDE-729 acid, with very 

low levels of halauxifen-methyl at or above the study LOAELs. Blood levels of XDE-729 acid 

in rats administered halauxifen-methyl were below the levels observed at comparable dietary 

doses of XDE-729 acid, consistent with the lack of observed kidney toxicity in studies on 

halauxifen-methyl and rapid excretion of the acid. The most abundant analyte in liver in the rat 

90-day study on halauxifen-methyl was XDE-729 acid, but halauxifen-methyl was detected in all 

treated groups and showed dose linearity. In males, but not females, XDE-729 acid as percent 

AD decreased at the highest dose. In rat and rabbit developmental studies on halauxifen-methyl, 

only XDE-729 acid was observed in maternal and fetal blood. Levels in fetal blood were lower 

than maternal blood in the rat (about 65% of maternal levels) and rabbit (5 – 27% of maternal 

levels). Toxicokinetic data from studies on XDE-729 acid showed linearity of the AUC except 

at high doses.

4.2.1 Dermal Absorption

Dermal absorption of halauxifen-methyl following a 10 hr exposure was evaluated in a rat in 

vivo dermal absorption study and a comparative in vitro dermal absorption study using rat and 

human skin samples. Both studies were conducted using a liquid concentrate formulation, GF2573, containing [14C]-halauxifen-methyl and applied undiluted or as two field dilutions, at 

concentrations of 75, 1.5 or 0.25 μg ai/cm2, respectively. Absorption was monitored in the in 

vivo study up to 144 hr (concentrate) or 192 hr (dilutions) and in the in vitro study, up to 24 hr.

In vivo percent dermal absorption (absorbed plus absorbable dose, excluding the two outermost 

layers of skin) was 13, 15 and 24% of the applied dose (highest to lowest concentration, 

respectively). 

Toxicokinetic evaluations were performed in the in vivo rat study and show that following 

absorption, internal exposure is to XDE-729 acid. Blood levels of radioactivity at 4 and 8 hr 

post-application showed that only XDE-729 acid was present, indicating rapid hydrolysis of the 

absorbed parent compound to the acid metabolite following dermal absorption. This is 

consistent with the findings of a comparative in vitro tissue S9 methyl ester hydrolysis study in 

which halauxifen-methyl was hydrolyzed to XDE-729 acid in the blood, although activity was 

lower than in the liver. Hydrolysis activity of the skin tissue itself was not examined.

The ratios of in vitro dermal absorption in human:rat skin samples were 0.18, 0.37 and 0.60

(highest to lowest dilution, respectively). 
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Based on the in vivo dermal absorption study, the data indicate that up to 24% of the applied 

dose was absorbed in the rat following a 10 hr exposure to halauxifen-methyl. However, given 

that no toxicity was observed in the rat 28-day dermal toxicity study, a dermal assessment is not 

required.

4.3 Toxicological Effects

Halauxifen-methyl is rapidly hydrolyzed in vivo at the methyl ester to its major metabolite, 

XDE-729 acid. The toxicity profiles of the two compounds in oral toxicity studies showed 

significant differences, because of the rapid demethylation of the parent compound to the acid. 

The major target organ of halauxifen-methyl following oral exposure is the liver, which results 

from direct, pre-systemic exposure of the liver from the enterohepatic circulation. In subchronic 

(28- and 90-day) dietary studies in the rat, increased liver weight, hepatocellular hypertrophy, 

increased hepatocyte mitotic figures, vacuolization consistent with fatty change and increased 

cholesterol levels were reported at the same dose levels in both studies. These findings were 

minimal in both studies, suggesting lack of progression in exposures of short- to intermediateduration. 

The liver effects were associated with significant increases (≥1500-fold above controls) in the 

expression of the cytochrome enzyme Cyp1a1. Mechanistic studies on halauxifen-methyl 

identified activation of the aryl hydrocarbon receptor (AhR) as the molecular initiating event 

(MIE) in the mode of action (MOA) leading to hepatic toxicity. The mechanistic data are 

summarized in greater detail below in Section 4.3.2, and additional details are provided in a 

separate document (TXR # 0051197). The identification of liver AhR activation as the MOA for 

liver toxicity is relevant to both systemic and carcinogenic effects from long-term exposure, as 

described below. Thyroid follicular cell hypertrophy was also observed at doses causing liver 

effects. Increased expression of liver UGT enzymes, measured in the rat 90-day oral study, 

suggests that the thyroid effects are secondary to increased liver metabolism of thyroid 

hormones. Other reported effects included decreased body weight/weight gain, and thymic 

atrophy with decreased thymus weight. Hyperplasia of erythroid cells in the bone marrow and 

slight increases in extramedullary hematopoiesis in the spleen were reported in the 28-day rat 

study. However, effects on the thymus and the hematopoietic system were only observed at high 

dose levels that were significantly greater than the doses showing liver or thyroid effects.

In contrast, following oral exposure to XDE-729 acid, the kidney is the major target organ. Due 

to the rapid hydrolysis of halauxifen-methyl in the liver following absorption, systemic exposure 

is to XDE-729 acid rather than the parent compound. Kidney effects were observed following 

exposures of all durations (28- and 90-day subchronic and 1- to 2-year chronic oral studies) and 

included increased renal tubule degeneration/regeneration in all species tested. In the rat, 

increased renal mitotic figures, necrosis, hypertrophy and/or vacuolization of collecting duct 

epithelial cells, and increased urinary volume were seen in the subchronic studies. Urinary 

bladder histopathology was observed in female mice after 90 days’ exposure. Increased kidney 

weights were seen in maternal animals in the rat developmental toxicity study, but 
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histopathology was not performed. The severity of the kidney and urinary tract effects increased 

with exposure duration in all species. In the mouse carcinogenicity and rat chronic 

toxicity/carcinogenicity studies, males showed increased mortality secondary to renal and 

urinary bladder lesions. In the dog chronic study, multifocal glomerulosclerosis in both sexes 

and in males, granulomas and fibrosis in the renal cortex, were observed. Following oral 

administration in all species, effects ranging from decreased body weight and/or body weight 

gain in rats and dogs to hematological changes in all species were also seen at doses eliciting 

nephrotoxicity. In the mouse 28-day study, focal necrosis and karyomegaly in the liver were 

observed at approximately the limit dose, but this was not reported in other mouse studies at 

similar dose levels. Decreased thymus weights were reported in the dog 28- and 90-day studies. 

No toxicity was observed in rats exposed dermally to XDE-729 acid for 28 days up to the limit 

dose. A dermal toxicity study with halauxifen-methyl was not conducted, but data from the rat 

in vivo dermal absorption study indicated that it is rapidly hydrolyzed to XDE-729 acid 

following absorption, based on lack of halauxifen-methyl in the blood following absorption. 

Despite the rapid conversion of halauxifen-methyl to XDE-729 acid in vivo, there was no 

evidence of kidney toxicity in the rat following exposure to halauxifen-methyl. A comparison of 

blood levels of XDE-729 acid in rats treated with halauxifen-methyl vs. XDE-729 acid for 28 or 

90 days showed that levels following halauxifen-methyl exposure were lower than the levels 

observed in studies on XDE-729 acid, even at comparable mg/kg/day dose levels (Tables 4.3.1.1 

and 4.3.1.2). Levels in the kidney were not examined, but the blood concentrations of XDE-729 

acid associated with kidney toxicity in studies on the acid were not achieved in rats exposed to 

halauxifen-methyl, consistent with the lack of kidney toxicity observed from exposure to 

halauxifen-methyl. 

Table 4.3.1.1. Comparison of Blood Levels of XDE-729 Acid Following Exposure to the Rat in 28-Day Studies on Halauxifenmethyl and XDE-729 Acid.

XDE-729 Acid1

M: 734 m/k/d

F: 1000 m/k/d

MRID 48557825

Halauxifen-Methyl2

M: 782 m/k/d

F: 782 m/k/d

MRID 48557826

XDE-729 Acid1

M: 250 m/k/d

F: 250 m/k/d

MRID 48557825

Halauxifen-Methyl2

M: 261 m/k/d

F: 261 m/k/d

MRID 48557826

Halauxifen-Methyl2

M: 52 m/k/d

F: 52 m/k/d

MRID 48557826

Time (hrs)3 XDE-729 Acid in Blood (μg/g)

Male 0 63.9 15.7 30 6.6 1.3

Male 3 47.6 11.8 24.4 4.6 0.8

Male 11 36.6 8.1 12 3 0.7

Female 0 103.8 4 32.9 7.4 1.6

Female 3 72.8 2.4 16.3 5.8 1.4

Female 11 44.5 0.3 6.5 2.2 0.6

1 NOAEL = 250 m/k/d. LOAEL = 734 m/k/d (boldface) based on kidney toxicity. Original dose for male was 1000 m/k/d but switched to 500 

m/k/d on day 11.

2 NOAEL = 10.8 m/k/d. LOAEL = 55.6 m/k/d based on liver toxicity.

3 The first time point (measured at 6 a.m.) is set as “0” and all other times points are in reference to the zero point. Samples were taken on the 

final day of dosing.
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Table 4.3.1.2. Comparison of Blood Levels of XDE-729 Acid Following Exposure to the Rat in 90-Day Studies on HalauxifenMethyl and XDE-729 Acid.

XDE-729 Acid1

M: 750 m/k/d

F: 750 m/k/d

MRID 48557828

Halauxifen-Methyl2

M: 500 m/k/d

F: 500 m/k/d

MRID 48557830

XDE-729 Acid1

M: 250 m/k/d

F: 250 m/k/d

MRID 48557828

Halauxifen-Methyl2

M: 261 m/k/d

F: 261 m/k/d

MRID 48557830

Halauxifen-Methyl2

M: 52 m/k/d

F: 52 m/k/d

MRID 48557830

Time (hrs)3 XDE-729 Acid in Blood (μg/g)

Male 0 105.7 17.5 29.3 10.4 12.4

Male 3 51.7 8.5 13.3 4.5 1.5

Male 11 30.8 5.1 9.5 2.9 0.6

Female 0 145 19.1 34.1 11.7 2.4

Female 3 67.3 7.6 14.7 5.3 0.8

Female 11 54.3 9.2 7.0 7.8 0.6

1 NOAEL = 252 m/k/d. LOAEL = 758 m/k/d (boldface) based on kidney toxicity.

2 NOAEL = 10 m/k/d. LOAEL = 52.3 m/k/d based on liver toxicity.

3 The first time point (measured at 6 a.m.) is set as “0” and all other times points are in reference to the zero point. The 3 hour time point for the 

XDE-729 methyl study was actually 5 hours. Samples were taken on the final day of dosing.

Halauxifen-methyl and XDE-729 acid did not show evidence of neurotoxicity. Slightly 

decreased activity at the highest two dose levels in the acute neurotoxicity study was considered 

to be an equivocal effect, based on the lack of effects on other parameters and the lack of 

findings in the subchronic study, and was not considered to demonstrate neurotoxicity. There 

were no clinical or histopathological indications of neurotoxicity in any of the other studies on 

the acid form or halauxifen-methyl, although the latter was not tested in neurobehavioral studies. 

No immunotoxicity was reported in rats treated with halauxifen-methyl, although increased liver 

weight and decreased thymus weight were observed. 

There was no evidence of increased qualitative or quantitative pre- and/or postnatal susceptibility 

for either halauxifen-methyl or XDE-729 acid. In the case of halauxifen-methyl, no 

developmental effects were observed at doses causing decreased maternal weight gain and food 

consumption toxicity in rats; in rabbits, slight developmental effects (decreased fetal body 

weight and delayed ossification of the pubis) were seen in the presence of maternal 

hepatotoxicity. For XDE-729 acid, decreased fetal weights and delays in ossification were 

observed in the developmental rat study at a dose that caused significant maternal toxicity 

leading to moribund sacrifice of some of the dams during the study. No developmental effects 

were seen in the rabbit at doses causing slightly decreased maternal body weight gain and food 

consumption. In a two-generation reproductive toxicity study conducted in the rat on XDE-729 

acid, there was no evidence of reproductive toxicity, and no toxicity to offspring was observed, 

although parental animals showed significant renal toxicity and effects on body weight. A 

reproductive toxicity study was not conducted on halauxifen-methyl, but as described below 

(Sections 4.4 and 4.5), the available data allow selection of endpoints that are protective of 

offspring.

There was no evidence of carcinogenicity in rat and mouse cancer studies on XDE-729 acid, or 

of genotoxic potential for either compound. XDE-729 acid is classified as “not likely to be 

carcinogenic to humans.” Carcinogenicity studies on halauxifen-methyl were not conducted. 

Systemic exposure from halauxifen-methyl is primarily to XDE-729 acid and therefore most 

tissues are not exposed to the parent compound. However, the mechanistic studies showed that 

pre-systemic exposure of the liver to halauxifen-methyl results in activation of the AhR receptor, 
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an effect that when sustained (e.g., with long-term exposure) may promote an increased 

incidence of liver tumors due to increased hepatocellular proliferation. The molecular marker for 

AhR activation, the molecular initiating event (MIE) for liver toxicity, is increased expression of 

hepatic Cyp1a1 (see Section 4.3.2 and TXR # 0051197). The chronic dietary endpoint for 

halauxifen-methyl is based on the point of departure (POD) for Cyp1a1 induction from a 

subchronic study. Since increased expression of Cyp1a1 is one of the early key events in the 

MOA leading to hepatotoxicity, although in itself it is not an adverse effect, a dose that is 

protective of this event will be protective of the potential risk for liver cancer with chronic 

exposure, based on the rapid onset of AhR activation following initiation of exposure, and the 

lack of evidence of temporal progression of liver toxicity in the available studies (28- and 90-

day). The MOA is considered relevant to human health risk assessment, but in vitro

transactivation and AhR receptor binding data suggest that humans are unlikely to be more 

sensitive than the rat, and furthermore, that halauxifen-methyl is a relatively weak AhR agonist 

due to rapid metabolism and excretion (see TXR #0051197 for further details). Based on a 

weight-of-the-evidence consideration, halauxifen-methyl is classified as “not likely to be 

carcinogenic to humans” at doses that do not induce liver Cyp1a1 expression. 

Both halauxifen-methyl and XDE-729 acid show low toxicity in acute oral and dermal lethality 

studies (Toxicity Category III or IV) and are not irritants or dermal sensitizers. Acute inhalation 

toxicity was not examined due to problems generating the test atmosphere.

4.3.2 Mode of Action for Liver Toxicity of Halauxifen-Methyl

As previously noted, the observation of liver effects (including increased liver weights, 

hepatocellular hypertrophy, mitotic figures in hepatocytes, and hepatocellular vacuolization) in 

rat subchronic dietary studies with halauxifen-methyl, but not with XDE-729 acid, led the 

registrant to perform in vitro and in vivo mechanistic studies to further understand the MOA. 

Based on the results of these studies, a MOA was proposed that involves activation of the AhR 

through the following key events: 

(1) pre-systemic liver exposure to halauxifen-methyl (following oral exposure); 

(2) AhR activation by halauxifen-methyl (relatively weak binding) as the MIE resulting 

in: 

(a) liver enzyme induction (Cyp1a1) and

(b) liver weight increases, hepatocyte hypertrophy and vacuolization, and: 

(3) hepatocellular proliferation. 

(4) unlike many AhR activators, halauxifen-methyl is rapidly metabolized and not 

persistent in the body; progression of liver toxicity (including carcinogenic potential) 

from sustained AhR activation is therefore not expected. 

The HED ToxSAC reviewed the MOA and determined that it is plausible and supported by the 

submitted studies (TXR No. 0056954). This conclusion is consistent with the determinations of 

the Global Partners for review of halauxifen-methyl (Canada PMRA, Australia OCS, and the 

EU). More detailed summaries of the individual mechanistic studies and the MOA framework 

are provided in the separate document (TXR # 0051197). Characterization of AhR as a MOA 
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for toxicity and carcinogenicity in the liver and other tissues has been previously described for 

other compounds (reviewed in Nebert et al., 2004; Cohen, 2010; Safe et al., 2013). 

Table 4.3.2.1, below, briefly summarizes the supporting data for each key event. 

Table 4.3.2.1. Summary of Key Events and Supporting Data for Liver Toxicity from Oral Exposure to 

Halauxifen-Methyl.

Key Event Data Supporting Key Event

Presystemic liver exposure to halauxifen-methyl

Rapid metabolism to XDE-729 acid; systemic 

exposure is to the acid.

Comparison to humans

Reversibility of findings

Levels of halauxifen-methyl in liver: A dose-related 

increase in levels of halauxifen-methyl in the liver was 

observed in toxicokinetic studies of halauxifen-methyl 

in liver cells in rat 28- and 90-day dietary studies. A 

sharp increase was seen after 7 days’ exposure to the 

rat at a high dose in a 7-day dietary study.

Toxicokinetic data show that XDE-729 acid but not 

halauxifen-methyl was found in the blood, except for 

very low levels of halauxifen-methyl that were seen at 

higher dose levels. Rat metabolism studies show a 

rapid half-life of elimination of radioactivity, primarily 

as XDE-729 acid. 

PBPK modeling based on in vitro metabolism in rat 

and human blood and liver S9 showed that halauxifenmethyl and XDE-729 acid achieve steady-state 

concentrations soon after exposure, clear rapidly from 

blood and liver, and are not expected to accumulate. 

Yes, the findings were reversible. Levels of 

halauxifen-methyl in liver decreased with time 

following exposure. Metabolism studies showed that 

excretion was rapid with minimal retention of 

radioactivity in tissues.

Activation of the AhR

Targeted gene expression

Binding of halauxifen-methyl: In vitro binding of 

halauxifen-methyl to the AhR was observed. 

Transactivation assays in mouse and human cell lines 

with a luciferase reporter vector showed that 

halauxifen-methyl was a weak AhR agonist in mouse 

but not human cells. In a ligand competition binding 

assay using rat liver cytosol, halauxifen-methyl 

competed weakly with the photoaffinity ligand for rat 

AhR. XDE-729 acid did not bind to AhR. 

Induction of Cyp1a1 expression: Analyses of gene 

expression were performed in 7-, 28- and 90-day 

dietary studies. Dose-related increases in levels of 

Cyp1a1 gene expression were observed for halauxifenmethyl but not XDE-729 acid. In vitro gene expression 

assays in rat, mouse and human hepatocytes also 

showed a dose-related increase that was most 

pronounced in rat hepatocytes. Increases correlated 

with the presence of halauxifen-methyl in the liver.
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Table 4.3.2.1. Summary of Key Events and Supporting Data for Liver Toxicity from Oral Exposure to 

Halauxifen-Methyl.

Key Event Data Supporting Key Event

Associated liver histopathological effects related to 

AhR activation

Reversibility of findings

Hepatocellular hypertrophy, vacuolization, increased 

liver weight, observed in dietary studies on halauxifenmethyl, but not XDE-729 acid. Liver effects were 

observed at dose levels that caused pronounced 

increases in Cyp1a1 expression. A mild increase in 

hepatocyte Cyp1a1 expression was observed at the 

NOAEL for liver toxicity, but not at the lowest dose 

tested, which was identified as a POD for increased 

Cyp1a1 expression.

Yes, the findings were reversible. In the 28-day dietary 

study, Cyp1a1 expression and liver 

weights/histopathology were comparable to controls 

following a 28-day recovery period.

Hepatocellular proliferation 

Reversibility of findings observed

Increased mitotic indices: Very slight increases in 

mitotic figures in the liver were observed at high doses 

in the rat 7-day study. In the rat 28-day study, a doserelated increase was observed (1/5 and 3/5 animals at 

52 and 261 mg/kg/day). No mitotic figures were 

observed in the rat 90-day study.

Increased BrdU incorporation: Slightly increased 

BrdU incorporation at 261 mg/kg/day in the rat 28-day 

study. Not observed at lower dose levels.

Yes, the findings were reversible. No increases in 

mitotic figures in the 28-day study following a 28-day 

recovery period. BrdU incorporation levels return 

towards control levels. 

Pre-systemic liver exposure to halauxifen-methyl was demonstrated in dietary studies on mice 

and rats. Pre-systemic exposure is also expected in humans based on the PBPK modeling. Data 

supportive of halauxifen-methyl mediated AhR activation included increased enzyme expression 

of AhR marker genes in vivo and transactivation/binding assays in vitro. Observations of liver 

weight increases and hepatocyte hypertrophy and vacuolization were seen in multiple rodent 

studies following treatment with halauxifen-methyl but not XDE-729 acid. Finally, increased 

hepatocellular proliferation was demonstrated by slightly increased bromodeoxyuridine (BrdU)

uptake by hepatocytes following dietary exposure to halauxifen-methyl in rats, along with a 

slight increase in mitotic figures at 7 and 28 days, but not at 90 days. At least partial recovery 

from induced effects was observed.

The key events showed temporal and dose-related concordance. In the subchronic rat toxicity 

studies, Cyp1a1 expression was increased by 52-fold at a dose that showed no evidence of 

hepatotoxicity (i.e., study NOAEL). In contrast, there was a marked induction of Cyp1a1 

(>1500-fold) at doses where liver effects were also observed; however, liver toxicity at the 

LOAEL was minimal and did not become more severe, or occur at a lower dose, with increased 

exposure duration. Taken together, the key events and MOA are plausible and supported by the 

data. Other possible MOAs for liver effects from halauxifen-methyl were also considered, but 
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the data did not support them. These included DNA reactivity, targeted gene expression of other 

genes such as CAR, PXR or PPARα, cytotoxicity/apoptosis, or other agents (infections, metals, 

statins, estrogens).

The MOA is considered relevant to humans based on the fact that the AhR pathway is conserved 

across species. PBPK modeling indicated that humans would have pre-systemic exposure to 

halauxifen-methyl, and halauxifen-methyl was able to activate CYP1A1 genes in human 

hepatocytes. While there are in vitro data indicating that humans may be less sensitive than the 

rat to activation of AhR, a quantitative correlation to in vivo exposure is not possible. The data 

suggest, however, that humans are unlikely to show greater sensitivity than the rat. 

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

The halauxifen-methyl risk assessment team has recommended that the FQPA Safety Factor be 

reduced to 1X. Exposure estimates rely on data that reasonably and conservatively account for 

potential exposures from dietary sources and occupational use. The dietary assessment is based 

on reliable data and will not underestimate exposure. 

There is no evidence of increased prenatal susceptibility to halauxifen-methyl or pre-/post-natal 

susceptibility to XDE-729 acid. Residual concerns regarding lack of information on metabolism 

of halauxifen-methyl during early post-natal development are low because actual dietary 

exposure is expected to be very low and a conservative endpoint, protective of AhR activation in 

the adult in the absence of actual liver toxicity, was selected for risk assessment. 

4.4.1 Completeness of the Toxicology Database

The toxicology database for halauxifen-methyl is considered adequate for assessment of 

developmental sensitivity/susceptibility and includes acceptable developmental toxicity 

(halauxifen-methyl and XDE-729 acid), reproductive toxicity (XDE-729 acid), acute and 

subchronic neurotoxicity (XDE-729 acid) and immunotoxicity (halauxifen-methyl) studies. The 

bridging and mechanistic studies on halauxifen-methyl, considered together with the 

reproductive toxicity study on XDE-729 acid, are considered adequate for assessment of postnatal effects.

4.4.2 Evidence of Neurotoxicity

There was no evidence of neurotoxicity in the available studies on halauxifen-methyl and XDE729 acid, including acute and subchronic neurotoxicity studies conducted on XDE-729 acid. In 

the acute neurotoxicity study, slightly reduced activity was observed but was considered an 

equivocal effect, based on lack of other observations, and was not considered evidence of a 

neurotoxic effect. The neurotoxicity data on XDE-729 acid are considered sufficient because 

systemic exposure is to the acid.

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal
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There was no evidence of increased prenatal quantitative or qualitative susceptibility. No 

developmental effects were observed in rats exposed to halauxifen-methyl or rabbits exposed to 

XDE-729-acid. In rats exposed to XDE-729 acid and rabbits exposed to halauxifen-methyl, fetal 

toxicity (decreased fetal weight, delays in bone ossification) was observed only at doses that 

caused significant maternal toxicity (hepatotoxicity and moribundity). Toxicokinetic evaluations 

showed that maternal and fetal systemic exposure following exposure to halauxifen-methyl was 

to XDE-729 acid, based on levels found in the blood.

There was no evidence of increased postnatal quantitative or qualitative susceptibility to XDE729 acid, based on lack of offspring or reproductive toxicity at doses causing parental toxicity in 

a rat two-generation reproductive toxicity study. In the absence of reproductive toxicity data on 

halauxifen-methyl, there is residual uncertainty for increased postnatal susceptibility from 

dietary exposure to halauxifen-methyl during early development (e.g. during weaning, via 

drinking water or solid foods) due to potential differences in liver metabolism and toxicity, 

which may result from differential expression of liver Cyp1a1 expression (lower) or AhR levels 

(higher) in young offspring compared to adults. For example, decreased hepatic Cyp1a1 activity 

may result in a slower rate of metabolism, leading to greater hepatic and systemic exposure to 

the parent compound. However, concern for increased susceptibility to halauxifen-methyl is low

for several reasons. The selected POD for all exposure assessments of halauxifen-methyl is 

based on a dose that is well below doses that cause minimal liver toxicity in adults. Halauxifenmethyl was demonstrated to be a relatively weak agonist of the AhR receptor, due to its rapid 

metabolism and excretion, and is unlikely to be a more potent inducer of Cyp1a1 expression in 

humans compared to rats. Furthermore, actual dietary exposure to halauxifen-methyl is 

anticipated to be extremely low, due to low residue levels and degradation of parent compound 

in the environment.

4.4.4 Residual Uncertainty in the Exposure Database

Exposure to halauxifen-methyl will not be underestimated in this assessment. There are no 

exposure data gaps. The dietary exposure assessment relies upon conservative assumptions and 

modelling data, together with a conservative dose and endpoint. The dietary risk estimates were 

based on tolerance level residues which reflect that measurable residues are not expected in any 

foods, no processing factors due to the lack of concentration in processed commodities, 100% 

crop treated assumption and modeled drinking water levels.

Residue chemistry data indicate that exposure in the diet (food and drinking water) is very low 

and primarily to XDE-729 acid, and that levels of halauxifen-methyl itself are expected to be 

minimal due to environmental degradation. Dietary exposure to residues from halauxifen-methyl 

is primarily from drinking water. Residues in the proposed food crops show levels of 

halauxifen-methyl and metabolites of concern that are below the LOQ.

4.5 Toxicity Endpoint and Point of Departure Selections

4.5.1 Dose-Response Assessment
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Dietary exposure to halauxifen-methyl and its metabolites/degradates is very low, primarily from 

drinking water contaminants. Residues in treated crops are below the LOQ. Separate dietary 

endpoints were selected for halauxifen-methyl and XDE-729 acid due to their distinct toxicities. 

Most other, minor degradates are hydrolyzed at the methyl ester; their toxicity is expected to be 

similar to XDE-729 acid and they are considered together with the acid. Occupational endpoints 

were selected only for halauxifen-methyl, the ai in end-use products. No residential endpoints 

were selected because there are no proposed residential uses.

Halauxifen-Methyl:

For acute dietary exposure, no hazard was identified for halauxifen-methyl acid that could be 

attributed to a single exposure. There are no single-dose studies on halauxifen-methyl, but liver 

toxicity from AhR activation, the most sensitive effect, is not considered a single dose effect. 

No chronic studies are available for halauxifen-methyl. For assessment of chronic dietary 

exposure, the POD of 3.0 mg/kg/day, based on increased liver Cyp1a1 expression (biomarker for 

AhR activation) at 10 mg/kg/day in the rat 90-day oral toxicity study, was selected. This is an 

early event in the adverse outcome pathway and therefore protective of liver toxicity for all 

exposure durations. The selected dose and endpoint are the most sensitive available and 

protective of all effects from halauxifen-methyl, as well as from XDE-729 acid (see below). It is 

noted that at the level of Cyp1a1 expression (52-fold above controls) seen at 10 mg/kg/day, no 

actual adverse effects were identified, and that the mild liver effects identified at the study 

LOAEL of 53 mg/kg/day were associated with a significant increase (>1500-fold) in its 

expression. Selection of 3.0 mg/kg/day as the POD is protective of potential liver effects that 

may result from sustained long-term exposure, since toxicity will not occur in the absence of the 

MIE. The selected POD is protective of developmental toxicity (NOAEL in rabbit 18.5 

mg/kg/day, in rat, ≥323 mg/kg/day).

An uncertainty factor (UF) of 100x was applied (interspecies factor of 10x and intraspecies 

factor of 10x). An additional UF for extrapolation from subchronic to chronic data (UFS) was 

not added based on: (1) dose and endpoint were based on the MIE for AhR activation, a rapidly 

occurring event following exposure, and are therefore protective of liver toxicity from exposures 

of all durations; (2) rapid in vivo metabolism to XDE-729 acid, for which chronic data are 

available, and lack of bioaccumulation; (3) lack of progression of liver toxicity in rat 28- and 90-

day studies and (4) low dietary exposure. The selected dose and endpoint are therefore 

protective of liver toxicity from exposures of all durations.

For short- and intermediate-term dermal (occupational) exposure, an endpoint of concern was 

not identified. A dermal toxicity study on halauxifen-methyl was not conducted. No toxicity 

was observed in a rat 28-day dermal toxicity study on XDE-729 acid up to the limit dose; 

however, the dermal absorption of XDE-729 acid is not known. In an in vivo rat dermal 

absorption study on halauxifen-methyl, toxicokinetic analysis showed that it was rapidly 

hydrolyzed to XDE-729 acid in the blood following absorption; therefore, internal exposure is to 

the acid. Dermal absorption of halauxifen-methyl was determined to be 24%. It is reasonable,

given the similarities, to assume similar dermal absorption for XDE-729 acid and halauxifenmethyl. The toxicokinetic data for dermal absorption of halauxifen-methyl support the 
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conclusion of XDE-729 acid as the toxic moiety. As noted above, no adverse effects were 

observed in the dermal toxicity study with XDE-729 acid. Considering these findings together, 

HED does not expect toxic effects from dermal exposure to halauxifen-methyl. 

For short- and intermediate-term (occupational) inhalation exposure, an oral study was selected 

because inhalation data were not available. The POD of 3 mg/kg/day for increased liver Cyp1a1 

expression at 10 mg/kg/day in the rat 90-day toxicity study on halauxifen-methyl, was selected. 

The study is of an appropriate duration. The selected dose and endpoint are protective of 

potential toxicity from ingestion of large, nonrespirable particles. Additionally, since the lung is 

a metabolically active tissue and susceptible to AhR activation and Cyp1a1 induction, the 

selected dose and endpoint are considered protective of potential portal-of-entry effects. 

Toxicity via the inhalation route is assumed to be equivalent to toxicity via the oral route.

For inhalation risk assessment, the level of concern (LOC) is a MOE of 100, based on combined 

interspecies (10x) and intraspecies (10x) UFs. An additional UFDB for lack of inhalation data 

was not added because the data were waived, halauxifen-methyl has low volatility, and 

inhalation MOEs calculated using the oral POD were adequately protective (≥1000).

XDE-729 Acid:

For acute dietary exposure, no endpoint was identified for XDE-729 acid that could be attributed 

to a single dose. In the rat acute neurotoxicity study on XDE-729 acid, transiently decreased 

body weight was observed only at the limit dose of 2000 mg/kg. Slight decreases in activity 

level at 750 and 2000 mg/kg did not show a dose-response and were not considered to be 

toxicologically significant effects in the absence of other, associated neurological findings.

For chronic dietary exposure to XDE-729 acid, the NOAEL of 20.3 mg/kg/day from the rat 

chronic oral toxicity/carcinogenicity study was selected, based on kidney toxicity in females at 

the LOAEL of 101 mg/kg/day. This study is of the appropriate duration and is protective of all 

effects seen in other studies, including developmental effects observed in the rat. A NOAEL of 

16.7 mg/kg/day was observed in the chronic oral dog study, based on kidney toxicity in females 

at 89.9 mg/kg/day. However, the NOAELs and LOAELs from the two studies are similar and 

the difference is considered to be the result of dose spacing, rather than increased sensitivity in 

the dog. 

An UF of 100x was applied (interspecies factor of 10x and intraspecies factor of 10x) for chronic 

dietary assessment of XDE-729 acid. The 10x FQPA SF was reduced to 1x, based on the 

completeness of the database and lack of pre- and/or post-natal susceptibility.

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

When there are potential occupational and residential exposures to a pesticide, the risk 

assessment must address exposures from three major sources: oral, dermal and inhalation. It 

must be determined whether or not the individual exposures have the same toxicological effects. 

If they do, then they can be combined. For halauxifen-methyl, there are no residential uses 

which could result in oral exposure, and a dose and an endpoint was not identified for dermal 
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exposure. Therefore, only inhalation exposure from agricultural uses of halauxifen-methyl will 

be quantitatively assessed, and no combined exposures are warranted. 

4.5.3 Cancer Classification and Risk Assessment Recommendation

Separate cancer classifications were assigned to halauxifen-methyl and the metabolite XDE-729 

acid, since each compound shows significantly different toxicity (with a MOA described for liver 

toxicity from halauxifen-methyl).

XDE-729 acid is classified as “not likely to be carcinogenic to humans.” There was no evidence 

of carcinogenicity in acceptable mouse carcinogenicity and rat chronic toxicity/carcinogenicity 

studies conducted on XDE-729 acid. Genotoxicity studies for XDE-729 acid were negative.

Halauxifen-methyl is classified as “not likely to be carcinogenic to humans” at doses below those 

that induce liver Cyp1a1 expression – a key early event in the MOA for liver toxicity and, with 

sustained activation of AhR from long-term exposure, carcinogenicity. Genotoxicity studies for 

halauxifen-methyl were negative. Rat chronic toxicity/carcinogenicity and mouse 

carcinogenicity studies were not conducted on halauxifen-methyl itself, but when considered 

together, the rat subchronic dietary and mechanistic studies on halauxifen-methyl, along with the 

chronic studies on XDE-729 acid, provide sufficient information to assess potential 

carcinogenicity (see Sections 4.3.1 and 4.3.2). The POD for increased liver Cyp1a1 expression 

is protective of effects to the liver that, with time, may induce increased hepatocellular 

proliferation and tumors. Exposure to other tissues will be to XDE-729 acid, which shows no 

evidence of carcinogenicity in the long-term dietary studies.

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

Assessment

Table 4.5.4.1. Summary of Toxicological Doses and Endpoints for Halauxifen-Methyl for Use in Dietary 

and Non-Occupational Human Health Risk Assessments.

Exposure/ 

Scenario

Point of 

Departure

Uncertainty/FQP

A Safety Factors

RfD, PAD, 

Level of 

Concern for 

Risk 

Assessment

Study and Toxicological Effects

Acute Dietary 

(All 

Populations, 

including 

Infants and 

Children and 

Females age 

13-49)

No hazard from a single exposure was identified in the available database; therefore, no risk is 

expected from this exposure scenario.
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Table 4.5.4.1. Summary of Toxicological Doses and Endpoints for Halauxifen-Methyl for Use in Dietary 

and Non-Occupational Human Health Risk Assessments.

Exposure/ 

Scenario

Point of 

Departure

Uncertainty/FQP

A Safety Factors

RfD, PAD, 

Level of 

Concern for 

Risk 

Assessment

Study and Toxicological Effects

Chronic 

Dietary (All 

Populations)

POD = 3.0

mg/kg/day

UFA= 10x

UFH=10x

FQPA SF= 1x

Chronic RfD 

= 0.03

mg/kg/day

cPAD = 0.03

mg/kg/day

90-day oral toxicity in the rat 

(halauxifen-methyl)(MRID 48557830)

At the study NOAEL of 10 mg/kg/day, 

increased hepatic Cyp1a1 expression 

(MIE for liver toxicity from AhR 

activation) was observed (endpoint 

selected for risk assessment). The lowest 

dose of 3.0 mg/kg/day was selected to be 

protective of potential long-term effects 

from low-level but sustained increased 

AhR expression in the liver.1

Study LOAEL = 53 mg/kg/day, based on 

mild liver enlargement and pathology.

Incidental 

Oral ShortTerm (all 

durations)

There are no residential uses for halauxifen-methyl and therefore an endpoint was not 

identified.

Dermal ShortTerm (all 

durations)

There are no residential uses for halauxifen-methyl and therefore an endpoint was not 

identified.

Inhalation (all 

durations)

There are no residential uses for halauxifen-methyl and therefore an endpoint was not 

identified.

Cancer (oral, 

dermal, 

inhalation)

Classification: Not likely to be carcinogenic to humans at dose levels that do not induce 

Cyp1a1 expression. The cRfD is considered protective of potential cancer effects because it 

protects for the MIE for hepatocellular proliferation (AhR activation) that, over time, may result 

in promotion of liver tumors.

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

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

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

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

sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. PAD = 

population adjusted dose (a = acute, c = chronic). RfD = reference dose. MIE = molecular initiating event

1 The POD selected for risk assessment was based on a non-adverse finding, increased liver Cyp1a1 expression in a 

rat 90-day dietary study, which was observed below the study NOAEL of 10 mg/kg/day for liver toxicity. This 

effect is a biomarker for activation of AhR, which causes liver toxicity and hepatocellular proliferation. The POD 

reflected a mild increase in Cyp1a1 expression and was selected to be protective of potential liver effects resulting 

from chronic dietary exposure to halauxifen-methyl. Other tissues and organs will not be exposed to halauxifenmethyl due to rapid conversion to XDE-729 acid. The POD is protective of effects from exposure to XDE-729 acid.
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Table 4.5.4.2. Summary of Toxicological Doses and Endpoints for Halauxifen-Methyl for Use in 

Occupational Human Health Risk Assessments.

Exposure/ 

Scenario

Point of 

Departure

Uncertainty 

Factors

Level of Concern 

for Risk 

Assessment

Study and Toxicological Effects

Dermal ShortTerm (1-30 

days) and 

IntermediateTerm (1-6 

months)

No hazard from dermal exposure was identified in the available database; therefore, no risk is 

expected from this exposure scenario. 

Inhalation 

Short-Term 

(1-30 days) 

and 

Intermediateterm (1-6 

months)

POD = 3.0 

mg/kg/day

[Absorption 

via the 

inhalation 

route assumed 

equivalent to 

oral 

absorption]

UFA=10x

UFH=10x

Occupational 

LOC for MOE = 

100

90-day oral toxicity in the rat 

(halauxifen-methyl)(MRID 48557830)

At the study NOAEL of 10 mg/kg/day, 

increased hepatic Cyp1a1 expression 

(MIE for toxicity from AhR activation) 

was observed the (endpoint selected for 

risk assessment). The lowest dose of 3.0 

mg/kg/day was selected to be protective 

of potential long-term effects from lowlevel but sustained increased AhR 

expression in the liver.1

Study LOAEL = 53 mg/kg/day, based 

on liver enlargement and pathology at 

53 mg/kg/day.

Cancer (oral, 

dermal, 

inhalation)

Classification: Not likely to be carcinogenic to humans at dose levels that do not induce 

Cyp1a1 expression. The cRfD is considered protective of potential cancer effects because it 

protects for the MIE for hepatocellular proliferation (AhR activation) that, over time, may 

result in promotion of liver tumors.

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

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

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

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

sensitivity among members of the human population (intraspecies). MOE = margin of exposure. LOC = level of 

concern. MIE = Molecular Initiating Event.

1 The POD selected for risk assessment was based on a non-adverse finding, increased liver Cyp1a1 expression in a 

rat 90-day dietary study, which was observed below the study NOAEL of 10 mg/kg/day for liver toxicity. This 

effect is a biomarker for activation of AhR, which causes toxicity and proliferation in the liver but also other tissues 

including the lung, which is metabolically active and contains Cyp1a1. The POD reflected a mild increase in 

Cyp1a1 expression and was selected to be protective of potential lung effects resulting from inhalation exposure to 

halauxifen-methyl. Other tissues and organs will not be exposed to halauxifen-methyl following inhalation 

exposure, due to rapid conversion to XDE-729 acid. The POD is protective of potential portal of entry effects in the 

respiratory tract from exposure to XDE-729 acid because it protects for the MIE.
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Table 4.5.4.3. Summary of Toxicological Doses and Endpoints for XDE-729 acid for Use in Dietary and 

Non-Occupational Human Health Risk Assessments.

Exposure/ 

Scenario

Point of 

Departure

Uncertainty/FQP

A Safety Factors

RfD, PAD, 

Level of 

Concern for 

Risk 

Assessment

Study and Toxicological Effects

Acute Dietary 

(All 

populations, 

including 

Infants and 

Children and 

females age 

13-49)

No hazard from a single exposure was identified in the available database; therefore, no risk is 

expected from this exposure scenario.

Chronic

Dietary (All 

Populations)

NOAEL = 

20.3

mg/kg/day

(females)

UFA= 10x

UFH=10x

FQPA SF= 1x

Chronic RfD 

= 0.20

mg/kg/day

cPAD = 0.20

mg/kg/day

Rat two-year dietary chronic 

toxicity/carcinogenicity study

NOAEL = 101/20.3 mg/kg/day [M/F]

LOAEL = 404/101 mg/kg/day [M/F] 

based on increased mortality, altered 

urinalysis parameters, decreased body 

weight, increased kidney weights, 

adrenal zone glomerulosa hypertrophy, 

increased degeneration and regeneration 

of renal tubules and kidney stones, and 

bladder pathology in males; in females, 

hyperplasia of pelvic epithelium of the 

kidney. 

Incidental 

Oral ShortTerm (all 

durations)

There are no residential uses for XDE-729 acid and therefore an endpoint was not identified.

Dermal ShortTerm (all 

durations)

There are no residential uses for XDE-729 acid and therefore an endpoint was not identified.

Inhalation (all 

durations) There are no residential uses for XDE-729 acid and therefore an endpoint was not identified.

Cancer (oral, 

dermal, 

inhalation)

Classification: Not likely to be carcinogenic to humans. 

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

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

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

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

sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. PAD = 

population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. 

5.0 Dietary Exposure and Risk Assessment 

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies
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Appendix B summarizes the results of the plant and animal metabolism studies, including the 

structures of the metabolites and degradates. Studies depicting the metabolism of halauxifenmethyl in wheat and turnips as primary crops are adequate to delineate the residues of concern 

for the currently proposed uses. The studies were conducted at approximately the maximum 

application rate. In the wheat metabolism study, residues of parent and all metabolites were 

found at levels below 10% of the total radioactive residue (TRR). Wheat matrices harvested and 

analyzed for residues included immature forage and hay and mature straw and grain. In the 

turnip metabolism study, residues above 10% of the TRR were found in turnip tops. Compounds 

that were found at this level were parent and the glucose conjugates of parent and XDE-729 acid. 

As the registrant is only proposing tolerances for residues in wheat and barley, the tolerance 

expression should include parent only at this time. 

In the confined rotational crop study, the TRR in all commodities at all plantback intervals was 

0.001 ppm or less. Because of the very low TRR levels, none of the radioactivity in the tissue 

samples was characterized further. Based on these very low residues, rotational crop tolerances 

do not need to be established. A field rotational crop study was not conducted and is not 

required for the uses associated with this petition. 

In the cattle feeding study, X11449757 was the only metabolite found above the LOQ in any 

livestock commodity analyzed (i.e., milk, muscle, liver, kidney, fat). Residues were found in 

liver, kidney, and fat. In the goat metabolism study, X11449757 was found in significant levels 

along with its sulfate conjugate, XDE-729 acid, the sulfate conjugate of X11406790, and the Nor O-glucuronic acid conjugate of X11406790. These metabolites were found in milk, liver, 

kidney, and fat. 

In the poultry metabolism study, quantifiable levels of X11449757, XDE-729 acid, and the 

sulfate conjugate of X11406790 were found in liver and skin with fat. A poultry feeding study 

was not conducted and is not required for this use petition. 

5.1.2 Summary of Environmental Degradation

In the environment, halauxifen-methyl hydrolysis is pH dependent, with the compound being 

relatively stable to hydrolysis at neutral pH and the hydrolysis rate increasing at lower or higher 

pHs. The estimated single first-order (SFO) hydrolysis half-lives for halauxifen-methyl at pH 4, 

7, and 9 are approximately 81 days, 155 days, and 3 days, respectively. The only hydrolysis 

product reported is XDE-729 acid, which was produced quantitatively at each pH condition 

during the 30-day study, suggesting that XDE-729 acid is stable to hydrolysis. 

Halauxifen-methyl degraded rapidly in an aqueous photolysis study, with a half-life of less than 

10 minutes, with major degradates of XDE-729 acid, as well as degradates 1, 2, 4, 10, 11, and 

14. In the soil photolysis study, rapid halauxifen-methyl transformation was observed in the dark 

control (faster than the irradiated samples); therefore, the phototransformation of halauxifenmethyl in soil could not be appropriately assessed as a route of dissipation in the soil under the 

conditions used in the submitted study. Halauxifen-methyl is assumed to be stable to soil 

photolysis or at least photolysis is expected to be slower than aerobic soil metabolism. 

Transformation products observed in this soil photolysis study were XDE-729 acid and 
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X11449757. In an aerobic soil metabolism study, the major degradates observed were XDE-729 

acid and X11449757, with half-lives ranging between 1.1 to 5.3 days. In the anaerobic soil 

metabolism study, the major degradates were XDE-729 acid and X11449757, with a half-life of 

3.1 to 7.1 days. The major degradates under anaerobic aquatic conditions included XDE-729 

acid, X11449757, and X11406790. 

Halauxifen-methyl is characterized as moderately mobile; however, significant degradation of

halauxifen-methyl was observed during an adsorption and desorption equilibrium experiment.

Therefore, the mobility of halauxifen-methyl cannot be accurately assessed. Transformation 

products XDE-729 acid and X11449757 are considered to be mobile to moderately mobile. 

Based on available data from a fish bioconcentration study, there is little concern of halauxifenmethyl bioaccumulation. All nine identified environmental transformation products have the 

potential to reach drinking water as they were major degradates in one or more biodegradation 

studies. Two degradates (XDE-729 acid and X11449757) were also observed in the terrestrial 

field dissipation study. Six degradates, degradates 1, 2, 4, 10, 11, and 14, were only detected in 

the aqueous photolysis study. It is unclear if the extraction procedures were suitable for 

quantitative recovery of these degradation products as well as minor transformation products. 

Deg 11 and 14 are different in structure than parent and the other identified transformation 

products. These two transformation products no longer retain the picolinic acid moiety.

5.1.3 Comparison of Metabolic Pathways

Parent halauxifen-methyl undergoes the same demethylation in wheat, turnips, goats, hens, and 

rats. In addition, many of the primary metabolites seen in the metabolism studies are also seen in 

the environmental degradation studies. Halauxifen-methyl contains two methoxy groups (i.e., 

-OCH3 groups). One of these methoxy groups forms the methyl ester moiety on the chemical 

and the other methoxy group is attached to the phenyl ring. The primary metabolic pathways 

involve conversion of either one or both of these two methoxy groups to hydroxyl groups. When 

the methoxy group that forms the methyl ester is converted to a hydroxy group, the moiety 

becomes an acid. This compound is the XDE-729 acid metabolite. When the methoxy group on 

the phenyl ring is converted to a hydroxyl group, the compound becomes a phenol (the 

X11406790 metabolite). Both of these metabolites can then be converted to the metabolite with 

both the acid and phenol moieties (the X11449757 metabolite). In plants and animals, these 

metabolites can conjugate with various moieties, depending on the plant or animal species.

Rat metabolism studies were performed with both the parent compound as well as the XDE-729 

acid metabolite. Rats metabolized the compounds in a comparable manner. Absorption and 

excretion were extensive and rapid. XDE-729 acid was the primary metabolite of the parent 

compound. When rats were dosed with XDE-729 acid, the limited metabolism that did occur 

was due to acyl-glucuronidation or O-demethylation to form X11449757, followed by 

glucuronide or sulfate conjugation. Supplemental metabolism studies were done with dogs and 

mice. Metabolism was extensive and rapid in these species as well. Rats administered 

halauxifen-methyl formed the same demethylated metabolites that were seen in the plant 

metabolism studies and in the environmental degradation studies: XDE-729 acid and 

X11449757.
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5.1.4 Residues of Concern Summary and Rationale

Holman, E. 20 May 2014, D416125

HED’s Residues of Concern Knowledgebase Subcommittee (ROCKS) has recommended that the 

residue of concern in primary crops for both tolerance enforcement and risk assessment be parent 

halauxifen-methyl only. The committee recommended that the residue of concern in ruminants 

and poultry for both tolerance enforcement and risk assessment be the metabolite X11449757. 

These conclusions are summarized below in table 5.1.4. 

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

Tolerance Expression.

Matrix Residues Included in Risk Assessment Residues Included in Tolerance 

Expression for Compliance 

Monitoring

Plants Primary Crop Halauxifen-methyl Halauxifen-methyl

Rotational Crop Not Applicable Not Applicable

Livestock Ruminant X11449757 X11449757

Poultry X11449757 X11449757

Drinking Water Halauxifen-methyl + XDE-729 Acid + 

X11449757 + X11406790 + Degradates 

1, 2, 4, 10, 11, and 14

Not Applicable

Plants (Primary Crops): For dietary risk assessment and tolerance enforcement, the parent 

compound should be the only residue included as a residue of concern. Halauxifen-methyl was 

the only quantifiable residue observed in the crop field trials, specifically in wheat straw and 

forage, with no quantifiable residues of halauxifen-methyl in wheat grain or any barley matrix. 

XDE-729 acid was not quantifiable in any of the crop field trials. 

Rotational Crops: In the confined rotational crop study, the TRR in all commodities at all 

plantback intervals was 0.001 ppm or less. Because of the very low TRR levels, none of the 

RAC samples were characterized further. Therefore, the residues of concern in rotational crops 

are not applicable at this time, nor do rotational crop tolerances need to be set at this time. 

Livestock: For dietary risk assessment and tolerance enforcement, metabolite X11449757 (4-

amino-3-chloro-6-(4-chloro-2-fluoro-3-hydroxyphenyl)-pyridine-2-carboxylic acid) should be 

the only residue of concern. X11449757 was the only quantifiable residue in the cattle feeding 

study, and was a major residue in the goat and poultry metabolism studies. Although 

quantifiable, the residues of XDE-729 acid, X11406790, and X11406790 conjugates were 

generally low (<0.01 ppm). Given the anticipated livestock dietary burden associated with the 

proposed uses, X11449757 is considered to be adequate for enforcement and risk assessment 

purposes. Should the dietary burden increase in the future due to proposed new uses, this 

decision may need to be revisited and additional data may be required, including a poultry 

feeding study. If poultry feeding studies are conducted in the future, then it is recommended that 

metabolites observed in the poultry metabolism study be analyzed, including XDE-729 acid, 

X11406790, and X11449757.
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Water: The ROCKS recommended including parent halauxifen-methyl, as well as 

transformation products XDE-729 acid, X11449757, X11406790, degradates 1, 2, 4, 10, 11, and 

14. Parent halauxifen-methyl in the environment is rapidly metabolized primarily to XDE-729 

acid, especially at low, environmentally relevant concentrations. Therefore, actual exposure is 

essentially to XDE-729 acid regardless of environmental exposure to either halauxifen-methyl or 

the XDE-729 acid. Therefore, grouping parent halauxifen-methyl with XDE 729-acid for 

drinking water is appropriate. 

From an exposure perspective, degradates 1, 2, 4, 10, 11, and 14 were only observed in the

aqueous photolysis study. Since aquatic photolysis is only likely to occur in clear shallow 

surface water and on foliar surfaces, exposure to these transformation products is expected to be 

primarily in surface water rather than groundwater. 

5.2 Food Residue Profile

Dotson, D., 11 July 2014, D416127

The submitted residue chemistry studies were generally well conducted and are adequate for 

supporting regulatory conclusions, establishing appropriate tolerance levels for enforcement, and 

for risk assessment. The number of trials is sufficient and the geographic distribution of the 

trials captures the major wheat and barley growing regions in the U.S., per OCSPP Series 860 

Guidelines. Data from the trials are representative of worst-case residues expected to result from 

application of halauxifen-methyl according to the proposed use patterns, and the residue data are 

supported by adequate analytical method performance and storage stability data. For regulatory 

enforcement, analysis of residues can be accomplished through standard analytical techniques, 

and residues do not show any trends for dissipation during frozen storage.

In the wheat field trials, parent halauxifen-methyl was the only residue present in forage, grain, 

and straw (Table 5.2). Residues of parent were below the LOQ of 0.01 ppm in all samples of 

grain. Residues of XDE-729 acid were below the LOQ of 0.01 ppm in all samples of wheat 

forage, hay, grain, and straw. In the barley field trials, residues of both parent and XDE-729 acid 

were below the LOQ of 0.01 ppm in all samples of wheat forage, hay, grain, and straw. 

Under the currently proposed use pattern, tolerances are not needed for rotational crop 

commodities; however, HED notes that if future uses have higher application rates, limited field 

rotational crop data may be required. 

The cattle feeding study is adequate and can be used to determine the need for tolerances for

ruminant commodities. No data deficiencies are associated with the cattle feeding study. Based 

on the dietary burdens that were constructed and the results of the cattle feeding study and the 

hen metabolism study, tolerances are not needed for livestock commodities. There is no 

reasonable expectation of finite residues in livestock commodities (40CFR §180.6(a)(3). As 

noted previously, should the dietary burden increase in the future due to proposed new uses, this 

decision may need to be revisited and additional data may be required, including a poultry 

feeding study. 

Tolerances are not needed for wheat or barley processed commodities. In the wheat processing 

study, an exaggerated application rate of 5x was used. All samples of unprocessed grain and 
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processed commodities contained residues below the limit of detection. In addition, in all wheat 

and barley field trials, residues were below the LOQ in all samples of grain. Therefore, HED 

concludes that the processing data are adequate, and that tolerances are not needed for processed 

commodities.

Table 5.2. Summary of Residues from the NAFTA Crop Field Trials with Halauxifen-Methyl.

Proposed Use: 0.0088 lb ai/acre (9.8 g/ha) total application rate

1-day PHI for Forage

21-day PHI for Hay

60-Day PHI for Grain and Straw

Crop Matrix

Applic. Rate

lb ai/A

(g ai/ha)

PHI 

(days)

Residues (ppm)

n Min Max LAFT HAFT1 Median Mean.

Std. 

Dev.2

Wheat

Wheat Forage

0.0087-0.0099

(9.7-11)

1 27 0.036 0.389 0.043 0.384 0.157 0.159 0.08

Wheat Hay 17-31 27 <0.01 0.026 <0.01 0.025 <0.01 0.011 0.004

Wheat Grain 42-73 27 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA

Wheat Straw 42-73 27 <0.01 0.013 <0.01 0.012 <0.01 <0.01 0.0004

Barley

Barley Hay

0.0089-0.0098

(9.98-11)

17-33 18 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA

Barley Grain 43-83 18 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA

Barley Straw 43-83 18 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NA

1: HAFT = Highest Average Field Trial.

2: Std. Dev.: Standard Deviation. Blank cell indicates that all field trial samples are below the LOQ.

5.3 Water Residue Profile

Bohaty, R. 12 June 2014, D406773

EFED provided estimated drinking water concentrations (EDWCs) for the proposed uses 

(D406773, R. Bohaty, 06/12/2014). EFED provided values for parent, acid metabolite, and the 

total toxic residues in both surface and groundwater. In all three cases (parent, acid metabolite, 

and total toxic residues) the groundwater values were higher than the surface water numbers. 

For parent halauxifen-methyl, EFED used the PRZM-GW Model and generated a maximum 

groundwater value of 0.007 ppb. For risk assessment, the drinking water residues of concern 

include parent, the acid metabolite, and various other metabolites. For the XDE-729 acid dietary 

exposure assessment, all residues of concern were included as the total toxic residue, yielding an 

EDWC value of 19.5 ppb.

5.4 Dietary Risk Assessment

Dotson, D. 11 July 2014, D417128
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5.4.1 Description of Residue Data Used in Dietary Assessment

As discussed previously, the toxicological effects caused by parent halauxifen-methyl and the 

XDE-729 acid metabolite are different. Therefore, separate chronic dietary exposure 

assessments were performed for the two compounds. For parent halauxifen-methyl, an 

assessment was performed for food and drinking water. Due to the extremely low residues of 

halauxifen-methyl in drinking water, a drinking water only assessment yielded no exposure 

(Doston D., D417128). The assessment that included food was based on tolerance level 

residues (LOQ-level of 0.01 ppm) and the assumption that 100% of all barley, wheat, and 

triticale consumed will be treated with halauxifen-methyl. For the acid metabolite, the 

assessment was based on drinking water alone because the acid metabolite is not a residue of 

concern in food, but it is a residue of concern in water. 

Processing factors were not used for any commodities in the assessments. The submitted 

processing study demonstrated that residues do not concentrate in cereal grain processed 

commodities. In addition, there are no default processing factors for wheat or barley processed 

commodities. 

5.4.2 Dietary Exposure Risk Estimates

Dietary exposure (food + drinking water) and risk estimates were developed for the following 

population subgroups: the general U.S. population, all infants (<1 year old), children 1-2, 

children 3-5, children 6-12, youth 13-19, adults 20-49, females 13-49, and adults 50+ years old. 

Generally, HED is concerned when dietary risk estimates exceed 100% of the populationadjusted dose (PAD) for either acute or chronic exposure scenarios. For halauxifen-methyl and 

XDE-729 acid, all dietary risk estimates were well below 100% of either PAD (Table 5.4.3). In 

both of the chronic dietary exposure assessments that were performed, the general U.S. 

population and all population subgroups used <1% of the chronic population adjusted dose 

(cPAD). 

Halauxifen-methyl is classified as “not likely to be carcinogenic to humans at dose levels that do 

not induce liver toxicity or Cyp1a1 expression.” As a result, cancer risk is not of concern for this 

chemical.

5.4.3 Dietary Risk Assessment Summary Table

Table 5.4.3. Summary of Dietary Exposure and Risk Estimates for Parent Halauxifen-Methyl 

and XDE-729 Acid.

Population 

Subgroup

Parent Halauxifen-Methyl:

Food Plus Water

XDE-729 Acid Metabolite:

Drinking Water Only

Exposure 

Estimate, 

mg/kg/day

% cPAD

cPAD 

mg/kg/day

Exposure Estimate

mg/kg/day % cPAD

U.S. Population 0.000017 <1 0.20 0.000408 <1

All infants* 0.001010 <1 0.20 0.001053 <1

Children 1-2 yrs 0.000040 <1 0.20 0.000589 <1
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Table 5.4.3. Summary of Dietary Exposure and Risk Estimates for Parent Halauxifen-Methyl 

and XDE-729 Acid.

Population 

Subgroup

Parent Halauxifen-Methyl:

Food Plus Water

XDE-729 Acid Metabolite:

Drinking Water Only

Exposure 

Estimate, 

mg/kg/day

% cPAD

cPAD 

mg/kg/day

Exposure Estimate

mg/kg/day % cPAD

Children 3-5 yrs 0.000041 <1 0.20 0.000496 <1

Children 6-12 yrs 0.000030 <1 0.20 0.000358 <1

Youth 13-19 yrs 0.000018 <1 0.20 0.000298 <1

Adults 20-49 yrs 0.000015 <1 0.20 0.000407 <1

Adults 50-99 yrs 0.000012 <1 0.20 0.000403 <1

Females 13-49 yrs 0.000013 <1 0.20 0.000406 <1

*Most highly exposed population subgroup

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

At this time there are no proposed residential uses of halauxifen-methyl. 

6.1 Residential Bystander Post-Application Inhalation Exposure

Volatilization of pesticides may be a source of post-application inhalation exposure to 

individuals nearby pesticide applications. The agency sought expert advice and input on issues 

related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act 

Scientific Advisory Panel (SAP) in December 2009, and received the SAP’s final report on 

March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html). The 

agency has evaluated the SAP report and has developed a Volatilization Screening Tool and a 

subsequent Volatilization Screening Analysis 

(http://www.regulations.gov/#!docketDetail;D=EPA-HQ-OPP-2014-0219). During Registration 

Review, the agency will utilize this analysis to determine if data (i.e., flux studies, route-specific 

inhalation toxicological studies) or further analysis is required for halauxifen-methyl. 

6.2 Spray Drift

Spray drift is a potential source of exposure to those nearby pesticide applications. This is 

particularly the case with aerial application, but, to a lesser extent, spray drift can also be a 

potential source of exposure from the ground application methods (e.g., aerial and groundboom)

employed for halauxifen-methyl. The agency has been working with the Spray Drift Task Force 

(a task force composed of various registrants which was developed as a result of a Data Call-In 

issued by EPA), EPA Regional Offices and State Lead Agencies for pesticide regulation and 

other parties to develop the best spray drift management practices (see the agency’s Spray Drift 

website for more information). The Agency has also developed a policy on how to appropriately 

consider spray drift as a potential source of exposure in risk assessments for pesticides. The 

potential for spray drift will be quantitatively evaluated for each pesticide during the Registration 

Review process which ensures that all uses for that pesticide will be considered concurrently. 
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The approach is outlined in the revised (2012) Standard Operating Procedures for Residential 

Risk Assessment (SOPs) - Residential Exposure Assessment Standard Operating Procedures 

Addenda 1: Consideration of Spray Drift. This document outlines the quantification of indirect 

non-occupational exposure to drift. 

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and 

risks from three major sources: food, drinking water, and residential exposures. In an aggregate 

assessment, exposures from relevant sources are added together and compared to quantitative 

estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When 

aggregating exposures and risks from various sources, HED considers both the route and 

duration of exposure.

As previously noted, there are no residential uses for halauxifen-methyl at this time; therefore, 

dietary exposure is the only pathway appropriate for assessment. Aggregate risk estimates are 

equivalent to the dietary risk estimates discussed in Section 5 and are below HED’s level of 

concern.

8.0 Cumulative Exposure/Risk Characterization

Unlike other pesticides for which EPA has followed a cumulative risk approach based on a 

common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as 

to halauxifen-methyl or XDE-729 acid and any other substances, and halauxifen-methyl or XDE729 acid does not appear to produce a toxic metabolite produced by other substances. For the 

purposes of this tolerance action, therefore, EPA has not assumed that halauxifen-methyl or 

XDE-729 acid has a common mechanism of toxicity with other substances. For information 

regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity 

and to evaluate the cumulative effects of such chemicals, see the policy statements released by 

EPA’s Office of Pesticide Programs concerning common mechanism determinations and 

procedures for cumulating effects from substances found to have a common mechanism on 

EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

Figueroa, Z. 22 July 2014, D416129

There is a potential for occupational exposure associated with handler activities (i.e., mixing, 

loading, and applying). Post-application exposure is expected for workers re-entering treated 

areas. Based on the proposed labels’ use pattern/directions (e.g., 1-2 applications per season), 

short- term (1 to 30 days) and intermediate-term (1 to 6 months) durations of exposure are 

expected for handlers. Long-term exposures (greater than 6 months) are not anticipated.

9.1 Short- and Intermediate-Term Handler Risk

It is HED policy to use the best available data to assess handler exposure. Sources of generic 

handler data, used as surrogate data in the absence of chemical-specific data, include the 

Pesticide Handlers Exposure Database Version 1.1 (PHED 1.1) and the Agricultural Handler 
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Exposure Task Force (AHETF) database, or other registrant-submitted occupational exposure 

studies. Some of these data are proprietary (e.g., AHETF data), and subject to the data 

protection provisions of FIFRA. Default assumptions established by HED’s Science Advisory 

Council for Exposure were used for parameters such as body weight and area treated .

The maximum single application rate on the proposed labels was used to assess the handler 

exposure scenarios. Refer to Table 3.3 for a complete use pattern description. The inputs for 

area treated were based on information in ExpoSAC Policy 9.1. 

Since no short- or intermediate-term dermal endpoints of concern were identified, a dermal 

exposure assessment was not conducted. Occupational assessments for halauxifen-methyl are 

based on inhalation exposures only.

Table 9.1 presents the estimated short- and intermediate-term inhalation risk estimates for 

occupational handlers. All occupational handler scenarios for the proposed uses resulted in 

inhalation risk estimates that are greater than the LOC of 100 (i.e., not of concern) at some level 

of PPE and/or risk mitigation (i.e., baseline attire with no respirator; engineering control for 

aerial applications (enclosed cockpits)). The lowest inhalation MOE, identified for handlers 

conducting mixer/loader activities with a water dispersible granule (WDG) formulation for aerial

applications, was 2,500; thus providing an ample margin to account for any uncertainties in the 

route-to-route extrapolation. No additional PPE recommendations are needed on the proposed 

labels. 

Table 9.1. Short- and Intermediate-Term Occupational Handler Exposure and Risk Estimates for Halauxifen-methyl.

Exposure Scenario Mitigation 

Levela

Inhalation Unit 

Exposure b

(μg/lb ai)

Application Ratec Area TreateddInhalation 

Dosee

(mg/kg/day)

Inhalation 

MOEf

Mixer/Loader

Mixing/Loading WDG for 

Aerial 

Baseline 8.96 0.0091 lb ai/A 1,200 A 0.001220 2,500

0.0049 lb ai/A 0.000659 4,600

Mixing/Loading WDG for 

Groundboom 

Baseline 8.96 0.0091 lb ai/A 200 A 0.000204 15,000

0.0049 lb ai/A 0.000110 27,000

Mixing/Loading Liquids 

for Aerial 

Baseline 0.219 0.0058 lb ai/A 1,200 A 0.000019 160,000

Mixing/Loading Liquids 

for Groundboom 

Baseline 0.219 0.0058 lb ai/A 200 A 0.00000318 940,000

Applicator

Applying Sprays for 

Aerial Application

Engineering 

control 

(enclosed 

cockpits)

0.0049 0.0091 lb ai/A 1,200 A 0.00000067 4,500,000

0.0049 lb ai/A 0.00000036 8,300,000

0.0058 lb ai/A 0.00000043 7,000,000

Applying Sprays for 

Groundboom Application 

Baseline 0.34 0.0091 lb ai/A 200 A 0.00000774 390,000

0.0049 lb ai/A 0.00000416 720,000
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Table 9.1. Short- and Intermediate-Term Occupational Handler Exposure and Risk Estimates for Halauxifen-methyl.

Exposure Scenario Mitigation 

Levela

Inhalation Unit 

Exposure b

(μg/lb ai)

Application Ratec Area TreateddInhalation 

Dosee

(mg/kg/day)

Inhalation 

MOEf

0.0058 lb ai/A 0.00000493 610,000

Flagger

Applying Sprays for 

Aerial Application 

Baseline 0.35 0.0091 lb ai/A 350 A 0.0000139 220,000

0.0049 lb ai/A 0.0000075 400,000

0.0058 lb ai/A 0.00000889 340,000

a. Mitigation level or PPE.

b. Unit Exposures based on AHETF/PHED. 

c. Application rates are based on proposed use on labels; for GF-2685 = 0.0091 lb ai/A, for GF-2686 and GF-2687 = 0.0049 lb ai/A; and GF2688=0.0058 lb ai/A.

d. Exposure Science Advisory Council Policy No. 9.1.

e. Inhalation Dose (mg/kg/day) = Daily Unit Exposure (μg/lb ai) x Application Rate (lb ai/acre) x Area Treated / Body Weight (80 kg). 

f. Inhalation MOE = HED (3.0 mg/kg/day) / Inhalation Daily Dose (mg/kg/day). LOC = 100. 

9.2 Short- and Intermediate-Term Post-Application Risk

Inhalation

There are multiple potential sources of post-application inhalation exposure to individuals 

performing post-application activities in previously treated fields. These potential sources 

include volatilization of pesticides and resuspension of dusts and/or particulates that contain 

pesticides. The Agency sought expert advice and input on issues related to volatilization of 

pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel 

(SAP) in December 2009, and received the SAP’s final report on March 2, 2010 

(http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html). The Agency has 

evaluated the SAP report and has developed a Volatilization Screening Tool and a subsequent 

Volatilization Screening Analysis (http://www.regulations.gov/#!docketDetail;D=EPA-HQOPP-2014-0219). During Registration Review, the Agency will utilize this analysis to determine 

if data (i.e., flux studies, route-specific inhalation toxicological studies) or further analysis is 

required for halauxifen-methyl.

In addition, the Agency is continuing to evaluate the available post-application inhalation 

exposure data generated by the Agricultural Reentry Task Force. Given these two efforts, the 

Agency will continue to identify the need for and, subsequently, the way to incorporate 

occupational post-application inhalation exposure into the agency's risk assessments.

Although a quantitative occupational post-application inhalation exposure assessment was not 

performed, an inhalation exposure assessment was performed for occupational/commercial 

handlers. Handler exposure resulting from application of pesticides outdoors is likely to result in 

higher exposure than post-application exposure. Therefore, it is expected that these handler 

inhalation exposure estimates would be protective of most occupational post-application 

inhalation exposure scenarios. 

Dermal
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Quantitative occupational post-application risk estimates for agricultural workers following 

treatments to agricultural crops were not assessed, since no dermal endpoint was identified.

The REI specified on the proposed label is based on the acute toxicity of halauxifen-methyl. 

Halauxifen-methyl is classified as Toxicity Category IV via the dermal route and for skin 

irritation, and Toxicity Category III for eye irritation potential. It is not a skin sensitizer. Under 

40 CFR 156.208 (c) (2) (iii), ai’s classified as Acute III or IV for acute dermal, eye irritation and 

primary skin irritation are assigned a 12-hour REI. Therefore, the [156 subpart K] Worker 

Protection Statement interim REI of 12 hours is adequate to protect agricultural workers from 

post-application exposures to halauxifen-methyl.

10.0 References

Hansen, L. 2 March 2016, D406773. Halauxifen-Methyl: Summary of the Mechanistic Data 

and a Framework Analysis for the Proposed Mode of Action for Liver Toxicity.

Hansen, L. 2 March 2016. D 430415. Halauxifen-Methyl: Toxicology Data Evaluation 

Reports for Studies Submitted to Support the Proposed Registration of New Technical 

Active Ingredient (Joint Chemical Review with Canada and Australia).

Dotson, D. 11 July 2014, D416127. Halauxifen-Methyl. Petition for the Section 3 

Registration and the Establishment of Tolerances on Wheat, Barley, and Triticale. 

Summary of Analytical Chemistry and Residue Data

Dotson, D. 11 July 2014, D416128. Halauxifen-Methyl Chronic Aggregate Dietary (Food 

and Drinking Water) Exposure and Risk Assessment for the Section 3 Registration 

Action on Barley, Wheat, and Triticale. 

Figueroa, Z. 22 July 2014, D416128. Halauxifen-methyl. Occupational and Residential 

Exposure and Risk Assessment to Support the Registration of the New Active 

Ingredient on Wheat, Barley, and Triticale.

Holman, E. 20 May 2014, D416125. Halauxifen-methyl. Report of the Residues of Concern 

Knowledgebase Subcommittee (ROCKS). 

Bohaty, R. 12 June 2014, D406773. Halauxifen-methyl: New Chemical Drinking Water 

Assessment

Safe, S., Lee, S.-O. and Jin, U-H. (2013) Role of the Aryl Hydrocarbon Receptor in 

Carcinogenesis and Potential as a Drug Target. Toxicol. Sciences online 

doi:100.1093/toxsci/kft128, June 14, 2013

Cohen, S.M. (2010) Evaluation of Possible Carcinogenic Risk to Humans Based on Liver Tumors 

in Rodent Assays: The Two-Year Bioassay is No Longer Necessary. Toxicol. Pathol. 

38:487-501

Nebert, D.W., Dalton, T.P, Okey, A.B. and Gonzalez, F.J. (2004) Role of Aryl Hydrocarbon 

Receptor-mediated Induction of the CYP1 Enzymes in Environmental Toxicity and 

Cancer. J. Biolog. Chem. 279 (23):23847-23850, June 4, 2004.
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Appendix A. Toxicology Profile and Executive Summaries

A.1 Toxicology Data Requirements

The requirements (40 CFR 158.340) for food use for halauxifen-methyl are shown below in Table 1. Use of the new 

guideline numbers does not imply that the new (1998) guideline protocols were used.

Study

Technical

Required Satisfied

870.1100 Acute Oral Toxicity.......................................................

870.1200 Acute Dermal Toxicity..................................................

870.1300 Acute Inhalation Toxicity..............................................

870.2400 Primary Eye Irritation....................................................

870.2500 Primary Dermal Irritation..............................................

870.2600 Dermal Sensitization .....................................................

yes1

yes1

yes1

yes1

yes1

yes1

yes

yes

yes

yes

yes

yes

870.3100 Oral Subchronic (rodent)...............................................

870.3150 Oral Subchronic (nonrodent).........................................

870.3200 28-Day Dermal..............................................................

870.3250 90-Day Dermal..............................................................

870.3465 90-Day Inhalation..........................................................

yes1

yes2

yes2

no3

 no3

yes

yes

yes

-

-

870.3700a Developmental Toxicity (rodent) ..................................

870.3700b Developmental Toxicity (nonrodent) ............................

870.3800 Reproduction .................................................................

yes1

yes1

yes2

yes

yes

yes

870.4100a Chronic Toxicity (rodent)..............................................

870.4100b Chronic Toxicity (nonrodent)........................................

870.4200a Oncogenicity (rat)..........................................................

870.4200b Oncogenicity (mouse) ...................................................

870.4300 Chronic/Oncogenicity (rat)............................................

yes2,4

yes2

yes2,4

yes2

yes2

yes

yes

yes

yes

yes

870.5100 Mutagenicity—Gene Mutation - bacterial.....................

870.5300 Mutagenicity—Gene Mutation - mammalian................

870.5xxx Mutagenicity—Structural Chromosomal Aberrations...

870.5xxx Mutagenicity—Other Genotoxic Effects.......................

yes1

yes1

yes1

yes1

yes

yes

yes

yes

870.6100a Acute Delayed Neurotoxicity (hen)...............................

870.6100b 90-Day Neurotoxicity (hen) ..........................................

870.6200a Acute Neurotoxicity Screening Battery (rat).................

870.6200b 90-Day Neurotoxicity Screening Battery (rat) ..............

870.6300 Develop. Neurotoxicity .................................................

no

no

yes2

yes2

no

-

-

yes

yes

-

870.7485 General Metabolism ......................................................

870.7600 Dermal Penetration........................................................

870.7800 Immunotoxicity .............................................................

yes1

yes5

yes5

yes

yes

yes

Special Studies for Ocular Effects

Acute Oral (rat)...........................................................

Subchronic Oral (rat)..................................................

Six-month Oral (dog)..................................................

no

no

no

-

-

-

1 Studies were conducted on both halauxifen-methyl and XDE-729 acid

2 Study was conducted on XDE-729 acid

3 Study waiver granted

4 Requirement satisfied by 870.4300, Chronic/Oncogenicity study in the rat

5 Study was conducted on halauxifen-methyl
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 43 of 126

A.2 Toxicity Profiles

Acute Toxicity of Halauxifen-methyl (XDE-729 methyl)

Guideline No./Study Type MRID Results Toxicity 

Category

870.1100 Acute oral toxicity (rat) 48557814 LD50 > 5000 mg/kg IV

870.1200 Acute dermal toxicity (rat) 48557816 LD50 > 5000 mg/kg IV

870.1300 Acute inhalation toxicity (rat) 48557818 Study not submitted based on 

inability to generate test 

atmosphere. 

-

870.2400 Acute eye irritation (rabbit) 48557820 Mild eye irritant III

870.2500 Acute dermal irritation (rabbit) 48557822 Not a dermal irritant IV

870.2600 Skin sensitization (guinea pig) 48557824 Not a dermal sensitizer NA

NA = Not Applicable

Acute Toxicity of XDE-729, acid (metabolite of halauxifen-methyl, or XDE-729 methyl)

Guideline No./Study Type MRID Results Toxicity 

Category

870.1100 Acute oral toxicity (rat) 48557834 LD50 > 5000 mg/kg IV

870.1200 Acute dermal toxicity (rat) 48557815 LD50 > 5000 mg/kg IV

870.1300 Acute inhalation toxicity (rat) 48557817 Study not submitted based on 

inability to generate test 

atmosphere. 

-

870.2400 Acute eye irritation (rabbit) 48557819 Mild eye irritant III

870.2500 Acute dermal irritation (rabbit) 48557821 Not a dermal irritant IV

870.2600 Skin sensitization (guinea pig) 48557823 Not a dermal sensitizer NA

NA = Not Applicable
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 44 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Subchronic Studies

870.3050 

28-day oral toxicity 

(mice)

Study conducted only for 

XDE-729 acid 

870.3050 

28-day oral toxicity 

(F344/DuCrl rats)

48557826 (2011)

Acceptable/guideline

Dietary concentrations 

equivalent to:

0, 10.8, 55.6, 296, or 822 

mg/kg/day for males 

0, 10.7, 55.5, 277, or 825

mg/kg/day for females

NOAEL = 10.8/55.5 mg/kg/day (M/F) 

LOAEL = 55.6 mg/kg/day in males, based on increased 

liver weight, hepatocellular hypertrophy, increased 

mitotic figures in hepatocytes, hepatocellular 

vacuolization (consistent with fatty change) and 

hypertrophy of thyroid gland follicular cells. The 

female LOAEL is 277 mg/kg/day, based on decreased 

body weight gains and food consumption, increased 

liver weight, increased cholesterol levels and 

hepatocellular hypertrophy. 

At the highest dose slight thymic atrophy, acinar cell 

hypertrophy of the submandibular salivary gland, 

hyperplasia of erythroid cells in the bone marrow, 

increased incidence of very slight extramedullary 

hematopoiesis (erythroid cell) in the spleen, and 

decreased size of the cervix, uterus and vagina

(females) were observed.

Toxicokinetics: XDE-729 acid was present in blood, 

but not halauxifen-methyl. Less than 0.1% AD 

halauxifen-methyl was present in urine; remainder 

excreted as XDE-729 acid. Saturation of either 

absorption or metabolism possible.

870.3050 

28-day oral toxicity 

(Beagle dog)

Study conducted only for 

XDE-729 acid form
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 45 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3100

90-Day oral toxicity 

(F344/DuCrl rats)

48557830 (49124405, 

amended report) (2011)

Acceptable/guideline

Dietary concentrations 

equivalent to:

0, 3.1, 10.3, 53.4, 270 or 513 

mg/kg/day in males and 0, 

3.0, 10.1, 52.3, 263 or 504 

mg/kg/day

NOAEL = 10.0/10.1 mg/kg/day [M/F]

LOAEL = 53.4/52.3 mg/kg/day [M/F], based on 

increased hepatocellular vacuolization (males) and 

increased cholesterol and liver weights (females). 

At 270/263 mg/kg/day, lower % neutrophils and 

increased lymphocytes (females), increased cholesterol 

(males), increased absolute and relative liver weight, 

hypertrophy with cytoplasmic eosinophilia, increased 

absolute and relative thyroid weight and thyroid 

hypertrophy were observed.

At 513/504 mg/kg/day, decreased body weight and 

weight gain and decreased thymic weight and atrophy 

(females) were observed.

Toxicokinetic studies: Halauxifen-methyl showed 

dose-dependent levels in liver at all doses, and in blood 

≥10 mg/kg/day. XDE-729 acid metabolite was present

as the major moiety in blood, liver, urine, along with 

other minor conjugated metabolites. Data suggest 

nonlinearity in metabolism/elimination of halauxifenmethyl, with increasing conjugation of metabolites as 

dose increased, and induction of hepatic metabolic 

enzymes.

Gene expression studies: Cyp1a1 expression in liver 

showed a dose-related increase. At 3 mg/kg/day 

expression was slightly higher but similar to controls 

(3.6 to 5.7-fold) but showed a more pronounced 

increase (52-149-fold) at the NOAEL of 10 mg/kg/day 

and a significant increase (1495-2268-fold) at the 

LOAEL of 53 mg/kg/day.

870.3150

90-Day oral toxicity

(Beagle dog)

Study conducted only for 

XDE-729 acid form.

870.3200

28- or 90-day dermal 

toxicity (rats)

Study conducted only for 

XDE-729 acid form. Waiver 

for XDE-729 methyl granted 

based on lack of systemic 

exposure to methyl ester 

following dermal exposure 

(acid form found in blood in 

dermal absorption study).

870.

28- or 90-day 

inhalation toxicity 

(rats)

Waiver granted due to 

inability to generate test 

atmosphere. 

Developmental and Reproduction Studies
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 46 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3700a

Prenatal 

developmental probe 

study

[(Crl:CD(SD) rats]

48557881 (2012)

Acceptable/nonguideline

Dietary concentrations of 0, 

521, 2083, 4167 or 833 ppm, 

equivalent to 0, 40.4, 169, 

303 or 490 mg/kg/day 

Administered GD 6 through 

21

Maternal

NOAEL = 169 mg/kg/day

LOAEL = 303 mg/kg/day based on decreased food 

consumption and body weight gains. 

Dams given 490 mg/kg/day were sacrificed on GD 9 

due to excessive body weight loss.

Developmental

NOAEL/LOAEL = not determined, fetal examination 

not performed. There were no significant cesarean 

findings. 

870.3700a

Prenatal 

developmental study

[Crl:CD(SD) rats]

48557836 (2012)

Acceptable/guideline

Dietary concentrations of 

521, 2083 or 4167 ppm, 

equivalent to 0, 41.1, 159 or 

323 mg/kg/day

Administered GD 6 through 

21

Maternal 

NOAEL = 159 mg/kg/day

LOAEL = 323 mg/kg/day based on decreased body 

weight gains and food consumption, increased liver 

weight and hepatocyte histopathology (altered 

centrilobular and midzonal cytoplasmic homogeneity 

and hepatocellular vacuolization consistent with fatty 

change).

Developmental 

NOAEL ≥323 mg/kg/day

No dose related effects were seen, thus the 

developmental LOAEL was not determined.

870.3700b

Prenatal 

developmental probe 

study

(NZW rabbits)

48557839 (2012)

Acceptable/nonguideline

Dietary concentrations of 0, 

521, 2083, 4167. 8333, 

10417, 15625 or 24,500 

ppm, equivalent to:

0, 21.5, 83.3, 157, 244, 371, 

426 or 727 mg/kg/day

Administered GD 7 through 

28

Maternal 

NOAEL = 157 mg/kg/day

LOAEL = 244 mg/kg/day based on decreased body 

weight, weight gain and food consumption. 

Dams given 727 mg/kg/day were prematurely sacrificed 

on prior to GD 15 due to excessive weight loss.

Developmental 

NOAEL/LOAEL = not determined, fetal examination 

not performed. There were no significant cesarean 

findings.

No dose related effects were seen, thus the 

developmental LOAEL was not determined.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 47 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3700b

Prenatal 

developmental

(NZW rabbits)

48557840 (2012)

Acceptable/guideline

Dietary concentration of 0, 

122, 391 or 1539 ppm, 

equivalent to 0, 5.8, 18.5 or 

71.6 mg/kg/day

Administered GD 7 through 

28

Maternal 

NOAEL = 5.8 mg/kg/day

LOAEL = 18.5 mg/kg/day based on increased liver 

weight and hepatocellular histopathology (hypertrophy 

with increased cytoplasmic eosinophilia of periportal 

hepatocytes, increased number of mitotic figures in 

hepatocytes, increased altered cytoplasmic homogeneity 

of centrilobular/midzonal hepatocytes.

At 71.6 mg/kg/day, increased panlobular vacuolization 

of hepatocytes consistent with fatty change was also 

observed.

Developmental 

NOAEL = 18.5 mg/kg/day

LOAEL = 71.6 mg/kg/day, based on decreased fetal 

body weights and delayed ossification of the pubis. 

870.3800

Reproduction and 

fertility effects

(rats)

Study conducted on XDE729 acid

Chronic Toxicity Studies

870.4100b

Chronic toxicity 

(Beagle dogs)

Study conducted on XDE729 acid

Chronic/Carcinogenicity Studies

870.4200b

Carcinogenicity 

(mice)

Study conducted on XDE729 acid

870.4300

Chronic/

Carcinogenicity -

(rats)

Study conducted on XDE729 acid

Mutagenicity Studies

Gene Mutation

870.5100

Bacterial Reverse 

Mutation Test (preincubation assay)

48557846 (2011)

Acceptable/guideline

0, 50, 10, 500, 1500, or 5000 

μg/plate +/- S9

Negative in Salmonella typhimurium strains TA 1535, 

TA1537, TA98, and TA100 and Escherichia coli Wp2 

uvrA up to the limit concentration with evidence of 

compound insolubility but no cytotoxicity.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 48 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Gene Mutation

870.5100

Bacterial Reverse 

Mutation Test (plateincorporation assay)

48557845 (2011)

Acceptable/guideline

0, 50, 10, 500, 1500, or 5000 

μg/plate +/- S9

Negative in Salmonella typhimurium strainsTA1537, 

TA98, and TA100 and Escherichia coli Wp2 uvrA up to 

the limit concentration with no evidence of compound 

insolubility or cytotoxicity.

Gene Mutation

870.5300 

In vitro mammalian 

cell forward gene 

mutation, Chinese 

hamster ovary (CHO) 

cells, HGPRT locus 

48557851 (2012) 

Acceptable/guideline 

0, 15.625, 31.25, 62.5, 125, 

250 or 500 μg/mL -S9

activation 

and 

0, 18.75, 37.5, 75, 150, 300 

or 500 μg/mL +S9 activation

Negative for the induction of mutant colonies over 

background up to precipitating concentrations

Gene Mutation

870.5300 

In vitro mammalian 

cell forward gene 

mutation, Chinese 

hamster ovary (CHO) 

cells, HGPRT locus 

48557849 (2011) 

Acceptable/guideline 

0, 3.13, 6.25, 12.5, 25 or 50 

μg/mL+/- S9 activation

Negative for the induction of mutant colonies over 

background up to precipitating concentrations.

Cytogenetics 

870.5375 

In vitro mammalian 

cell chromosomal 

aberration assay, 

human peripheral 

lymphocytes

48557854 (2012) 

Acceptable/guideline 

0, 15.624, 31.25, 62.5, 125, 

250 or 500 μg/mL +/- S9

activation 

Negative for induction of chromosomal aberrations over 

background up to cytotoxic and precipitating 

concentrations.

Cytogenetics 

870.5375 

In vitro mammalian 

cell chromosomal 

aberration assay, 

primary lymphocyte 

cultures (Sprague 

Dawley rats)

48557853 (2012) 

Acceptable/guideline 

0, 0.78, 1.56, 3.13, 6.25, 

12.5, 25 or 50 μg/mL - S9

activation and 

0, 0.39, 0.78, 1.56, 3.13, 

6.25, 12.5, 25 or 50 μg/mL + 

S9 activation

Negative for induction of chromosomal aberrations over 

background up to precipitating concentrations.

Neurotoxicity Studies

870.6200a Acute 

Neurotoxicity 

Screening Battery 

(rats)

Conducted using XDE-729 

acid

870.6200b Subchronic 

Neurotoxicity Study 

(rats)

Conducted using XDE-729 

acid

Other Studies
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 49 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.7485

Metabolism and 

pharmacokinetics

(rats)

48557860 (2010)

Acceptable/guideline

See MRID 48557860, XDE729

870.7600

Dermal penetration in 

vivo (rats)

48557911 (2011)

Acceptable/guideline

[

14C]-XDE-729 methyl in EC 

Formulation GF-2573 was 

applied to shaved rat skin as 

concentrate, field 1 and field 

dilution 2 (75, 1.5 and 0.25 

μg/cm2, respectively).

Exposure time was 10 hours, 

followed by measurement of 

absorption at 24, 48, 72 or 

144 hr (concentrate) and 24, 

48, 96 and 192 hr (dilutions).

Dermal absorption increased with dose but not 

proportionally. The percentage of the dose absorbed 

decreased with the increasing administered dose. The 

total body burden could be expected to rapidly decrease 

due to excretion via urine and feces. 

Mean dermal absorption for the 10-hour exposure 

interval (at 144/192 hr post-exposure) was 13, 15 and 

24% in the concentrate, field dilution 1 and field 

dilution 2 dose groups, respectively.

Blood measurements demonstrated that systemic 

exposure was to XDE-729 acid due to rapid hydrolysis 

of the parent following absorption.

Nonguideline study

Dermal penetration, 

comparative in vitro

study on rat and 

human skin

48557910 (2011)

Acceptable/nonguideline

[

14C]-XDE-729 methyl in EC 

Formulation GF-2573 was 

applied to rat and human 

skin samples as concentrate, 

field 1 and field dilution 2 

(75, 1.5 and 0.25 μg/cm2, 

respectively).

Exposure time was 10 hours, 

followed by measurement of 

absorption at 24 hr.

Percent dermal absorption for rat skin samples was 4.9, 

41.1 and 42.7% of the applied dose.

Percent dermal absorption for human skin samples was 

0.8, 14.9 and 26.5% of the applied dose.

The ratio of in vitro to in vivo dermal absorption in the 

rat was 0.5, 3.0 and 2.2 (24 hr in vivo absorption used 

for comparison).

The ratio of human to rat in vitro absorption (highest to 

lowest concentration tested) is 0.16, 0.36 and 0.62, 

respectively.

870.7800

Immunotoxicity

(F344/DuCrl rat, 

females only)

48557864 (2012)

Acceptable/guideline

Dietary concentrations of 0, 

147, 643 or 6323 ppm, 

formulated to provide 0, 10, 

52, or 500 mg/kg/day

Immunotoxicity NOAEL = 500 mg/kg/day 

Immunotoxicity LOAEL = No dose related effects were 

seen, thus the immunotoxicity LOAEL was not 

determined.

Systemic NOAEL = 52 mg/kg/day

Systemic LOAEL = 500 mg/kg/day based on decreased 

thymus weight and increased liver weight.

Non-Guideline Studies
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 50 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Hepatic gene 

expression and 

biomarker analyses 

(male F344/DuCrl 

rats)

48557879 (2012)

Acceptable/nonguideline

Dietary concentrations 

formulated to provide 

0, 766 mg/kg/day XDE-729 

methyl ester (halauxifenmethyl) or 672 mg/kg/day 

XDE-729 acid, administered 

for 7 days

XDE-729 acid toxicity: There were no effects on the 

liver or thyroid. Decreased body weight and food 

consumption were observed. 

XDE-729 methyl toxicity: Observed effects included 

increased liver weight, very slight panlobular 

hypertrophy of hepatocytes, very slightly increased 

numbers of mitotic figures in hepatocytes, increased 

thyroid weight, very slight hypertrophy of thyroid gland 

follicular cells. 

Toxicokinetics and gene expression: Test material was 

present in liver and blood. Most of excreted test 

material was as XDE-729 acid, with very little XDE729 methyl. In liver and urine, low levels of an acyl 

glucuronide conjugate of XDE-729, O-demethyl XDE729, sulfate conjugate of O-demethyl XDE-729 and 

glucuronide conjugates of O-demethyl XDE-729 and 

XDE-729 methyl were identified. Thyroid effects 

appeared to be secondary to liver alterations, based on 

observed activation of the AhR signaling pathway genes 

by XDE-methyl, based on increased hepatic Cyp1a1 

levels. Animals treated with XDE-729 acid did not 

show induction of liver metabolic genes and animals 

treated with XDE-729 methyl did not show significant 

activation of enzymes of the CAR pathway, or the PXR 

or PPAR-α signaling pathways.

Seven day dietary 

toxicity study probe 

study [Crl:CD(ICR) 

mice]

48557872 (2012)

Acceptable/nonguideline

Dietary concentrations 

formulated to supply:

0, 250 or 778 mg/kg/day in 

males and

0, 276 or 863 mg/kg/day in 

females,

Administered for seven days

Males and females at 778/863 mg/kg/day showed 

increased liver weights but no histopathological 

changes and increased Cyp1a1 mRNA expression (AhR 

activation). 

Parent and XDE-729 acid generally exhibited linear 

kinetics in liver, blood (6 am) & urine. XDE-729-

methyl was detected at low levels in ~50% of blood 

samples at all doses but represented ≤1% of the XDE729 levels (0.03-0.07 μg/mL). XDE-729 methyl was 

only in urine at the high dose (0.20-0.24 μg/mL). Acid 

was present in all treated animals at 100 and 9000-fold 

higher than methyl for blood and urine, respectively. In 

liver, methyl was 50 to 100-fold higher than blood 

(1.39-4.95 μg/g). In liver, acid was 2-fold higher than 

the methyl. 

Levels of both XDE-729 methyl and XDE-729 acid 

were generally lower in female mice compared to males 

in blood (6 am), liver and urine; the only exceptions 

were the terminal blood concentrations, where slightly 

higher XDE-729 methyl and XDE-729 acid levels were 

seen in females.

Kinetics were similar in male mice and rats. The rat is 

more sensitive than the mouse to XDE-729 methylinduced hepatic responses 
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 51 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

28-day dietary study 

on molecular and 

cellular changes in the 

liver 

[ male rats]

48557873 (2012)

Acceptable/nonguideline

Dietary concentrations of 0, 

57.7, 198, 1036 or 5156 ppm 

formulated to provide 0, 3, 

10, 55 or 271 mg/kg/day

Administered for 28 days 

plus additional recovery 

groups were given 0 or 10.3 

mg/kg/day and a 4-day 

recovery period, or 0, 53.7 or 

277 mg/kg/day with a 28-day 

recovery period.

No treatment-related findings were observed on 

survival, clinical signs, body weights, food consumption 

or kidney weights. 

At ≥3 mg/kg bw/day: No quantifiable halauxifenmethyl in liver at 3 mg/kg bw/day, ≤0.36 μg/g at 10 and 

52 mg/kg bw/day, 3.0 μg/g at 261 mg/kg bw/day

At ≥55 mg/kg bw/day: increased liver weight, increased 

Cyp1a1 mRNA expression (684, 121800-fold), 

increased hepatocellular hypertrophy with increased 

cytoplasmic eosinophilia, hepatocellular vacuolation, 

mitotic figures.

At 271 mg/kg bw/day: Marginal ↑ BrdU uptake in liver, 

↑ Cyp1a2 (22-fold), Ugt1a6 (11-fold) & Cyp2b1 (8-

fold) mRNA expression

Recovery: Complete reversal of changes in liver weight, 

liver histopathology, Cyp gene expression & 

quantifiable liver concentrations of methyl & acid

TK: Low levels of methyl in liver, which became 

supralinear between 54.8-271 mg/kg/day. Levels of 

methyl <LLQ in all blood samples; acid was present in 

all blood samples (minor metabolites not measured). 

Blood levels of methyl were sub-linear at 271 

mg/kg/day. In urine, ~0.05% dose was excreted as 

methyl and ~50% dose was excreted as acid. Total 

amount of acid in urine was sub-linear at 271 

mg/kg/day. 

Comparative in vitro 

AhR transactivation 

(Luciferase reporter 

and competition 

ligand binding assays) 

[Mouse (Hepa 1.1), 

human (Hep G2 40/6) 

liver cell lines]

48557878 (2012)

Acceptable/nonguideline

1) Cell-based Luciferase 

reporter assay: 0.001, 0.01, 

0.1, 1, 10, 50, 100 μM 

(XDE-729 methyl, ITE); 

0.01, 0.1, 1, 10, 50 μM 

(XDE-729 methyl ultrapure)

2) Competition ligandbinding assay: 0.01, 0.1, 1, 

10, 50 μM (XDE-729 methyl 

ultrapure)

In cells exposed to XDE-729 methyl at ≥10 μM, a weak 

increase inAhR transactivation activity was observed in 

mouse Hepa 1.1 reporter cell line only, compared to 

positive control ITE (not due to an impurity).

XDE-729 methyl ultrapure (99.7%) is an AhR ligand 

that weakly competes with the photoaffinity ligand for 

the rat Ah receptor (EC50 = 1.5x10-5(XDE-729 methyl), 

8.4x10-7(ITE)).

No AhR transactivation was observed with XDE-729

acid.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 52 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

In vitro AhR nuclear 

receptor activation & 

Cyp1a mRNA 

expression 

[Mouse, Rat, Human 

hepatocytes]

48557876 (2012)

Acceptable/nonguideline

0.01, 0.1, 1.0, 10, 30, 100 

μM XDE-729 methyl for 0, 

2, 4, 8, 12, 24 h

Positive controls: 

3-methylcholanthrene (3-

MC; 0-5 μM); phenobarbital 

(PB; 1 μM)

Exposure to XDE-729 methyl at concentrations ≥30 μM 

caused limited induction of Cyp1a1 and Cyp1a2 in 

mouse and human hepatocytes at multiple time-points 

up to 24 h compared to 3-MC. Human hepatocytes were 

less responsive than mouse and rat hepatocytes to 

Cyp1a1 induction but more responsive to Cyp1a2

induction compared to mice. Peak expression occurred 

from 8-12 h. Sensitivity to Cyp1a induction: 

Rat>Mouse>Human (Cyp1a1>Cyp1a2)

3-MC: Dose responsive ↑Cyp1a1 and Cyp1a2 in all 

species. Cyp1a1 was more responsive compared to 

Cyp1a2 with the mouse and human, but Cyp1a2 was 

induced to a greater extent in the rat. For Cyp1a1

induction, mouse was the most sensitive, followed by 

the human where all but 1 donor had a higher CYP1A1

induction than rat. Rat was most responsive to 3-MC 

with respect to Cyp1a2 induction, human was the next 

most responsive and mouse had the least amount of 

Cyp1a2 induction.

PB: Induction of Cyp2b10 (mouse; modest response), 

Cyp2b6 (rat) and CYP2B6 (human) in hepatocytes
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 53 of 126

Subchronic, Chronic, and Other Toxicity Studies for Halauxifen-methyl (XDE-729 methyl)

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Comparative in vitro

methyl ester 

hydrolysis, PBPK 

modeling

[Mouse, rat and 

human tissues; whole 

blood and synthetic 

gastric fluid (SGF)]

48557874 (2012)

Acceptable/nonguideline

Liver S9: 

0.5, 1.0, 2.0, 5.0, and 10.0 

μM XDE-729 methyl - 

sampling at 0, 3, 8, 15, and 

30 minutes, post-fortification 

(rat, human) 

0, 3, 8, 15, and 30, 60, and 

120; 0.25, 0.5, 1.0, 5.0, and 

10.0 μM-0, 3, 8, 15, 30, 60, 

and 120 minutes (mouse)

Whole blood: 

1 and 100 μM XDE-729 

methyl-0, 0.08, 0.25, 0.5, 1, 

2, 4, 8, 24 hours, postfortification. 

0.25, 0.5, 1, 5, and 10 μM- 0, 

0.05, 0.13, 0.25, 0.5, 1, and 2 

hours post-fortification

SGF: 

1, 100 μM XDE-729 methyl 

at pHs 1.2, 3.0, 4.0-0, 0.08, 

0.25, 0.5, 1, 2, 4, 8 and 24 

hours, post-fortification

Rapid hydrolysis to XDE-729 acid by liver S9 was 

observed in all species, with the relative rate of 

metabolism in order of (fastest to slowest) human>> 

rat>mouse. In human liver S9, XDE-729 methyl was 

not present at >LOQ after 8 min of incubation. XDE729 methyl was metabolized in whole blood by 1st 

order kinetics (fastest to slowest): rat>mouse>>human

The rate of metabolism in synthetic gastric fluid was the 

slowest. The fastest rate of conversion at pH 1.2 (i.e. pH 

of human GIT) = t1/2 100 h was much slower than liver 

S9 & whole blood.

The predicted hydrolysis of XDE-729 methyl was most 

dependent on the rate of metabolism in liver tissue for 

both species (~60% of total hydrolysis in rat and >95% 

in human). Model simulations (adapted to simulate a 

28-day exposure) indicate that XDE-729 methyl and 

XDE-729 acid achieve steady-state concentrations soon 

after exposure is begun, the levels clear rapidly from 

blood and liver following the cessation of exposure, and 

that neither XDE-729 methyl nor XDE-729 would be 

expected to accumulate in either liver or blood 

following exposures to XDE-729 methyl. Modelderived peak concentrations (and 24 h AUCs) of XDE729 methyl in liver and blood are comparable between 

rats and human. 

Peak concentrations from simulated 28 d exposure to 

XDE-729 methyl at 0.001 mg/kg bw/day (relevant 

exposure level based on residue data) resulted in levels 

10000-15000-times lower than LOQ (LOQs: in blood 

~0.01 μg XDE-729 methyl/g; in liver ~0.04 μg XDE729 methyl/g).
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 54 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Subchronic Studies

870.3050 

28-day oral toxicity 

[Crl:CD-1(ICR) mice]

48557827 (2009)

Acceptable/guideline

Dietary concentrations of 10, 

50, 250 or 1000 ppm, 

equivalent to:

0, 10.2, 54.0, 269 or 

1025 mg/kg/day for males 

and 0, 9.9, 48.7, 241 or 958 

mg/kg/day for females

NOAEL = 269/241 mg/kg/day [M/F] 

LOAEL = 1025/958 mg/kg/day [M/F], based on 

decreased numbers and mean volume of red blood cells, 

decreased alanine aminotransferase activity, decreased 

weight and an increased incidence of tubule 

degeneration in the kidney, and increased incidences of 

multifocal karyomegaly and focal necrosis in the liver.

870.3050 

28-day oral toxicity 

(F344/DuCrl rats) 

48557825 (2009) 

Acceptable/guideline 

Dietary concentrations 

equivalent to:

0, 10.5, 52.7, 270 or 734 

mg/kg/day for males 

0, 10, 50.3, 250 or 982 

mg/kg/day for females, 

respectively

NOAEL = 270/250 mg/kg/day M/F

LOAEL = 734/982 mg/kg/day M/F , based on 

decreased body weight, body weight gains and food 

consumption, increased urinary volume, decreased urine 

specific gravity, cloudy urine and phosphate crystals in 

urine sediment (females only), increased relative kidney 

(males only) weights, renal histopathology 

(degeneration and hypertrophy of renal tubules and 

vacuolization of collecting duct epithelium) and splenic 

extramedullary hematopoiesis. 

870.3050 

28-day oral toxicity 

(Beagle dog)

48557871 (2010)

Acceptable/guideline

Dietary concentrations of 0, 

300, 3000, 15,000 (females 

only) or 30,000 ppm (males 

only), equivalent to:

0, 11, 80 or 635 mg/kg/day 

in males

0, 9, 85 or 323 mg/kg/day in 

females

NOAEL = 11/85 mg/kg/day [M/F] 

LOAEL = 80/323 mg/kg/day [M/F], based on 

degeneration and regeneration of renal tubules, and in 

females, body weight loss, reduced food consumption,

and thymic atrophy (likely secondary to stress).

At 635 mg/kg/day in males, body weight loss, reduced 

food consumption, decreased RBC parameters (one 

male), increased cholesterol and decreased thymus 

weight were observed.

870.3100 

90-Day oral toxicity

[Crl:CD-1(ICR) mice]

48557829 (2010)

Acceptable/guideline

Dietary concentrations of 0, 

50, 250, 500 or 1000 ppm, 

equivalent to:

0, 49.4, 247, 495, or 989 

mg/kg/day for males and 0, 

50.2, 258, 512, or 1008 

mg/kg/day for females

NOAEL = 495/1008 mg/kg/day [M/F] 

LOAEL = 989 mg/kg/day [M], based on increased 

neutrophils and urinary bladder histopathology. A 

LOAEL in females was not established (>1008 

mg/kg/day).
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 55 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3100

90-Day oral toxicity 

(F344/DuCrl rats)

48557828 (2010)

Acceptable/guideline

Dietary concentrations 

equivalent to:

0, 10.5, 52.4, 262, or 

782 mg/kg/day for males and 

0, 10.1, 50.3, 252, or 

758 mg/kg/day

NOAEL = 262/252 mg/kg/day [M/F]

LOAEL = 782/758 mg/kg/day [M/F], based on 

decreased body weight and body weight gains (males 

only), increased urinary volume, decreased urine 

specific gravity and renal histopathology (necrosis, 

increased mitotic figures, hypertrophy of collecting duct 

epithelial cells, vacuolization of collecting duct 

epithelium in the papilla of the kidney, tubular 

dilatation and degeneration) and increased amorphous 

crystals in the urine sediment (females only).

870.3150

90-Day oral toxicity

(Beagle dog)

48557831 (2011)

Acceptable/guideline

Dietary concentrations of 0, 

500, 2500 or 12,500 ppm, 

equivalent to: 

0, 16.75, 80.64 or 

423.81mg/kg/day in males 

0, 18.71, 79.88 or 414.74 

mg/kg/day in females

NOAEL = 80.64/79.88 mg/kg/day [M/F] 

LOAEL = 423.81/414.74 mg/kg/day [M/F], based on 

decreased body weights, decreased body weight gains, 

reduced food consumption (females), reduced red cell 

parameters (red blood cells, hemoglobin, hematocrit 

levels) and additional hematological effects, decreased 

thymus weights, thymic atrophy, renal toxicity 

(including degeneration and regeneration of renal 

tubules) and extramedullary hematopoiesis of liver and 

spleen (females).

870.3200

28-Day dermal 

toxicity (F344/DuCrl 

rats)

48557834 (2010)

Acceptable/guideline

0, 100, 300, or 1000 

mg/kg/day for 6 hrs/day, 7 

days/week, 28 days

Systemic NOAEL = 1000 mg/kg/day

Systemic LOAEL = not observed

Dermal NOAEL = 1000 mg/kg/day

Dermal LOAEL = not observed

Developmental and Reproduction Studies

870.3700a

Prenatal 

developmental

[Crl:CD(SD) rats]

48557835 (2010)

Acceptable/guideline

Dietary concentrations of 0, 

500, 200 or 8000 ppm, 

equivalent to 0, 34.1, 140 or 

526 mg/kg/day

Administered GD 6 through 

21

Maternal

NOAEL = 140 mg/kg/day

LOAEL = 526 mg/kg/day based on increased mortality 

(moribund sacrifice), decreased fecal output and 

increased perinasal red soiling, decreased gravid uterine 

weight, and increased relative kidney weights.

Developmental

NOAEL = 140 mg/kg/day 

LOAEL = 526 mg/kg/day based on decreased fetal 

weights and increased incidence of delayed ossification 

of thoracic centra.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 56 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3700b

Prenatal 

developmental probe

(NZW rabbit)

48557838 (2011)

Acceptable/nonguideline

Dietary concentrations of 0, 

5000, 10,000, 15,000 or 

23,000 ppm, 

equivalent to 0, 196, 358, 

495 or 929 mg/kg/day 

Administered GD 7 through 

28

Maternal 

NOAEL = 929 mg/kg/day

LOAEL = No dose related effects were seen, thus the 

maternal LOAEL was not determined.

Developmental 

NOAEL = 929 mg/kg/day

LOAEL = No dose related effects were seen, thus the 

developmental LOAEL was not determined.

870.3700b

Prenatal 

developmental

(NZW rabbit)

48557837 (2011)

Acceptable/guideline

Dietary concentrations of 0, 

4000, 10,000 or 28292 ppm, 

Equivalent to 0, 170, 434 or 

1094 mg/kg/day

Administered GD 7 through 

28

Maternal 

NOAEL = 434 mg/kg/day

LOAEL = 1094 mg/kg/day based on marginally 

decreased body weight gains and food consumption 

during gestation.

Developmental 

NOAEL = 1094 mg/kg/day

LOAEL = No dose related effects were seen, thus the 

developmental LOAEL was not determined.

870.3800

Reproduction and 

fertility effects probe

[Crl:CD(SD) rats]

48557880 (2010)

Acceptable/nonguideline

Dietary concentrations 

formulated to provide doses 

equivalent to (premating 

values):

0, 53.4, 255 or 754 

mg/kg/day in males 

and

0, 53.9, 269 or 535 

mg/kg/day in females

A group administered a 

target dose of 1000 

mg/kg/day was terminated 

on Day 8 due to excessive 

weight loss in some animals. 

The targeted dose of 750 

mg/kg/day for both sexes 

was reduced to 500 

mg/kg/day for both sexes 

due to excessive weight loss.

Parental/Systemic 

NOAEL = 255/269 mg/kg/day [M/F]

LOAEL = 754/535 mg/kg/day based on decreased 

gestation body weight and weight gain, kidney 

histopathology (multifocal hypertrophy/hyperplasia of 

collecting duct epithelial cell lining, nuclear 

karyomegally, necrosis of individual collecting duct 

epithelial cells, increased number of mitotic figures 

within collecting duct cells and tubule dilatation with 

inflammation)

Reproductive

NOAEL = 754/536 mg/kg/day [M/F]

LOAEL = No dose related effects were seen, thus the 

reproductive LOAEL was not determined.

Offspring

NOAEL = 754/536 mg/kg/day

LOAEL = No dose related effects were seen, thus the 

offspring LOAEL was not determined.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 57 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.3800

Reproduction and 

fertility effects

[Crl:CD(SD) rats]

48557841 (2011)

Acceptable/guideline

Dietary concentrations 

formulated to provide:

P generation premating: 0, 

20.7, 104 or 465 mg/kg/day 

in males and 0, 20.5, 103 or 

465 mg/kg/day in females. 

F1 generation: 0, 20.3, 101 or 

459 mg/kg/day in males and 

0, 19.5, 97.7 or 443 

mg/kg/day in females.

Parental/Systemic 

NOAEL = 104/103 mg/kg/day [M/F]

LOAEL = 465/443 mg/kg/day [M/F] based on renal 

histopathological observations (collecting duct 

epithelial cell hypertrophy and hyperplasia, hyperplasia 

of pelvic epithelium, mitotic figures of the collecting 

duct epithelium) in both sexes; clinical signs, decreased 

body weight gains and food consumption during late 

gestation/early lactation, increased kidney weights, 

increased incidences of mineralization of the aorta, 

renal arteries, renal tubular basal lamina and mammary 

gland artery in females.

Reproductive

NOAEL = 465/443 mg/kg/day [M/F]

LOAEL = No dose related effects were seen, thus the 

reproductive LOAEL was not determined.

Offspring

NOAEL = 465/443 mg/kg/day [M/F]

LOAEL = No dose related effects were seen, thus the 

offspring LOAEL was not determined.

Chronic Toxicity Studies

870.4100b

Chronic toxicity 

(Beagle dog)

48557832 (2012)

Acceptable/guideline

Dietary concentrations of 0, 

500, 2500 or 10,000 ppm, 

equivalent to:

13.90, 82.42, and 354.75 

mg/kg/day in males and 

16.72, 89.85 and 219.80 

mg/kg/day in females

NOAEL = 82.42/16.72 mg/kg/day [M/F]

LOAEL = 354.75/89.85 mg/kg/day [M/F], based on 

degeneration with regeneration of the renal proximal 

and distal tubules, degeneration of collecting ducts, 

multifocal glomerulosclerosis and in males only, 

granulomas and fibrosis in the renal cortex.

At 219.80 in females, transient body weight decrease 

was also observed.

Toxicokinetic evaluation: XDE-729 showed 

supralinear kinetics in plasma of males at 10,000 ppm at 

13 and 26 weeks; at 52 weeks, there were insufficient 

quantifiable samples to assess linearity. Quantifiable 

levels were not available at terminal sacrifice (fasting), 

indicating a short half-life. Most of the dose was 

excreted intact in urine; levels were sublinear (not 

significant) in males at 10,000. 

Chronic/Carcinogenicity Studies
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 58 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.4200b

Carcinogenicity 

[Crl:CD-1(ICR) mice]

48557843 (2012)

Acceptable/guideline

Dietary concentrations of 0, 

50, 250 or 750 (males only) 

or 1000 (females only) ppm, 

equivalent to:

0, 50, 251, or 751 mg/kg/day 

for males, and 0, 50.1, 251, 

or 1004 mg/kg/day for 

females.

NOAEL = 50 mg/kg/day, both sexes

LOAEL = 251 mg/kg/day (both sexes), based 

treatment-related subacute to chronic inflammation and 

microscopic calculi in the lumen of the kidneys of 

males and decreased body weight gains and increased 

hypertrophy of the intercalated cells in kidneys of 

females.

At 751 mg/kg/day in males, treatment-related increases 

in mortality due to renal and urinary bladder lesions,

decreased kidney weight, urinary bladder toxicity 

(thickened bladder wall, hyperplasia of the transitional 

epithelium lining of the mucosa, increased numbers of 

mitotic figures within the epithelium, ulceration of the 

epithelium, necrosis of individual transitional epithelial 

cells; decreased aggregates of mononuclear cells 

secondary to inflammation) and renal toxicity 

(degeneration with regeneration of cortical tubular 

epithelium, tubular dilatation, hypertrophy of the 

intercalated cells of the collecting ducts) were also 

observed.

No evidence of carcinogenicity

870.4300

Chronic/

Carcinogenicity 

(F344/DuCrl rats)

48557842 (2012) 

Acceptable/guideline

Dietary concentrations 

formulated to provide dose 

equivalent to:

0, 20.2, 101, 404 and 633 

mg/kg/day, males and

0, 20.3, 102, 407 or 750 

mg/kg/day, females.

NOAEL = 101/20.3 mg/kg/day [M/F]

LOAEL = 404/101 mg/kg/day [M/F] based on effects 

in males of increased mortality, increased urinary 

volume, decreased urine specific gravity, decreased 

terminal body weights, increased kidney weights, renal 

histopathology, hypertrophy of the zona glomerulosa of 

the adrenal glands, presence of calculi in the renal 

pelvis of the kidney, degeneration and regeneration of 

renal tubules and bladder histopathology (including 

calculi); and in females, based on hyperplasia of pelvic 

epithelium of the kidney.

At 407 mg/kg/day in females, increases in chronic 

progressive glomerulonephropathy (12 months only) 

and chronic interstitial inflammation were noted in the 

kidneys. 

At 633 mg/kg/day, increased mortality was observed in 

males. Body weight/weight gain were decreased in 

both sexes. Females given 750 mg/kg/day were 

terminated on test day 518 due to excessive mortality 

(56%) and body weight, indicating exceeding the

maximum tolerated dose (MTD); these animals were 

subsequently removed from the study and no 

histopathological assessments were conducted. 

No evidence of carcinogenicity
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 59 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Mutagenicity Studies

Gene Mutation

870.5100

Bacterial Reverse 

Mutation Test (Preincubation assay)

48557844 (2010)

Acceptable/guideline

0, 50, 10, 500, 1500, or 5000 

μg/plate +/- S9

Negative in Salmonella typhimurium strains TA 1535, 

TA1537, TA98, and TA100 and Escherichia coli Wp2 

uvrA up to the limit concentration with no evidence of 

compound insolubility or cytotoxicity.

Gene Mutation

870.5300 

In vitro mammalian 

cell forward gene 

mutation, Chinese 

hamster ovary (CHO) 

cells, HGPRT locus 

48557848 (2010) 

Acceptable/guideline 

0, 187.5, 375, 750, 1500, 

2000, 2500, 3000 or 3400 

μg/plate +/- S9

Negative for induced mutant colonies at the HGPRT 

locus up to precipitating and cytotoxic concentrations.

Cytogenetics

870.5375

In vitro mammalian 

cell chromosomal 

aberration assay, 

primary lymphocytes 

(Sprague-Dawley rats)

48557852 (2010)

Acceptable/guideline

0, 800, 1500 or 2000 μg/mL 

in the absence of mammalian 

metabolic activation (S9) ; 

0, 400, 800 or 1500 μg/mL

in the absence of mammalian 

metabolic activation

Negative for induction of chromosomal aberrations up 

to cytotoxic concentrations.

870.5395 

Cytogenetics 

In vivo mammalian 

micronucleus assay 

[Crl:CD1(ICR) mice]

48557856 (2010) 

Acceptable/guideline

0, 500, 1000 or 2000 mg/kg

Negative for increased frequency of micronucleated 

reticulocytes in peripheral blood up to a dose causing 

systemic toxicity.

Neurotoxicity Studies

870.6200a Acute 

Neurotoxicity 

Screening Battery 

(F344/DuCrl rats)

48557857 (2010)

Acceptable/guideline

0, 250, 750, or 2000 mg/kg

(gavage)

Neurotoxicity NOAEL = 2000 mg/kg (HDT)

Neurotoxicity LOAEL not established (>2000 mg/kg)

Systemic NOAEL = 2000 mg/kg

Systemic LOAEL not established (>2000 mg/kg), 

870.6200b Subchronic 

Neurotoxicity Study 

(F344/DuCrl rats)

48557858 (2011)

Acceptable/guideline

Dietary concentrations 

equivalent to 0, 50, 250, or 

750 mg/kg/day

Neurotoxicity NOAEL = 750 mg/kg/day (HDT)

Neurotoxicity LOAEL not established (>750

mg/kg/day)

Systemic NOAEL = 750 mg/kg/day

Systemic LOAEL not established (>750 mg/kg/day), 

Other Studies
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 60 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.7485

Metabolism and 

pharmacokinetics

probe study

[F344/DuCrl rats) and 

Crl:CD1(ICR) mice]

48557859 (2009)

Acceptable/nonguideline

100 mg/kg single dose 

(gavage)

radiolabeled XDE-729 acid, 

[Phenyl-U14C] label 

XDE-729 acid was rapidly and well-absorbed from the 

GI tract at comparable rates for mice and rats. 

Elimination was primarily via the urine (65-75% AD in 

rats and 65-66% AD in mice). Fecal elimination 

accounted for 12-21% AD in rats and 19-20% AD in 

mice. Radioactivity was not found in expired air. 

Kinetics was comparable for male and females. Peak 

plasma concentrations (Cmax) was achieved at 0.5 hr in 

both species, plasma half-life (T1/2α) was 0.14 hr and 

terminal elimination half-life (T1/2ß) was 3.37 hr. AUC 

was 529 μg/h/g-1in males and 428 μg/h/g-1in females. 

In both species and in males and females, XDE-729 was 

mainly excreted as unchanged parent compound, with 

six minor peaks observed in urine. Of these, 

glucuronide and sulfate conjugates of O-demethyl 

XDE-729, O-demethyl XDE-729 and an acylglucuronide conjugate were identified. Parent XDE729 was also the major radiolabeled component in rat 

and mouse plasma at Cmax. Total recovery was 89-90% 

in rats and 92-93% in mice. 
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 61 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.7485

Metabolism and 

pharmacokinetics

[F344/DuCrl rats)

48557860 (2010)

Acceptable/guideline

radiolabeled XDE-729 acid, 

sodium salt [Phenyl14C]

label: Single oral dose, 10 

mg/kg; single oral dose, 750 

mg/kg; 14-day oral repeat 

dose, 10 mg/kg/day; IV dose, 

10 mg/kg; 

radiolabeled XDE-729 acid, 

sodium salt [Pyridine14C]

label: Single oral dose, 10 

mg/kg;

radiolabeled XDE-729 

methyl ester, [Phenyl-U14C]

label: 10 mg/kg single 

gavage dose, 10 mg/kg

XDE-729 sodium salt was rapidly and well-absorbed 

from the GI tract (at least 68-92% based on recovery in 

urine and non-GI tissues). Relative oral bioavailability 

was about 100% based on comparison of the areas 

under the curve of the oral and IV 10 mg/kg dose 

groups. Kinetics and metabolite profiles were similar

for males and females. Maximum plasma concentration 

was achieved in 0.2 hr. The initial elimination half-life 

at 10 mg/kg (T1/2α) was 0.5-0.9 hr, followed by a 

slower phase (T1/2ß) of 6-16 hr. The initial elimination 

phase at 750 mg/kg was slower (2.7-4.3 hr) but not the 

terminal phase (5.6-8.7 hr). The AUC for the high dose 

was 2.0-2.6 times higher than would be expected for 

dose proportionality. Kinetics of radioactivity in RBC 

were less than plasma. Elimination was primarily via 

the urine (68-92% AD), with some excretion in the 

feces (11-29% AD). Excretion was rapid, with most of 

the oral dose excreted within 24 hr. Biliary excretion 

was observed, based on fecal excretion of 11-15% AD 

after IV dosing. Radioactivity in tissues was very low, 

with only 0.3% and 0.03% AD remaining in tissues by 

168 hours for the oral and IV low dose, respectively. 

The highest levels were generally found in the skin or 

GI tract. XDE-729 sodium salt was poorly metabolized 

and primarily excreted as unchanged parent. Minor 

metabolites of consisted of the acyl-glucuronide 

conjugate of parent XDE-729 (present in selected 

samples at greater than 5% of the administered dose) 

and O-demethylated XDE-729 and the corresponding 

sulfate and glucuronide conjugates. Recovery was 

close to 100% for all groups.

XDE-729 methyl: The absorption, tissue distribution, 

elimination kinetics and metabolite profiles were 

similar to XDE-729 sodium salt, indicating rapid 

hydrolysis of the methyl ester in vivo, resulting in 

bioequivalence. Peak plasma concentration was 

achieved by 0.4-0.5 hr. Blood samples collected at or 

near peak showed that the major component was XDE729 acid. XDE-methyl showed an increased compared 

to XDE-729 sodium salt, but AUCs were the same.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 62 of 126

Subchronic, Chronic, and Other Toxicity Studies for XDE-720 Acid Form

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.7485

Metabolism and 

pharmacokinetics

probe study

(Beagle dog)

48557870 (2012)

Acceptable/nonguideline

50 mg/kg single dose 

(gavage)

radiolabeled XDE-729 acid, 

sodium salt [Phenyl-U14C]

label 

XDE-729 acid was rapidly and well-absorbed from the 

GI tract in male and female beagle dogs. Plasma Cmax

was achieved at 0.7 hr in females (143 μg-eq./g) and 1.3 

hr in males (118 μg-eq./g). Elimination from the blood 

was biphasic, with an initial half-life (T1/2α) of 1 hr and 

terminal half-life (T1/2ß) of 9-12 hr. Excretion was 

primarily via the urine (about 80% AD), mostly within 

12 hr, with about 10-14% excreted in feces, mostly by 

48 hr. XDE-729 was excreted primarily as unchanged 

parent compound. Minor metabolites in urine, feces or 

plasma included O-demethyl XDE-729 and the sulfate 

and glucose conjugates of O-demethyl XDE-729, 

hydroxy XDE-729, and an acyl glucuronide conjugate 

of XDE-729.Total recovery by 168 hr was 95-98% AD.

Non-Guideline Studies

See XDE-729 methyl 

for Mechanism of 

Action (MOA) studies 

for liver effects

Subchronic, Chronic, and Other Toxicity Studies for Metabolite X11449757

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

Mutagenicity Studies

Gene Mutation

870.5100

Bacterial Reverse 

Mutation Test (Preincubation assay)

48557847 (2012)

Acceptable/guideline 

0, 156.25, 312.5, 625, 1250, 

2500 or 5000 μg/plate - S9

activation 

and

0, 51.2, 128, 320, 800, 2000 

or 5000 μg/plate + S9

activation

Negative in Salmonella typhimurium strains TA 1535, 

TA1537, TA98, TA100 and TA102 and Escherichia 

coli Wp2 uvrA up to the limit concentration with no 

evidence of compound insolubility or cytotoxicity.

Gene Mutation

870.5300 

In vitro mammalian 

cell forward gene 

mutation, Chinese 

hamster ovary (CHO) 

cells, HGPRT locus 

48557855 (2012) 

Acceptable/guideline 

0, 100, 200, 400, 800, 1600 

or 3210 μg/mL +/- S9

activation

Negative for induced mutant colonies over background 

at the HGPRT locus up to precipitating and cytotoxic 

concentrations.
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

Page 63 of 126

Subchronic, Chronic, and Other Toxicity Studies for Metabolite X11449757

Guideline No./ Study 

Type

MRID No. (year)/ 

Classification /Doses

Results

870.5375 

Cytogenetics 

In vitro mammalian 

cell chromosomal 

aberration assay, 

human peripheral 

lymphocytes

48557850 (2012) 

Acceptable/guideline 

0, 25.3, 50.6, 101.3, 202.5, 

405 or 810 μg/mL +/- S9

activation

Negative for increased chromosomal aberrations over 

background up to cytotoxic and precipitating 

concentrations.

A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Reference Dose (aRfD) - Females age 13-49

An appropriate endpoint was not identified for a single exposure to females age 13-49. No effects 

from a single-dose exposure were identified for either halauxifen-methyl or XDE-729 acid.

A.3.2 Acute Reference Dose (aRfD) - General Population

An appropriate endpoint was not identified for a single exposure to the general population. No 

effects from a single-dose exposure were identified for either halauxifen-methyl or XDE-729 

acid.

A.3.3 Chronic Reference Dose (cRfD)

Halauxifen-Methyl:

Study Selected: 90-day oral toxicity in the rat

MRID No.: 48557830

Executive Summary: See Appendix A, Guideline §870.3100a

Dose and Endpoint for Risk Assessment: POD = 3.0 mg/kg/day based on increased expression 

of Cyp1a1 in the liver at 10.3 mg/kg/day. 

Comments about Study/Endpoint/Uncertainty Factors: The rat 90-day oral study was selected 

because long-term oral toxicity studies are not available for halauxifen-methyl. The study NOAEL 

is 10.3 mg/kg/day, based on increased liver weight, hypertrophy and vacuolization consistent with 

fatty change at the LOAEL of 53.4 mg/kg/day. Liver effects at the LOAEL were of low severity. 

A marked increase (1500-fold above controls) in Cyp1a1 expression was also observed at the 

LOAEL. Mechanistic studies on halauxifen-methyl identified activation of liver AhR as the MOA 

for liver toxicity, for which increased expression of Cyp1a1 in the liver is a biomarker for AhR 

activation, the MIE. Although there were no liver effects at the study NOAEL, a 52-fold increase 

in Cyp1a1 expression was observed. Long-term effects on the liver from this lower level increase 

are not known in the absence of chronic data. Therefore, in order to be protective of potential 

adverse effects on the liver from lower-level increases in Cyp1a1 expression at longer exposure 
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durations, the POD of 3 mg/kg/day was selected, based on increased expression of liver Cyp1a1 

(52-fold) at 10 mg/kg/day. Cyp1a1 expression at 3 mg/kg/day was comparable to controls in both 

the 28- and 90-day studies (1.2- and 3.6-fold higher than controls, respectively), indicating that 

there is not expected to be a biologically significant activation of the AhR receptor at this dose 

level over time. The dose and endpoint are considered conservative, but protective of residual 

uncertainty due to the lack of chronic data, and are also protective of chronic effects from XDE729 acid, which are observed at higher doses. A UF of 100 is based on the combined interspecies 

(10x) and intraspecies (10x) UFs. An additional 10x UFs for lack of chronic data was not applied 

for the following reasons: (1) progression of toxicity was not observed in the 28- and 90-day 

dietary studies in the rat: the NOAELs and LOAELs for both studies were the same, and the 

severity of the findings was minimal at both exposure durations; (2) evaluation of Cyp1a1 

expression in the rat 7, 28 and 90-day studies indicated that at the selected POD, which is below 

the NOAELs for these studies, there is no expectation of significant AhR activation that could lead 

to liver toxicity; (3) halauxifen-methyl is rapidly metabolized to the acid, and neither 

bioaccumulate; (4) based on in vitro studies, humans are not anticipated to be more sensitive to 

liver effects of halauxifen-methyl, and (5) dietary exposure to humans is expected to be very low 

(<1% of the cPAD) due to the low application rates and environmental degradation. 

XDE-729 acid:

Study Selected: Chronic toxicity/carcinogenicity in the rat

MRID Nos.: 48557842 (rat)

Executive Summary: See Appendix A, Guidelines §870.4300 (rat)

Dose and Endpoint for Risk Assessment: NOAEL = 20.3 mg/kg/day based on hyperplasia of 

the renal pelvic epithelium in females at 101 mg/kg/day. 

Comments about Study/Endpoint/Uncertainty Factors: The selected study is of the 

appropriate route and duration for assessment of chronic dietary exposure. A similar NOAEL 

and LOAEL of 16.7 and 89.9 mg/kg/day, respectively, based on renal tubule toxicity in females, 

were identified in the one year oral dog study: the rat study is considered protective based on 

comparison of dose-spacing in the two studies. The NOAEL of 20.3 is protective of 

developmental effects, observed in the rat at 526 mg/kg/day (NOAEL = 140 mg/kg/day), and of 

maternal toxicity in both the rat (LOAEL = 526 mg/kg/day) and rabbit (LOAEL 1094 

mg/kg/day). Parental toxicity in the rat two-generation reproductive toxicity study was observed 

at 443 mg/kg/day (NOAEL 103 mg/kg/day), but no offspring or reproductive toxicity was 

reported. A UF of 100 is based on the combined interspecies (10x) and intraspecies (10x) UFs. 

 

A.3.4 Incidental Oral Exposure (Short- and Intermediate-Term)

Residential post-application exposure is not anticipated because there are no proposed residential 

uses. An endpoint was therefore not selected at this time.

A.3.5 Dermal Exposure (Short-, Intermediate- and Long-Term) – Halauxifen-methyl

An appropriate dose and endpoint for dermal exposure were not identified. In a rat 28-day 

dermal toxicity on XDE-729 acid, no toxicity was observed. However, occupational exposure 

will be to halauxifen-methyl in end-use products. Although a dermal toxicity study on 
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halauxifen-methyl was not conducted, dermal absorption data on halauxifen-methyl are 

available. In vivo absorption of halauxifen-methyl in the rat was 24% of the applied dose, with 

toxicokinetic data showing that XDE-729 acid, but not halauxifen-methyl, was present in the 

blood, indicating rapid hydrolysis following absorption. Application of the DAF of 24% to the 

LOAELs from rat studies on XDE-729 acid of the most sensitive rat studies of the appropriate 

duration (rat developmental, rat two-generation reproductive toxicity) indicated that toxicity 

would be expected at doses approximately twice the dermal limit dose of 1000 mg/kg/day, and 

adjusted NOAELs were 410-640 mg/kg/day. Therefore, since toxicity from dermal exposure 

would only be expected at exposures far exceeding potential human dermal exposure, an 

endpoint was not selected. A 28- or 90-day dermal toxicity study on halauxifen-methyl was 

therefore not needed (HASPOC memorandum, TXR No. 0056954). 

A.3.6 Inhalation Exposure (Short- and Intermediate-Term) – Halauxifen-methyl

Study Selected: 90-day oral toxicity in the rat, halauxifen-methyl

MRID No.: 48557830

Executive Summary: See Appendix A, Guideline §870.3100a

Dose and Endpoint for Risk Assessment: POD = 3.0 mg/kg/day based on increased expression 

of liver Cyp1a1 at 10 mg/kg/day.

Comments about Study/Endpoint/Uncertainty Factors: Inhalation exposure will be to 

halauxifen-methyl in end-use products. No inhalation studies are available. Therefore an oral 

study, the rat 90-day toxicity study on halauxifen-methyl, was selected for assessment of 

inhalation exposure. The selected oral study is of an appropriate exposure duration for 

assessment of short- and intermediate-term exposure risk. The study NOAEL was 10.3 

mg/kg/day, based on slightly increased liver weight, hepatocellular vacuolization and cholesterol 

at the LOAEL of 53 mg/kg/day. At the LOAEL, a marked increase in Cyp1a1 expression was 

observed (1500-fold) but a much lower increase (52-fold) was also seen at the NOAEL of 10.3 

mg/kg/day. Therefore, in the absence of information on the toxicity of halauxifen-methyl to the 

respiratory tract, a POD of 3 mg/kg/day, based on the endpoint of increased Cyp1a1 expression 

in the liver at 10 mg/kg/day, was selected. The selected POD is the most sensitive dose available 

and is protective of AhR activation, an adverse outcome pathway that may occur in the lung as 

well as the liver. The dose is also protective of maternal toxicity in the rabbit, the more sensitive 

species of the developmental studies (NOAEL of 5.8 mg/kg/day, based on liver enlargement and 

hepatocellular histopathology at the maternal LOAEL of 18.5 mg/kg/day) as well as 

developmental toxicity (decreased fetal body weights and delayed ossification, observed at 72 

mg/kg/day). 

Toxicity via the inhalation route is assumed to equivalent to toxicity via the oral route. The UF 

of 100x is based on the combined interspecies (10x) and intraspecies (10x) UFs. An additional 

UFDB was not included for lack of inhalation exposure data because the selected dose is 

conservative, is below the NOAEL for adverse effects in the rat oral 90-day study and is 

protective of AhR activation/Cyp1a1 induction. It is also noted that halauxifen-methyl has a low 

vapor pressure (4.4 x 10-11 mmHg at 25°C), reducing the likelihood of significant inhalation 

exposure. MOEs for inhalation exposure calculated using the oral POD were adequately 

protective (>1000). In addition, HED has determined that a route-specific inhalation study is not 

needed for halauxifen-methyl.
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A.4 Executive Summaries

Halauxifen-Methyl (XDE-729 Methyl)

A.4.1 Subchronic Toxicity

870.3050 28-Day Oral Toxicity – Rat

In a subchronic oral toxicity study (MRID 48557826, PMRA 2226461), five F344/DuCrl 

rats/sex/group were at test diets formulated to supply 0, 10, 52, 261, or 782 mg XDE-729 methyl

(97.2% a.i., lot no. E2837-51, TSN031117-0004)/kg body weight/day for 30 days. Average 

intake of XDE-729 methyl corresponded to time-weighted average doses of 0, 10.8, 55.6, 296, or 

822 mg/kg bw/day for males and 0, 10.7, 55.5, 277, or 825 mg/kg bw/day for females, 

respectively. In addition to standard toxicity parameters, blood (three time points) and urine 

samples (overnight collection) were collected during the fourth week of treatment and 

toxicokinetic analysis was performed.

There were no treatment-related effects on survival, clinical signs, urinalysis parameters or gross 

pathology. Treatment-related decreases in body weight gains (11-17%) and food consumption 

(7-20%) were observed at ≥277 mg/kg bw/day in females. Hematological effects consisted of 

decreases in red blood cell count, hematocrit, and/or hemoglobin concentration (2-6% each) in 

males and females at 822/825 mg/kg bw/day; these changes were considered to be treatmentrelated but non-adverse by Australia OCS. These alterations corresponded with slight erythroid 

cell hyperplasia of the bone marrow (incidences: ♂ 1, 1, 0, 0, 4; ♀ 0, 1, 1, 0, 2) and increased 

erythrocytic extramedullary hematopoiesis of the spleen (incidences: ♂ 1, 1, 0, 0, 4; ♀ 1, 1, 3, 1, 

4). Clinical chemistry effects consisted of higher cholesterol concentrations in males and 

females at ≥277 mg/kg bw/day (♂ 50-115%, ♀ 83-95%) and higher aspartate aminotransferase 

(AST) activities (♂ 35%, ♀ 24%) in high-dose males and females. 

Treatment-related statistically identified increases in the mean absolute and/or relative liver 

weights (absolute/relative ♂ 7-41/8-46%, ♀ 13-18/8-29%) were noted in males and females at 

55.6/55.5 mg/kg bw/day M/F relative to controls. In males at ≥55.6 mg/kg bw/day, there was 

associated hepatocellular hypertrophy (incidences: 0, 0, 3, 5, 5), increased mitotic figures in 

hepatocytes (incidences: 0, 0, 1, 3, 4) and hepatocellular vacuolization (consistent with fatty 

change) (incidences: ♂ 0, 0, 3, 4, 5). In females, the first two liver histopathological changes 

were observed at 277 and 825 mg/kg bw/day (incidences: ♀ 0, 0, 0, 5, 5 and 0, 0, 0, 0, 1 

respectively). The other primary histopathological effect was hypertrophy of collecting duct 

epithelial cells in the kidneys of two high-dose males. 

Males at 296 and 822 mg/kg bw/day had treatment-related higher absolute (16-29%) and relative 

(15-33%) thyroid weights, which were considered to be toxicologically relevant as this change 

was associated with treatment-related hypertrophy of thyroid gland follicular cells at ≥55.6 

mg/kg bw/day (incidences: 0, 0, 2, 5, 5). Males and females at 822/825 mg/kg bw/day,

respectively, had treatment-related lower absolute (♂ 17%, ♀ 14-21%) and relative (♂ 15%, ♀ 

10-14%) thymus weights. Males and females at 825 mg/kg bw/day had treatment-related acinar 
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cell hypertrophy of the submandibular salivary gland (incidences: ♂ 0, 0, 0, 0, 1; ♀ 0, 0, 0, 0, 4) 

and lymphoid atrophy of the thymus. The thymic lymphoid atrophy was a minimal effect that 

was only present in 2 males and 2 females from the high-dose group. Treatment-related 

decreases in the size of the cervix, uterus and vagina were observed in high-dose females

(incidences: 2, 1, 2 vs. 0 in controls, respectively). 

The male LOAEL is 55.6 mg/kg bw/day, based on increased liver weight, hepatocellular 

hypertrophy, increased mitotic figures in hepatocytes, hepatocellular vacuolization 

(consistent with fatty change) and hypertrophy of thyroid gland follicular cells. The female 

LOAEL is 277 mg/kg bw/day, based on decreased body weight gains and food 

consumption, increased liver weight, increased cholesterol levels and hepatocellular 

hypertrophy. The NOAEL in males is 10.8 mg/kg bw/day and the NOAEL in females is 

55.5 mg/kg bw/day.

After 4 weeks of exposure to XDE-729-methyl in the diet, XDE-729-methyl was not present at 

detectable levels in the whole blood of most male and female rats, except at the highest dose in 

males at low levels. However, XDE-729 acid was present in blood samples from both sexes at 

all four doses and almost all time points. Both XDE-729 acid and XDE-729-methyl were present 

in the urine of male and female rats collected over 24 hours on the same day as the blood sample 

collection. Less than 0.066% of the administered dose was excreted as XDE-729-methyl in the 

24 hour urine, and 23-43% of the administered dose was excreted as the metabolite XDE-729

acid. 

Daily systemic exposures (AUC24h) for XDE-729 acid and the total amounts excreted in the 24 

hour urine were dose-proportional (linear) at all doses in males, but only up to the mid-high dose 

in females. The daily systemic exposures for XDE-729 acid and the total amount of XDE-729

acid and XDE-729-methyl excreted over 24 hours in urine in females of the high-dose group 

were both less than dose-proportional (sublinear), suggesting possible saturation of absorption 

and/or metabolism at the high dose in female rats.

This subchronic toxicity study in the rat is classified as acceptable/guideline and satisfies the 

guideline requirement (OECD 407, OPPTS 870.3050) for a 28-day repeat-dose oral study in rats.

870.3100 90-Day Oral Toxicity – Rat

In a subchronic oral toxicity study (MRIDs 48557830, amended report 49124405; PMRA 

2315015), XDE-729 Methyl (97.2% a.i., lot no. E2837-51; TSN031117-0004) was administered 

to 10 DuCrl/F344 rats/sex/group in test diets formulated to supply 0, 3, 10, 52, 261, or 500 

mg/kg bw/day for at least 90 days. The actual doses to the animals equaled 0, 3.1, 10.3, 53.4, 270 

or 513 mg/kg bw/day in males and 0, 3.0, 10.1, 52.3, 263 or 504 mg/kg bw/day in females. In 

addition to standard guideline parameters, males were evaluated for liver gene expression and 

toxicokinetic analysis of the test material in liver, blood and urine.

There were no treatment-related effects on survival, clinical signs, functional tests, ophthalmic

parameters, food consumption, thyroid hormones, urinalysis parameters or gross pathologic 

observations.
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Treatment-related decreases in body weights (♂ 5%, ♀ 7%) and body weight gains (♂ 8%, ♀ 

14%) were observed in high-dose males and females throughout the study but were not 

considered of sufficient magnitude to be of toxicological significance.

Females at 263 and 504 mg/kg bw/day had lower percent neutrophils (27-31%) and higher 

percent lymphocytes (7-8%), relative to controls that were interpreted to be treatment-related. 

Males at ≥ 270 mg/kg bw/day and females at ≥ 52.3 mg/kg/day had treatment-related higher 

cholesterol concentrations (♂ 41-73%, ♀ 24-59%). Males and females at 270/263 or 513/504 

mg/kg bw/day M/F had treatment-related lower alkaline phosphatase activities (♂ 21-25%, ♀ 

14-20%) and high-dose males had lower aspartate aminotransferase activities (18%). 

Males at 270 or 513 mg/kg bw/day and females at 52.3, 263, or 504 mg/kg bw/day had 

treatment-related increases in absolute and relative liver weights (absolute/relative ♂ 22-34%/22-

40%, ♀ 12-26%/12-36%), which were associated with panlobular hepatocellular hypertrophy 

with altered tinctorial properties (increased cytoplastic eosinophilia) in both sexes at the two 

highest dose levels in all animals. Other treatment-related liver effects consisted of increased 

numbers of mitotic figures (hepatocytes in mitosis) in females at 263 or 504 mg/kg bw/day 

(incidences: 0, 0, 0, 0, 2, 5), and hepatocellular vacuolization (consistent with fatty change) in 

males at ≥ 52.3 mg/kg bw/day and females at ≥ 263 mg/kg bw/day (incidences: ♂ 0, 0, 0, 5, 10, 

10; ♀ 0, 0, 0, 0, 8, 5). A subsequent 7-day mode-of-action (MOA) study was performed in male 

F344 rats given a similar high dose of XDE-729 methyl or XDE-729 (acid) to compare liver 

gene expression responses for Cyp1a1, Cyp2b1, Cyp3a23, and Cyp4a22 as biomarkers for 

activation of the AhR, CAR, PXR, and PPAR- signaling pathways, respectively. Results from 

this study indicated that XDE-729 methyl, but not XDE-729 (acid), was activating the AhR 

signaling pathway as indicated by a robust induction in Cyp1a1 transcript levels. The appearance 

of histopathological apical effects in the liver at ≥ 53.4/52.3 mg/kg bw/day M/F correlated with a 

dose-responsive induction in Cyp1a1 and Ugt1a6 gene expression, which is consistent with 

activation of the AhR signaling pathway. Transcript levels for Cyp1a1 demonstrated the highest 

induction of 1495 and 2268-fold at 53.4/52.3 mg/kg bw/day in males and females, respectively. 

In addition, levels of XDE-729 methyl in the liver were increasing in a dose responsive manner 

across these treatment groups. Induction of Cyp1a1 transcript levels occurred in male and 

female livers in a dose-responsive manner at 53.4/52.3 mg/kg/day and above M/F. Therefore, 

increases in Cyp1a1 transcript levels of greater than 1000-fold were associated with the 

appearance of histopathological and apical effects in the liver at 53.4/52.3 mg/kg bw/day M/F. 

At the two highest doses, further increases in Cyp1a1 transcript levels were observed which were 

associated with increasing liver weights and a greater severity in the histopathological responses. 

In addition to liver effects, the thyroid hypertrophy observed in males and females at ≥ 270/263

mg/kg bw/day M/F was associated with a concomitant dose responsive induction of liver Ugt1a6

transcript levels. 

Males and females at 270/263 or 513/504 mg/kg bw/day M/F had treatment-related increased 

absolute and relative thyroid weights (absolute/relative ♂ 14-23%/13-28%; ♀19-25%/26-28%), 

which corresponded to microscopic thyroid follicular cell hypertrophy (incidences: ♂ 1, 0, 0, 1, 

9, 9; ♀ 0, 0, 1, 0, 8, 10). The thyroid hypertrophy observed in males and females at the two 

highest dose levels was associated with a dose responsive induction of liver Ugt1a6 transcript 

levels. There were no treatment-related effects on thyroid hormone levels at necropsy; however, 
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the sampling time likely occurred after a compensatory mechanism by the hypothalamicpituitary-thyroid (HPT) axis.

The high-dose females had lower absolute and relative thymus weights (absolute/relative 

30%/24%), with corresponding atrophy of thymic lymphoid tissue (incidences: 0, 0, 0, 1, 0, 6). 

These changes were considered to be treatment-related.

The LOAEL in males is 53.4 mg/kg bw/day, based on increased hepatocellular vacuolation 

(consistent with fatty change) and increased Cyp1a1 gene expression. The LOAEL in 

females is 52.3 mg/kg bw/day, based on increased liver weights and increased cholesterol 

levels and increased Cyp1a1 gene expression. The NOAEL in males and females is 10.3 and 

10.1 mg/kg bw/day, respectively. However, it is noted that a mild increase in the induction 

of Cyp1a1 gene expression (52- and 149-fold above controls, males and females) was 

observed at the study NOAEL of 10.3 mg/kg bw/day. At 3.1 mg/kg bw/day, there was only 

a slight increase (3.6- and 5.7-fold, males and females).

Toxicokinetic analysis results indicated that the parent, XDE-729 methyl, was present at 

quantifiable levels in the liver (all doses) and the blood (≥10 mg/kg bw/day males; 52 and 500 

mg/kg/day females). Consistent with hepatic induction of the AhR signaling pathway by XDE729 methyl, levels of XDE-729 methyl in the liver were increasing in a dose-responsive manner 

across these treatment groups.

The primary metabolite, XDE-729 acid, was present in blood, liver, and urine samples from both 

male and female rats at all doses and almost all time-points. Five additional metabolites were 

also detected in the blood, liver, and urine of most animals. The sulfate conjugate of Odemethylated XDE-729 acid appeared to be the most prevalent of these in males, while the 

glucuronide conjugates of O-demethyl XDE-729 methyl and XDE-729 acid were the most 

prevalent in females. 

In the liver, XDE-729 methyl was quantifiable at low levels from all treated animals and 

exhibited kinetic linearity. However, the primary metabolite, XDE-729 acid, was the most 

abundant and exhibited sublinearity in males at ≥270 mg/kg bw/day. Similarly, the O-demethyl 

XDE-729 metabolite was sublinear (both sexes) at ≥270/263 mg/kg bw/day M/F, while the 

glucuronide conjugate of this O-demethyl XDE-729 metabolite exhibited supralinearity in males 

at these dose levels. 

In whole blood, XDE-729 methyl was not present at quantifiable levels in a majority of males 

and females at all doses, except at the highest dose level. Similar to the liver samples, the 

primary metabolite XDE-729 acid was sublinear in male blood at ≥270 mg/kg bw/day. 

Analysis of the urine samples revealed that most of the administered dose of XDE-729 methyl 

was rapidly metabolized and excreted in the urine. XDE-729 methyl was not present above LLQ 

in urine, except in one high-dose urine sample. All of the metabolites exhibited supralinear 

kinetics in urine at ≥53.4/52.3 mg/kg/day M/F. Conversely, XDE-729 acid levels decreased with 

increasing doses in both sexes. These data suggested kinetic nonlinearity in the metabolism and 

elimination of administered XDE-729 methyl, with an increase in metabolic conversion to 

conjugated metabolites with increasing doses, which were in turn more rapidly eliminated in the 
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urine. These data are consistent with an increase hepatic induction of metabolism enzymes at 

≥53.4/52.3 M/F mg/kg bw/day.

This subchronic oral toxicity study in the rats is classified as acceptable/guideline and satisfies 

the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats. 

870.3050 28-Day Oral Toxicity - Mouse

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

870.3100 90-Day Oral Toxicity - Mouse

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

870.3150 90-Day Oral Toxicity - Dog

Study not conducted for halauxifen-methyl: see Executive Summaries for XDE-729 acid

870.3200 21/28-Day Dermal Toxicity – Rat

Study not conducted for halauxifen-methyl: see Executive Summaries for XDE-729 acid

870.3465 90-Day Inhalation – Rat

Study not conducted due to inability to generate acceptable test atmosphere

A.4.2 Prenatal Developmental Toxicity

Nonguideline Prenatal Developmental Rangefinding Study – Rat

In an oral range-finding developmental toxicity study (MRID 48557881; PMRA 2226497), 

XDE-729 methyl (97.2% a.i., lot no. E2837-51, TSN031117-0004) was administered to 5 timemated female Crl:CD(SD rats/dose in diet at dose levels of 0, 521, 2083, 4167, or 8333 ppm on 

gestation day (GD) 6 through 21, (equal to 0, 40.4, 169, 303, or 490 mg/kg bw/day, 

respectively). Limited parameters were examined; detailed fetal observations were not 

performed. Blood concentrations of XDE-729 methyl and the metabolite XDE-729 acid were 

determined in samples from all rats on GD 20 and 21 and their respective litters at necropsy.

The animals in the highest dietary concentration, 8333 ppm (490 mg/kg bw/day), were sacrificed 

prematurely based on excessive reductions in body weight gains (90% decrease from controls) 

from GD 6-9; this dose was considered to have exceeded the maximum tolerated dose. In the 

remaining animals at the lower dose levels, there were no treatment-related clinical signs or 

effects on body weights, gross pathology, organ weights or Caesarian section parameters. 

In the 303 mg/kg bw/day maternal animals, treatment-related decreases in body weight gains 

(89%) and food consumption (33%) were observed throughout the study. A detailed 
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developmental assessment was not conducted in this range-finding study. Blood concentrations 

showed linear kinetics across all three doses for both XDE-729 methyl, when present, and the 

metabolite XDE-729 acid. XDE-729 methyl was present above the lower limit of quantification 

(LLQ) during early sampling times on GD 20 and was absent in GD 21 dams. In contrast, the 

main metabolite, XDE-729 was present in all dams and fetal blood samples. When present, 

XDE-729 methyl concentrations were ~0.9% that of the metabolite XDE-729 acid. Fetal blood 

concentrations of XDE-729 acid were ~69% of the dam blood concentrations.

Canada PMRA, Australia OCS: No LOAELs or NOAELs were established in this 

supplemental range-finding study.

US EPA: Maternal NOAEL and LOAEL were selected for comparison with the definitive 

study. The maternal LOAEL is 303 mg/kg bw/day, based on decreased food consumption 

and body weight gains. The maternal NOAEL is 169 mg/kg bw/day. A developmental 

NOAEL/LOAEL was not determined since a fetal examination was not performed. 

The developmental toxicity study in the rat is classified as acceptable/non-guideline for a 

supplemental dose range-finding study that was not conducted to satisfy a guideline requirement 

for a rat developmental toxicity study. 

870.3700a Prenatal Developmental Toxicity Study – Rat

In an oral developmental toxicity study (PMRA 2226499, MRID 48557836), groups of 24 timemated female Crl:CD(SD) rats were administered diets containing 0, 521, 2083, or 4167 ppm 

XDE-729 methyl (97.2% a.i., lot no. E2837, TSN031117-0004) on gestation day (GD) 6 through 

21 which corresponded to time-weighted average doses of 0, 41.1, 159, or 323 mg/kg bw/day, 

respectively. In-life maternal study parameters included clinical observations, body weight, 

body weight gain and food consumption. On GD 21, all rats were euthanized and examined for 

gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along 

with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All 

fetuses were weighed, sexed and examined for external alterations. Approximately one half of 

the fetuses were examined for visceral and craniofacial alterations, while skeletal examinations 

were conducted on the remaining fetuses. Blood concentrations of XDE-729 methyl and the 

metabolite XDE-729 acid were analyzed in samples collected from four dams/group and their 

respective litters at necropsy.

Maternal toxicity: There were no treatment-related effects on survival, clinical signs, gross 

pathology or Caesarian section parameters in maternal animals. At 323 mg/kg bw/day, decreased 

body weight gains (23%) and food consumption (8%) were observed from GD 6-9 and were 

considered to be adverse. Treatment-related increases in liver weight (absolute/relative 

22%/21%), altered cytoplasmic homogeneity of centrilobular and midzonal hepatocytes

(incidences: 2, 3, 14, 21), and hepatocellular vacuolization consistent with fatty change 

(incidences: 3, 4, 3, 7) were also observed at this dose level. The increased liver weights and 

liver histopathology at 159 mg/kg bw/day were considered to be adaptive and non-adverse. 
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Canada PMRA/US EPA: The maternal LOAEL is 323 mg/kg bw/day based on decreased 

body weight gains and food consumption (GD 6-9), increased liver weight, altered 

cytoplasmic homogeneity of centrilobular and midzonal hepatocytes, and hepatocellular 

vacuolization consistent with fatty change. The maternal NOAEL is 159 mg/kg bw/day. 

Australia OCS: The maternal LOAEL is 159 mg/kg bw/day based on decreased body 

weight gains and food consumption (GD 6-9), increased liver weight and altered 

cytoplasmic homogeneity of centrilobular and midzonal hepatocytes. The maternal 

NOAEL is 41 mg/kg bw/day. 

Developmental toxicity: No adverse external, visceral or skeletal malformations or variations 

were noted after treatment with XDE-279 methyl. 

The developmental LOAEL was not established. The developmental NOAEL is 323 mg/kg 

bw/day. 

XDE-729 methyl was not present in any GD 21 dam and fetal blood samples. In contrast, the 

metabolite, XDE-729 acid was present at quantifiable levels (≥LLQ) in all of the blood samples 

from exposed animals. XDE-729 acid was present in fetal blood samples at concentrations ~ 

65% of the dam blood levels. XDE-729 acid blood concentrations were linear (doseproportional) at all three doses in dams and fetuses.

The developmental toxicity study in the rat is classified as acceptable/guideline and satisfies the 

guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.

Nonguideline Prenatal Developmental Rangefinding Study – Rabbit

In a range-finding developmental toxicity study (PMRA 2226502, MRID 48557839), groups of 

five time-mated female New Zealand White rabbits were administered XDE-729 methyl (97.2% 

a.i., lot no. E2837-51, TSN031117-0004) in the diet at concentrations of 0, 521, 2083, 4167, 

8333, 10417, 15625, or 24500 ppm on gestation days (GD) 7 through 28, or their date of early 

termination (≤GD 15), which corresponded to time weighted averages of 0, 21.5, 83.3, 157, 244, 

371, 426, or 727 mg/kg bw/day. In-life parameters evaluated for all groups included clinical 

observations, body weight, body weight gain, and feed consumption. On GD 28 all surviving 

rabbits were euthanized and examined for gross pathological alterations. Liver and kidney 

weights were recorded, along with the number of corpora lutea, implantations, resorptions, fetal 

body weights, and live/dead fetuses. Blood concentrations of XDE-729 methyl and the 

metabolite XDE-729 acid were determined in samples at two time points from all does on GD 27 

and at necropsy on GD 28.

Based on excessive body weight loss in does treated with dietary concentrations at ≥8333 ppm 

(244 mg/kg bw/day), these groups were prematurely sacrificed on GD 15 since the MTD was 

exceeded. In the remaining groups, there were no treatment-related effects on survival, clinical 

signs, kidney weights or gross pathology. Treatment-related decreases in body weight gains (23-

26%) and food consumption (≥12%) were observed in does at 83.3 and 157 mg/kg bw/day, but 

were not considered to be adverse due to lack of effects on mean body weight. Increased liver 
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weights (absolute 13-49%, relative 15-52%) were observed in females at 21.5, 83.3 and 157 

mg/kg bw/day. A histological assessment of these livers was not conducted in this range-finding 

study. Apart from decreased fetal weights (7-10%) at 83.3 and 157 mg/kg bw/day, there were no 

treatment-related effects on Caesarean section parameters. 

Blood toxicokinetic analysis demonstrated the presence of only the metabolite, XDE-729 acid, 

and not the parent molecule, XDE-729 methyl, in treated animals at concentrations above the 

analytical LLQ. Diurnal systemic exposure values for the metabolite XDE-729 acid exhibited 

linear kinetics through the highest analyzed dose group (4167 ppm).

Canada PMRA, Australia OCS: No LOAELs or NOAELs were established in this 

supplemental range-finding study.

US EPA: A maternal NOAEL and LOAEL was identified for comparison to the definitive 

study. The maternal LOAEL is 244 mg/kg bw/day, based on decreased body weight, 

weight gain and food consumption. The maternal NOAEL is 157 mg/kg bw/day. The 

developmental LOAEL/NOAEL was not determined as fetal examination was not 

performed. 

This study is classified as acceptable/non-guideline as a range-finding developmental toxicity 

study in rabbits. It was conducted to assist in dose selection for the definitive developmental 

study and was not conducted to support a guideline requirement.

870.3700b Prenatal Developmental Toxicity Study - Rabbit

In an oral developmental toxicity study (PMRA 2226501, MRID 48557840), groups of 24 timemated female New Zealand White rabbits were administered diets containing 0, 122, 391, or 

1539 ppm XDE-729 methyl (97.2% a.i., lot no. E2837-51, TSN031117-0004) on gestation days 

(GD) 7 through 28, which corresponded to time-weighted average doses of 0, 5.8, 18.5 or 71.6 

mg/kg bw/day, respectively. In-life maternal study parameters included clinical observations, 

body weight, body weight gain, and feed consumption. On GD 28, all rabbits were euthanized 

and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were 

recorded, along with the number of corpora lutea, uterine implantations, resorptions, and 

live/dead fetuses. Microscopic examination of the liver was conducted for all dose groups. All 

fetuses were weighed, sexed, and examined for external and visceral alterations. The heads 

were also examined for craniofacial alterations by serial sectioning in approximately one half of 

the fetuses in each litter, while skeletal examinations were performed on all fetuses. Blood 

concentrations of XDE-729 methyl and the metabolite XDE-729 acid were analyzed in samples 

collected from four does/group and their respective litters at necropsy.

There were no treatment-related effects on survival, clinical signs, body weights, food 

consumption or gross pathology in maternal animals. 

In does, treatment-related increased liver weights (absolute 9-27%, relative 13-29%), along with 

increased incidences of hypertrophy with altered tinctorial properties (increased cytoplasmic 

eosinophilia) of periportal hepatocytes (incidences: 1, 1, 14, 19), increased number of mitotic 
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figures in hepatocytes (incidences: 0, 0, 3, 7) and altered cytoplasmic homogeneity of 

centrilobular/midzonal hepatocytes (cytoplasmic glycogen) (incidences: 1, 0, 7, 12) were 

observed at 18.5 and 71.6 mg/kg bw/day. At 18.5 mg/kg bw/day, these changes were considered 

to be adaptive liver effects and non-adverse by Canada PMRA and US EPA, but were 

considered to be treatment-related by Australia OCS. In addition, the high-dose females had 

increased incidences of panlobular vacuolization of hepatocytes consistent with fatty change in 

the liver in addition to these findings. Dams in the high-dose group had decreased gravid uterine 

weights at necropsy.

Canada PMRA: The maternal LOAEL is 71.6 mg/kg bw/day, based on increased liver 

weight, increased incidences of hypertrophy with altered tinctorial properties (increased 

cytoplasmic eosinophilia) of periportal hepatocytes, increased number of mitotic figures in 

hepatocytes, increases in altered cytoplasmic homogeneity of centrilobular/midzonal 

hepatocytes (cytoplasmic glycogen), increased panlobular vacuolization of hepatocytes 

(consistent with fatty change), and decreased gravid uterine weights. The maternal 

NOAEL is 18.5 mg/kg bw/day. 

Australia OCS, US EPA: The maternal LOAEL is 18.5 mg/kg bw/day, based on increased 

liver weight, increased incidences of hypertrophy with altered tinctorial properties 

(increased cytoplasmic eosinophilia) of periportal hepatocytes, increased number of 

mitotic figures in hepatocytes and increases in altered cytoplasmic homogeneity of 

centrilobular/midzonal hepatocytes (cytoplasmic glycogen). The maternal NOAEL is 5.8

mg/kg bw/day. 

No treatment-related external, craniofacial or visceral malformations or variations were 

observed. Treatment-related increased incidences of delayed ossification of the pubis (a 

developmental variation; fetal (litter) incidences: 0(0), 1(1), 6(3), 11(8)) were observed at 71.6 

mg/kg bw/day. Fetuses in the high-dose group had treatment-related decreased body weights (7-

11%) upon Caesarean section but no other treatment-related effects on pregnancy rates, litter 

size, numbers of corpora lutea or implantations, percent pre-implantation loss or fetal sex ratios 

in any treated group were noted. Increases in resorptions/post-implantation loss (1.2, 2.5, 6.3, 

5.1%) were observed at 18.5 and 71.6 mg/kg bw/day; this finding was considered to be 

treatment-related by the Canada PMRA.

Canada PMRA: The developmental LOAEL is 18.5 mg/kg bw/day, based on increases in 

resorptions/post-implantation loss. The developmental NOAEL is 5.8 mg/kg bw/day.

US EPA, Australia OCS: The developmental LOAEL is 71.6 mg/kg bw/day, based on 

decreased fetal body weights and delayed ossification of the pubis. The developmental 

NOAEL is 18.5 mg/kg bw/day.

Mean daily XDE-729 methyl intake during the period of TK data collection (GD 27-28) was 6, 

17, and 73 mg/kg bw/day for rabbits in the 122, 391, and 1539 ppm groups, respectively. 

XDE-729 methyl was not detected in terminal blood samples of treated does or their litters. Doe 

blood from XDE-729 methyl-treated groups contained the metabolite, XDE-729 acid, at levels 

above the LLQ. XDE-729 acid was also present in blood from GD 28 fetuses at levels above the 

LLQ in the 391 and 1539 ppm groups, but not in the 122 ppm group. The blood concentrations 
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were dose-proportional across all three XDE-729 methyl concentrations in does as determined 

using the least squares regression statistical test. XDE-729 acid fetal blood concentrations were 

interpreted to be less than dose-proportional (sublinear) in the 1539 ppm group. While not 

statistically identified, the blood concentrations were similar to the 381 ppm group despite a 3.4-

fold higher applied dose. Fetal XDE-729 acid blood concentrations in the 391 and 1539 ppm 

groups were on average, 27 and 6% of dam blood concentrations, respectively. Thus, XDE-729 

acid exhibited kinetic linearity in GD 28 does, in all three treated groups, but kinetic nonlinearity 

in GD 28 fetuses at 1539 ppm.

This study is classified as acceptable/guideline and satisfies the guideline requirement for a 

Prenatal Developmental Study – Rabbits; OPPTS 870.3700; OECD 414. 

A.4.3 Reproductive Toxicity

870.3800 Reproduction and Fertility Effects - Rat

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

A.4.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity – Rat

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

870.4100b Chronic Toxicity – Dog

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

A.4.5 Carcinogenicity

870.4200a Carcinogenicity Study – rat

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

870.4200b Carcinogenicity (feeding) – Mouse

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

A.4.6 Mutagenicity

Gene Mutation

870.5100, Bacterial reverse mutation 

test, preincubation assay

MRID 48557846

Acceptable/guideline

0, 50, 10, 500, 1500, or 5000 μg/plate +/- S9

Negative in Salmonella typhimurium TA 1535, TA1537, TA98, and 

TA100 and Escherichia coli Wp2 uvrA up to the limit concentration with 

evidence of compound insolubility but no cytotoxicity.
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870.5100, Bacterial reverse mutation 

test, plate-incorporation assay

MRID 48557845

Acceptable/guideline

0, 50, 10, 500, 1500, or 5000 μg/plate +/- S9

Negative in Salmonella typhimurium strainsTA1537, TA98, and TA100 

and Escherichia coli Wp2 uvrA up to the limit concentration with no 

evidence of compound insolubility or cytotoxicity.

870.5300, in vitro mammalian cell 

forward gene mutation, Chinese 

hamster ovary (CHO) cells, HGPRT 

locus

MRID 48557851

Acceptable/guideline

0, 15.625, 31.25, 62.5, 125, 250 or 500 μg/mL -S9 activation 

and 0, 18.75, 37.5, 75, 150, 300 or 500 μg/mL +S9 activation

Negative for the induction of mutant colonies over background up to 

precipitating concentrations

870.5300, in vitro mammalian cell 

forward gene mutation, SD rat 

peripheral lymphocytes

MRID 48557849

Acceptable/guideline

0, 3.13, 6.25, 12.5, 25 or 50 μg/mL+/- S9 activation

Negative for the induction of mutant colonies over background up to 

precipitating concentrations.

Cytogenetics

870.5375, In vitro mammalian cell 

chromosomal aberration assay, 

human peripheral lymphocytes

MRID 48557854

Acceptable/guideline

0, 15.624, 31.25, 62.5, 125, 250 or 500 μg/mL +/- S9 activation

Negative for induction of chromosomal aberrations over background up 

to cytotoxic and precipitating concentrations.

870.5375, In vitro mammalian cell 

chromosomal aberration assay, 

primary lymphocyte cultures 

(Sprague Dawley rats)

MIRD 48557853

Acceptable/guideline

0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μg/mL - S9 activation and 

0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 μg/mL + S9 activation

Negative for induction of chromosomal aberrations over background up 

to precipitating concentrations.

A.4.7 Neurotoxicity

870.6100 Delayed Neurotoxicity Study - Hen

Study not required for halauxifen-methyl. 

870.6200 Acute Neurotoxicity Screening Battery

Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

870.6200 Subchronic Neurotoxicity Screening Battery
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Study not conducted for halauxifen-methyl. See Executive Summaries for XDE-729 Acid

870.6300 Developmental Neurotoxicity Study

Study not required for halauxifen-methyl. 

A.4.8 Metabolism

870.7485 Metabolism - Rat

See Executive Summaries for XDE-729 acid

870.7600 Dermal Absorption - Rat

In an in vivo dermal absorption study (MRID 48557911; PMRA No. 2226192), four male Wistar 

rats/dose group were dermally exposed for 10 hours to [14C]-XDE-729 methyl (radiochemical 

purity 98.5%; nonlabelled purity 98.9%, batch XW7-38246-49) formulated as GF-2573 (an 

emulsifiable concentrate), at three concentrations: 7.5 g acid equivalents (a.e.) L-1(concentrate), 

ca 0.15 g a.e.L-1(field dilution 1) and ca 0.025 g a.e.L-1 (field dilution 2). Test material in 100 

μL was applied to an area of shaved skin of 10 cm2to give dermal doses of 75, 1.5 and 0.25 

μg/cm2, respectively. Following exposure the test material was rinsed off and skin was tapestripped 20x to remove the stratum corneum. At each concentration, groups were designated for 

termination at 24, 48, 72, 144 (concentrate only) and 192 (field dilutions) hours post-dosing. 

Additionally, blood samples were collected at 4 and 10 hours post-dosing to evaluate levels of 

radioactivity, and further analysis was performed to determine whether XDE-729 methyl or its 

acid metabolite were present.

US EPA: Calculation of dermal absorption excluded radioactivity in the first two skin tape 

strips. In vivo percent dermal absorption (absorbed plus absorbable dose, excluding the 

outermost two layers of skin) at the end of 144-192 hours was 13, 15 and 24% of the applied 

dose (highest to lowest concentration tested, respectively). Based on the results of these studies, 

a dermal absorption factor (DAF) of 24% (192 hr post-exposure) from the lowest concentration 

field dilution was selected as the most appropriate value for risk assessment. The 10 hr exposure 

duration is considered relevant for occupational exposure and the selected value represents the 

maximum absorption observed in the rat in vivo absorption study. 

In vitro percent dermal absorption (MRID 48557910) in the rat skin at the end of 24 hr was 4.9, 

41.1 and 42.7%. The ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 3.0 and 2.2. 

Absorption in human skin after 24 hr was 0.8, 14.9 and 26.5%. The ratio of dermal absorption in 

human to rat skin was 0.16, 0.36 and 62.

Canada PMRA and Australia OCS: The first two tape strips were included in determination of 

the potentially absorbable dose for all test groups. Based on this calculation, in vivo percent 

dermal absorption at the end of 144-192 hours was 14, 18 and 30% (absorbed plus absorbable 

dose) of the applied dose (all values are given from highest to lowest concentration tested, 

respectively). A dermal absorption factor (DAF) of 30% (192 hr post-exposure) from the lowest 
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concentration field dilution in the rat in vivo absorption study was selected as the most 

appropriate value for risk assessment. 

In vitro percent dermal absorption (MRID 48557910) in the rat skin at the end of 24 hr was 5.0, 

42.5 and 45.9% of the applied dose (highest to lowest concentration, respectively). In 

comparison, at the end of 24 hr, in vivo dermal exposure in the rat was 9.9, 15.7 and 23.3%. The 

ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 2.7 and 2.0. Absorption in human 

skin after 24 hr was 0.9, 15.5 and 27.9%. The ratio of dermal absorption in human to rat skin 

was 0.18, 0.36 and 0.61.

Analysis of blood samples indicated that systemic exposure following dermal dosing is to XDE729, the acid metabolite. No parent (XDE-729 methyl) was identified. 

This study is considered acceptable/guideline and satisfies the requirement for an in vivo dermal 

absorption study in the rat (OPPTS 870.7600; OECD 427).

Nonguideline In Vitro Dermal Absorption – Rat and Human

In a comparative in vitro dermal absorption study (MRID 48557910, PMRA No. 2226190), the in 

vitro percutaneous absorption of [14C]-XDE-729 methyl (radiolabeled purity 98.5%, batch no. XS9-

100040-28; non-radiolabelled purity 98.9%, batch XW7-38246-49), formulated as GF-2573 (an 

emulsifiable concentrate formulation) through split-skin human and rat skin membranes was

evaluated using flow-through diffusion cells. The test substance was tested at three target 

concentrations (expressed as gram acid equivalents (a.e.) per liter): 7.5 g a.e.L-1(concentrate) and ca 

0.15 g a.e.L

-1

(field dilution 1) and ca 0.025 g a.e.L-1(field dilution 2). The concentrate represents the 

maximal concentration possible when handling the undiluted formulation, while the field dilutions 

reflect the concentrations recommended for use in the field. Skin samples were exposed to the test 

material for 10 hours, rinsed and then absorption was continued for an additional 14 hours. 

US EPA: The radioactivity in the first two skin tape strips was excluded from the determination of 

potentially absorbable dose for both the rat and human skin samples (as presented in the study 

report). Based on this calculation, the in vitro percent dermal absorption in the rat skin at the end of 

24 hr was 5.0, 41.1 and 42.7% of the applied dose (highest to lowest concentration, respectively). In 

comparison, at the end of 24 hr, in vivo dermal absorption in the rat was 9.5, 14 and 20%. The ratio 

of in vitro to in vivo dermal absorption in the rat was 0.5, 3.0 and 2.2 (based on the results of MRID 

48557911). Absorption in human skin after 24 hr was 0.9, 15 and 26% (highest to lowest 

concentrations, respectively). The ratio of dermal absorption in human to rat skin was 0.16, 0.36 and 

0.62.

Canada PMRA and Australia OCS: The first two skin tape strips were included in determination of 

the potentially absorbable dose. Based on this calculation, in vitro percent dermal absorption in the 

rat skin at the endo of 24 hr was 5.0, 42.5 and 45.9% of the applied dose (highest to lowest 

concentration, respectively). In comparison, at the end of 24 hr, in vivo dermal absorption in the rat 

was 9.9, 15.7 and 23.3%. The ratio of in vitro to in vivo dermal absorption in the rat was 0.5, 2.7 and 

2.0. Absorption in human skin after 24 hr was 0.9, 15.5 and 27.9%. The ratio of dermal absorption 

in human to rat skin was 0.18, 0.36 and 0.61.

This study is classified as acceptable/nonguideline. It was performed to provide information on 

comparative rat and human dermal absorption and not to satisfy a guideline requirement.
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A.4.9 Immunotoxicity

870.7800 Immunotoxicity

In an immunotoxicity study (MRID 48557864, PMRA 22265141), XDE-729 methyl (97.2% 

a.i., lot no. Lot # E2837-51, TSN031117-0004) was administered to ten female, F344/DuCrl 

rats/dose in diet at dose levels of 0, 127, 643 or 6323 ppm (0, 10.6, 53.6, or 505 mg/kg/day, 

respectively) for 29 days. An additional group of ten female F344/DuCrl rats were included to 

serve as immunosuppressive positive controls (IPC) and received intraperitoneal (i.p.) doses of 

20 mg/kg cyclophosphamide (CP) on study days 24-28. The immunotoxic potential of XDE729 methyl was assessed through the evaluation of the primary antibody response to sheep red 

blood cells (SRBC) using an enzyme-linked immunosorbant assay (ELISA) approach that 

measured the concentration of serum anti-SRBC IgM. There were no adverse effects on clinical 

observations, body weight, body weight gain, food consumption, hematology or gross necropsy 

observations.

Animals given 505 mg/kg bw/day had statistically significantly lower mean hemoglobin 

concentrations, hematocrit levels, and elevated numbers of reticulocytes when compared to 

those of the controls; however, these parameters were within the historical control 

ranges. Decreased absolute thymus weight and increased liver weights were observed at 505 

mg/kg bw/day. This observed response is consistent with previously conducted 28-and 90-day 

toxicity studies which demonstrated both decreased thymic weights and the histopathological 

correlate of very slight lymphoid atrophy of the thymus at doses of 728 mg/kg bw/day and 500 

mg/kg bw/day, respectively. Under conditions of this study, the systemic NOAEL is 53.6

mg/kg bw/day, the LOAEL was 505 mg/kg bw/day based on increased liver weights and

decreased thymus weights.

The anti-SRBC antibody responses were within the assay range and analysis of the data did not 

reveal a statistically significant difference between the three doses of XDE-729 methyl and the 

vehicle control. A high inter-individual variability was noted in all the treatment groups as well 

as in the control group. Evaluation of individual animal data of this study did not show any trend 

or distribution that would demonstrate significant suppression of anti-SRBC antibody response. 

The positive control cyclophosphamide produced a statistically significant and greater than 99% 

reduction in the anti-SRBC IgM response.

The Natural Killer (NK) cell activity was not evaluated in this study. Decreased thymus weights 

and histopathological findings were observed in a few studies. The effects in these studies were 

interpreted to be secondary to stress as the responses occurred in the presence of decreases in 

body weight, body weight gains and significant hepatic effects. The overall weight of evidence 

suggests that this chemical does not directly target the immune system. Under the US EPA HED 

guidance, a NK cell activity assay is not required at this time. The immunotoxicity NOAEL is 

505 mg/kg bw/day; the LOAEL was not established.

This study is classified as acceptable/guideline and satisfies the guideline requirement for an 

immunotoxicity study in the rat or mouse (OPPTS 870.7800).
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A.4.9 Special/Other Studies (All Nonguideline)

Seven Day Dietary Study in the Rat

This study (PMRA 2226494, MRID 48557879) was conducted to characterize the mode of

action for the XDE-729 methyl-induced liver and thyroid effects in the rat, and to compare the 

toxicokinetic profiles in rats administered XDE-729 methyl (97.2%, lot # E2837-51) versus 

XDE-729 acid (95.3%, lot # E2837-52). Four male F344/DuCrl rats per group were given test 

diets formulated to supply 0, 782 mg XDE-729 methyl/kg body weight/day, or 750 mg XDE-729

acid/kg body weight/day in the diet for seven days. Actual time-weighted average doses were 0, 

766 (XDE-729 methyl), or 672 mg/kg bw/day (XDE-729 acid). Parameters evaluated were daily 

cage-side observations, body weights, food consumption, organ weights and histopathological 

examination of the liver and thyroid, biomarker analysis of liver, blood, and urine, and targeted 

gene expression.

Concentrations of XDE-729 acid in the liver or blood from animals given XDE-729 acid were 

greater than animals given XDE-729 methyl at the corresponding acid equivalent dose. 

However, following administration of XDE-729 methyl most of the dose was converted to XDE729 acid with negligible amounts of XDE-729 methyl recovered in the blood at 30 minutes postdosing, and 24 hour urine (<0.03%), while 24% of the administered XDE-729 methyl dose was 

excreted in the urine as XDE-729 acid. The highest recovery of XDE-729 methyl was in the 

liver.

Concentrations of four identified metabolites were lower in blood, liver tissue and urine after 

administration of XDE-729 acid versus XDE-729 methyl. There was evidence of higher 

concentrations of O-demethyl metabolites in the liver and urine in XDE-729 methyl-treated 

males compared to those treated with XDE-729 acid. These metabolites consisted of an acyl 

glucuronide conjugate of XDE-729 acid, O-demethyl XDE-729 acid, a sulfate conjugate of Odemethyl XDE-729 acid, and a glucuronide conjugate of O-demethyl XDE-729 acid. An 

additional metabolite was identified following XDE-729 acid administration as a glucuronide 

conjugate of O-demethyl XDE-729 methyl. The data suggest that induction of O-demethylation 

after XDE-729 methyl treatment is greater than the levels seen after XDE-729 acid treatment. 

After XDE-729 acid treatment in males, there were no treatment-related effects on thyroid and 

liver weights or histopathology at 672 mg/kg bw/day in males. Minor treatment-related decreases 

in body weight gains and food consumption were observed. 

After XDE-729 methyl treatment in males at 766 mg/kg bw/day, there were no treatment-related 

effects on body weights, body weight gains, or food consumption in males. Males had treatmentrelated increases in liver weights and very slight panlobular hypertrophy of hepatocytes and very 

slight increased numbers of mitotic figures in hepatocytes. In addition, these animals had 

treatment-related increases in thyroid weights along with very slight hypertrophy of thyroid 

gland follicular cells and very slight increased numbers of mitotic figures in follicular cells.
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The early events in the proposed mode of action framework for XDE-729 methyl-induced liver 

toxicity have been further characterized. After administration of 766 mg/kg bw/day XDE-729 

methyl, the test material was present in the liver (3.99±1.04 μg/mL) and at very low 

concentrations in the blood (0.04±0.01 μg/mL). As discussed above, most of the administered 

XDE-729 methyl was converted to XDE-729 acid and the data suggest greater induction of Odemethylation after XDE-729 methyl treatment. Further, all five metabolites were detected in the 

liver tissue and urine after dosing with XDE-729 methyl, with the levels of metabolites in the 

liver ranging from 0.34-4.91 μg/mL. Gene expression profiling did reveal apparent changes

(>2.0-fold compared to control) in Cyp2b1, Cyp2b2, Cyp3a23, Cyp4a22, Ugt1a1, Ugt1a6 or 

Ugt2b1 mRNA expression in males treated with 672 mg/kg bw/day XDE-729 acid when 

compared to controls. Conversely, there was a minor (4.2-fold) increase in Cyp1a1 mRNA 

expression observed in XDE-729 methyl-treated animals at 766 mg/kg bw/day; this finding 

suggests that ligand-induced activation of the AhR pathway occurs in XDE-729 methyl-treated 

males but not in males treated with XDE-729 acid. The hepatic gene expression data for Cyp2b1

and Cyp2b2 also revealed a slight induction for animals administered XDE-729 methyl but not 

XDE-729 acid; however, the magnitude of induction is not consistent with a toxicologically 

significant activation of the CAR pathway. Cyp3a23 or Cyp4a22 transcripts were not induced in 

either XDE-729 methyl- or XDE-729 acid-treated rats indicating that the PXR and PPAR-

signaling pathways were not activated and are likely not involved in the observed hepatic 

responses. Similarly, no gene expression changes were noted for either XDE-729 methyl or 

XDE-729 acid for the phase II glucuronidation-associated Ugt1a1 or Ugt2b1 transcripts in livers 

of rats fed XDE-729 methyl. However, the phase II glucuronidation-associated Ugt1a6 was 

induced in XDE-729 methyl treated rats but not XDE-729 acid treated rats and was considered 

possibly mediated through the AhR signaling pathway.

XDE-729 methyl treatment resulted in effects of liver and thyroid weights and histopathological 

findings that were not observed with XDE-729. The observed thyroid effects were considered 

by the study authors to be likely secondary to treatment-related liver alterations from induction 

of the AhR signaling pathway, with induction of Ugt1a6 resulting in increased hepatic clearance 

of T3 and T4 thyroid hormones and disruption of thyroid homeostasis. This is supported by the 

molecular analysis, which indicated hepatic activation of the AhR signaling pathway and the 

presence of XDE-729 methyl and associated metabolites in the liver but not in blood 30 minutes 

post-dosing or 24 hour urine. In contrast, the males treated with XDE-729 acid did not have 

treatment-related hepatic or thyroid effects or correlative changes in gene expression, strongly 

suggesting that XDE-729 methyl is directly associated with these hepatic and secondary thyroid

effects following induction of the AhR signaling pathway.

This short-term mechanistic study in rats is acceptable/nonguideline as a supplemental study and 

provides additional information relevant to characterization of the MOA of liver toxicity of 

halauxifen-methyl.

Seven Day Dietary Toxicity Study in the Mouse

This study (PMRA 2226491, MRID 48557872) was conducted to characterize the toxicological 

effects of XDE-729 methyl (97.2%, lot #E2837-51) in the mouse, to further examine the mode 

of action for XDE-729 methyl-induced liver effects and to examine the toxicokinetic profile in
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mice after XDE-729 methyl treatment. Groups of five male and five female Crl:CD1(ICR) mice 

were given test diets formulated to supply 0, 261, or 782 milligrams XDE-729 methyl per 

kilogram body weight per day (mg/kg bw/day) for 7 days, which corresponded to time-weighted 

average doses of 0, 250, or 778 mg/kg bw/day for males and 0, 276, or 863 mg/kg bw/day for 

females, respectively. 

There were no treatment-related effects in cage-side observations, body weights, body weight 

gains, food consumption, clinical chemistry parameters, kidney weights or gross observations. 

After XDE-729 methyl treatment, males and females at 778/863 mg/kg bw/day M/F had higher 

liver weights than controls. The changes were considered to be treatment-related but nonadverse (adaptive) based on the lack of histopathological correlate and effects on clinical 

chemistry parameters. 

XDE-729 methyl was mostly converted to XDE-729 acid, which was present at quantifiable 

levels in all of the blood samples from treated animals. XDE-729 methyl was present above the 

lower limit of quantitation (LLQ) in ~50% of the blood samples from treated animals (taken at 

steady state), but at only ≤ 1% of levels of the primary metabolite XDE-729 acid. 

Concentrations of XDE-729 methyl were considerably higher in liver than in blood, while those 

of the metabolite XDE-729 in the liver were similar to those in blood. XDE-729 methyl was 

detected in the urine only from animals in the high-dose groups at negligible levels, while XDE729 acid was detected in all urine samples at levels markedly higher than XDE-729 methyl. 

XDE-729 methyl quantified as XDE-729 acid appeared to exhibit linear kinetics in both sexes in 

blood, liver, and urine. The only exception to this was in blood samples from males, taken at 6 

AM on day 7. Concentrations of XDE-729 acid appeared to be less than dose-proportional 

(sublinear) at the high dose in male mouse blood on day 7 (778 mg/kg bw/day). Levels of both 

XDE-729 methyl and XDE-729 acid were generally lower in female mice compared to males in 

blood (6 am), liver and urine, despite having consumed slightly larger doses of XDE-729 

methyl; the only exceptions were the terminal blood concentrations, where slightly higher XDE729 methyl and XDE-729 acid levels were seen in females. These data suggest female mice may 

eliminate XDE-729 methyl and its metabolites more efficiently compared to male mice;

however, in the absence of more robust toxicokinetic analyses, definitive conclusions on sex 

differences in toxicokinetic parameters in mice following administration of XDE-729 methyl 

cannot be made. 

Levels of the major metabolite, XDE-729 acid, in the urine of male mice were ~76% of those 

observed in the urine of male rats in a previous study in male rats after 7-day dietary treatment 

with XDE-729 methyl (PMRA 2226494, MRID 48557879). However, overall, the kinetics of 

XDE-729 methyl in male mice appears to be very similar to the kinetics of XDE-729 methyl 

observed in male rats.

The increased hepatic Cyp1a1 levels in XDE-729 methyl-treated mice and correlating 

treatment-related increase in liver weights (non-adverse) suggest that the ligand-induced 

activation of the aryl hydrocarbon receptor (AhR) pathway seen in rats also occurs in mice. The 

hepatic gene expression data for Cyp2b10 revealed a slight induction for animals administered 

XDE-729 methyl; however, the magnitude of induction is not consistent with a toxicologically 

significant activation of the CAR pathway. Cyp3a11 or Cyp4a22 transcripts were not induced 
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in either XDE-729 methyl mice indicating that the PXR and PPAR-α signaling pathways were 

not activated and are likely not involved in the observed hepatic responses. 

In mice, XDE-729 methyl only caused a marginal adaptive increase in liver weights in both 

sexes and there was no evidence of liver histopathology. This is contrasted to the study in rats 

where a much larger increase in liver weights were observed in addition to increased thyroid 

weights with correlative hepatic and thyroid histopathological effects observed after treatment 

with XDE-729 methyl but not XDE-729 acid. In rats, very slight panlobular hepatocellular 

hypertrophy and increased number of hepatocytes in mitosis were present in all animals and the 

magnitude of Cyp1a1 induction was markedly higher than in mice. These data suggest that rats 

are more sensitive to XDE-729 methyl-induced hepatic responses than mice. 

This short-term mechanistic study in mice is acceptable/nonguideline as a supplemental study 

investigating the mode of action of halauxifen-methyl liver toxicity.

Twenty Eight Day Dietary Toxicity Study in the Rat

The purpose of this short-term dietary study (PMRA 2226489. MRID 48557873) was to 

characterize the mode of action for XDE-729 methyl (97.2%, lot #E2837-51) induced liver 

effects in the rat. Groups of five male F344/DuCrl rats were exposed to 0, 3.1, 10.4, 54.8 or 271 

mg XDE-729 methyl/kg body weight/day (mg/kg bw/day) in the diet for 28 days. Recovery 

groups were selected from the control and low dose group (0 or 10.3 mg/kg bw/day) (4 day 

recovery group; n=3/dose level) and control and two highest dose groups (0, 53.7 or 277 mg/kg 

bw/day) (28-day recovery group; n=5/dose level) were switched to control diet following a 28 

day exposure before switching to control diet to investigate hepatic recovery after removal of 

treatment. 

There were no treatment-related effects on survival, clinical signs, body weights, food 

consumption or kidney weights. 

Treatment-related increases in absolute and relative liver weights, hypertrophy of 

centrilobular/midzonal hepatocytes with altered tinctorial properties (increased cytoplasmic 

eosinophilia), numbers of mitotic figures (hepatocytes in mitosis) and individual hepatocellular 

vacuolization (consistent with fatty change) were observed at 54.8 and 271 mg/kg bw/day 

relative to controls. At 271 mg/kg bw/day, a slight increase in BrdU labeling index in the 

centrilobular and midzonal regions was observed after 28 days of exposure, which was 

considered treatment-related when considering the related liver effects occurring at this dose 

and time-point. 

Examination of the gene expression responses for Cyp1a1 and Cyp1a2 revealed a doseresponsive induction profile correlating with the effects on liver weights and the appearance of 

histopathological apical effects in the liver consistent with ligand-induced activation of the AhR 

signaling pathway at 54.8 and 271 mg/kg bw/day, respectively. Transcript levels for Cyp1a1

demonstrated the highest induction of 683.8-, and 12,180.0-fold at doses of 54.8 and 271 mg/kg

bw/day, respectively. Similarly, the response and magnitude of the response for Ugt1a6 at 271 

mg/kg bw/day is consistent with induction of this gene in other repeat dose studies (7-day and 
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90-day studies in rats) and correlating alterations in thyroid weights and histopathology findings 

indicative of activation of the AhR signaling pathway and the hepatic induction for this gene.

Consistent with hepatic induction of the AhR signaling pathway by XDE-729 methyl, not 

quantifiable or low hepatic concentrations of XDE-729 methyl were observed at 3.2 and 10.6

mg/kg bw/day. Increasing hepatic concentrations of XDE-729 methyl were recovered in the 

liver at 54.8 and 271 mg/kg bw/day. Quantification of the analytes in the liver recovered six 

metabolites. XDE-729 acid was by far the most abundant analyte in all samples, followed by the 

glucuronide conjugate of XDE-729 methyl. Previous studies with XDE-729 methyl have 

demonstrated an induction of metabolizing enzymes, including phase II Ugt metabolizing 

enzymes with concurrent increases in liver weights with increasing dose. Consistent with this 

induction in the current study XDE-729 methyl administration resulted in increased levels of 

conjugated metabolites of XDE-729 methyl in the liver when compared to administration of 

XDE-729 acid, which are rapidly eliminated in the urine. Further, the amount of XDE-729 acid 

present as the major metabolite appeared to decrease significantly with increasing doses 

concurrent with increased recovery of conjugated metabolites with increasing dose. This is 

indicative of an increase in the rate of enzyme kinetics (induction of hepatic enzymes) with 

higher doses and is consistent with findings in the previous 90-day study in rats (PMRA 

2226466 (amended report: PMRA 2315015). MRID 48557830 (amended MRID 49124405)).

Parent XDE-729 methyl was not present above the lower limit of quantitation (LLQ) in any 

blood samples at necropsy. However, the major metabolite, XDE-729 acid, was present at 

quantifiable levels in all of the blood samples from exposed animals. Approximately 0.05% of 

the dose was excreted as intact parent XDE-729 methyl in 24 hour urine. Approximately 50, 52 

and 49% of the administered dose of XDE-729 methyl was excreted as XDE-729 acid in the 24 

hour urine at 10, 52 and 261 mg/kg bw/day, respectively. The total amount of XDE-729 

excreted in urine over 24 hours was slightly lower at 3 mg/kg bw/day (44% of the administered 

dose). 

XDE-729 methyl and all six of its measured metabolites were present in liver at levels above the 

lower limit of quantification (LLQ) in all but the low (3.1 mg/kg/day) dose group, which did not 

contain quantifiable levels of XDE-729 methyl or the sulfate conjugate of O-demethyl XDE729. The metabolites included de-methylated XDE-729 methyl (XDE-729), which was the 

major metabolite, and five minor metabolites, which were demethylated and/or conjugated 

XDE-729 methyl and/or XDE-729. XDE-729 was by far the most abundant analyte in all 

samples, followed by the glucuronide conjugate of XDE-729 methyl. At 3.1 mg/kg/day, XDE729, and all of the glucuronidated metabolites were present. XDE-729 methyl was not present 

above LLQ in liver at 3.1 mg/kg/day. XDE-729 methyl levels were approximately 10% of acid 

levels in liver at 10.4 and 54.8 mg/kg/day. In the 271 mg/kg/day dose group, XDE-729 methyl 

levels were 20% of XDE-729 acid levels and appeared to be supralinear. As observed 

previously, the percent of the administered dose present as the major metabolite, XDE-729, in 

the liver at the time of necropsy, decreased significantly with increasing doses. Statistically 

significant sublinearity in the dose-liver level relationship was identified in the 271 mg/kg/day 

dose group. Conversely, the percent of the administered dose present as conjugated metabolites 

appeared to increase with increasing dose, though the increase was not statistically significant. 

XDE-729 methyl was dose-proportional across all doses in liver and urine but appeared slightly 

supralinear at 261 mg/kg/day in liver. Parent XDE-729 methyl was not present above LLQ in 

any blood samples at necropsy. However, the major metabolite, XDE-729, was present at 
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quantifiable levels in all of the blood samples from exposed animals. Blood levels of XDE-729 

were significantly less than dose-proportional (sublinear) at 271 mg/kg/day. Approximately 

50% of the administered dose of XDE-729 methyl was excreted as XDE-729 in the urine. The 

total amount of XDE-729 excreted in urine over 24 hours declined with increasing doses and 

was statistically identified as sublinear at 271 mg/kg/day. 

The two recovery groups demonstrated complete reversal of changes in liver weight, liver 

histopathology, Cyp1a1, Cyp1a2, Cyp2b1 and Ugt1a6 mRNA expression at all doses tested after 

a 4 or 28 day recovery period. BrdU uptake in hepatocytes appeared to be still slightly elevated 

compared to the baseline range of labeling index (LI) values, but lower than the LI values 

following 28-days of treatment suggesting at least a partial recovery. However, based on 

discrepancies in the data, reversibility of the increased proliferative response at 277 mg/kg 

bw/day could not be adequately interpreted. There were no quantifiable liver concentrations of 

XDE-729 methyl or XDE-729 in the blood, liver or urine after the specified recovery period.

The data from this study further support an AhR-mediated mode of action for the hepatic 

responses observed after exposure of rats to XDE-729 methyl. The recovery for each of the 

responses highlights the transient and ligand-mediated nature of the hepatic effects due to a lack 

of sustained activation of the AhR signaling pathway. 

This study is considered acceptable/nonguideline as a supplemental study to provide information 

for characterization of the mode of action for liver toxicity from halauxifen-methyl.

In Vitro Transactivation Assay and Ligand Binding Study

In an in vitro transactivation assay and ligand binding study (PMRA 2226490, MRID 

48557878), XDE-729 acid (95.3%, lot # RSMP 1077053), XDE-729 methyl technical (97.2%, 

lot # RSMP 1076831,), XDE-729 methyl (99.1%, lot #RSMP 1076831), and XDE-729 methyl 

ultrapure (99.7% a.i., lot # RSMP 1082768) were tested in mouse and human stable cell lines 

containing a luciferase reporter vector driven by dioxin-responsive elements that are responsive 

to AhR agonists. 2-(1 ́H-indole-3 ́-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, 

99.9%), a potent AhR agonist, was included as a positive control and a reference for relative 

potency. XDE-729 acid was tested for AhR agonist activity in duplicate using Hepa 1.1 cells 

(mouse) and Hep G2 40/6 cells (human). In all experiments, XDE-729 acid failed to exhibit any 

agonist activity. XDE-729 methyl (97.2%) was assayed in duplicate using Hepa 1.1 cells 

(mouse) and in Hep G2 40/6 cells (human). In Hepa 1.1 cells, XDE-729 methyl technical 

(97.2%) exhibited significant agonist activity at 10 and 50 M. The XDE-729 methyl (99.1%) 

exhibited similar agonist activity to XDE-729 methyl (97.2%). In assays using Hep G2 40/6 

cells, both XDE-729 methyl (97.2%) and XDE-729 methyl (99.1%) failed to induce significant 

AhR transactivation activity. 

XDE-729 methyl ultrapure (99.7%) mediated induction of AhR activity in a dose-dependent 

manner at 10 and 50 μM in the mouse reporter cell line. In contrast, XDE-729 methyl ultrapure 

exhibits essentially no activity in the human reporter cell line at the doses tested. The data 

obtained with XDE-729 ultrapure (99.7%) was essentially the same as with XDE-729 methyl 

(97.2%) and XDE-729 methyl (99.1%). These data indicate that XDE-729 methyl and not an 
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impurity is a weak activator of the mouse AhR, but not of the human AhR. However, it must be 

noted that the positive control, ITE, also produced a much weaker response with the human Hep 

G2 40/6 reporter cell line. This makes it difficult to make comparisons of the ability of XDE-279 

methyl to be a weak agonist of the human AhR compared to the mouse AhR. Overall, the 

positive control (ITE) demonstrated a dose dependent increase in AhR activity at all 

concentrations confirming the sensitivity and validity of the assay.

To determine if XDE-729 methyl ultrapure (99.7%) is an actual AhR ligand that binds to the 

ligand-binding pocket, a competition AhR ligand binding assay was performed. The results 

indicate that XDE-729 methyl ultrapure (99.7%) is an AhR ligand that weakly competes with the 

photoaffinity ligand for the rat Ah receptor. These results are consistent with the results obtained 

in the reporter assays.

This in vitro mechanistic assay in mouse and human liver cells is acceptable/nonguideline as 

supplemental data submitted to characterize the mode of action of liver toxicity from halauxifenmethyl.

In Vitro Expression of Cyp Genes and AhR Activation in Cultured Hepatocytes 

from Mice, Rats and Humans

The objective of this in vitro mechanistic study (PMRA 2226487, MRID 48557876) was to 

assess the potential of XDE-729 methyl (99.1%, lot # not reported) to induce Cyp1a1/CYP1A1

and Cyp1a2/CYP1A2 in primary cultures of human, mouse and rat hepatocytes. Evaluation of 

the degree of CYP induction was performed by measuring and comparing mRNA levels between 

the various treatment groups by RT-PCR (TaqMan). Freshly isolated hepatocytes from six 

separate human donors were cultured on collagen-coated plates and incubated with control 

inducers, vehicle controls, or test article for varying amounts of time up to 24 hours. The same 

was done for mouse and rat hepatocytes using 3 separate pools of 3 mice and 3 rats each. 

Samples at 6 and 24 hours were also taken to assay for Lactate Dehydrogenase (LDH) release as 

a marker of toxicity using the Promega CytoTox-ONETM Homogeneous Membrane Integrity 

System. Gene expression of Cyp1a1/CYP1A1 and Cyp1a2/CYP1A2 were analyzed using RNA 

prepared at the specified time points. Control induction experiments were conducted using a dose 

response of 3-methylcholanthrene (3-MC) and 1 mM phenobarbital (PB). The 3-MC controls 

were quantitated for CYP1A or Cyp1a expression levels and the PB samples were assayed for 

CYP2B or Cyp2b expression levels to monitor induction in the isolated hepatocytes. 

A dose responsive increase in primarily Cyp1a1/CYP1A1 was observed with the 3-MC positive 

control. Cyp1a1/CYP1A1 was more responsive compared to Cyp1a2/CYP1A2 with the mouse 

and human, but Cyp1a2 was induced to a greater extent in the rat. For Cyp1a1/CYP1A1

induction, mouse was the most sensitive, followed by the human where all but 1 donor had a 

higher CYP1A1 induction than the rat. The rat was most responsive to 3-MC with respect to 

Cyp1a2/CYP1A2 induction, the human was the next most responsive and the mouse had the least 

amount of Cyp1a2 induction. After treatment with another positive control, PB, there was 

induction of Cyp2b10 in the mouse, Cyp2b6 in the rat and CYP2B6 in human hepatocytes. The 

response in the mouse was modest and markedly lower than expected. No further studies were 

conducted to discern a cause for the dampened response; however, based on the robust increase 
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in Cyp1a mRNA levels in the treatment groups, this deficiency is not expected to invalidate the 

methods used in this study. Overall, the positive control results confirmed the validity and 

sensitivity of the assay for Cyp1a/CYP1A and Cyp2a/CYP2A induction in mouse, rat and human 

hepatocytes.

XDE-279 methyl was also able to induce Cyp1a1/CYP1A1 and Cyp1a2 /CYP1A2 expression in 

hepatocytes of all species, but at levels lower than the positive control. The rat was the most 

responsive to Cyp1a1/CYP1A1 and Cyp1a2 /CYP1A2 induction compared to mouse and human 

hepatocytes that had limited induction of Cyp1a1/CYP1A1 and Cyp1a2/ CYP1A2 with XDE-729 

methyl across multiple dose levels up to 100 μM and multiple time-points up to 24 hours 

compared to the positive control, 3-MC. All species were generally more sensitive to 

Cyp1a1/CYP1A1 induction compared to Cyp1a2/CYP1A2 induction following XDE-729 methyl 

exposure. Rat hepatocytes (most responsive to XDE-729 methyl) had Cyp1a1 and Cyp1a2

maximum inductions slightly lower in responsiveness compared to the positive control. Average 

induction at the 2 highest concentrations was 32-fold and 13-fold for Cyp1a1 and Cyp1a2, 

respectively. Human hepatocytes were less responsive than mouse and rat hepatocytes to 

Cyp1a1/CYP1A1 induction with an average of 4-fold induction increase at the two highest 

concentrations compared to negative controls but were slightly more responsive to Cyp1a2/

CYP1A2 induction compared to mice with an average of 1.5-fold increase at the two highest 

concentrations compared to negative controls. Peak expression of the examined Cyp1a1/CYP1A1

isoforms occurred from 8-12 hours. All species were generally more sensitive to

Cyp1a1/CYP1A1 induction compared to Cyp1a2/ CYP1A2 induction. No significant cytotoxic 

effect was observed in any species with any treatment as demonstrated by the lack of lactate 

dehydrogenase (LDH) leakage. 

This in vitro mechanistic assay in mouse, rat and human hepatocytes is acceptable/nonguideline

as supplemental data. It provides additional information to characterize the mode of action for 

liver toxicity from halauxifen-methyl.

In Vitro Hydrolysis Rates, Liver S9, Blood and Synthetic Gastric Fluid of Mouse, Rat, 

and Human and PBPK Simulations of Systemic Exposure in Rats and Humans

In this in vitro mechanistic study (PMRA 2226488, MRID 48557874), rates of metabolism of 

XDE-729 methyl (97.2%, Lot# E2837-51) to XDE-729 acid (95.3%, Lot # E2837-52) were 

determined by conducting in vitro incubations of the XDE-729 methyl in key physiological 

matrices (liver S9 cell fraction, whole blood, and synthetic gastric fluid (SGF)) of mouse, rat, 

and human. These rates were then incorporated into a whole-body, physiologically-based 

pharmacokinetic (PBPK) model to predict systemic exposures to XDE-729 methyl and XDE-729 

in blood and liver tissue of rats and human. 

XDE-729 methyl was rapidly hydrolyzed to XDE-729 acid by liver S9 of all three species at 

concentrations up to 10 μM. Metabolism of XDE-729 methyl by liver S9 appeared to follow 

(saturable) Michaelis-Menten kinetics; thus, Km and Vmax terms were derived to mathematically 

define the metabolism of XDE-729 methyl in liver tissue. The data demonstrated relative rates 

of metabolism decreasing in order of (fastest to slowest) human>>rat>mouse. The rate of 
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metabolism was so rapid in incubations with human liver S9 that XDE-729 methyl was not 

present at levels exceeding the limit of quantitation after 8 minutes of incubation. 

The mass balance data indicated that XDE-729 acid was the primary metabolite of XDE-729 

methyl, and furthermore, that there was minimal downstream metabolism, by liver S9, of XDE729 acid. This was also observed in analyses of whole blood.

XDE-729 methyl was metabolized in whole blood according to 1st order kinetics, with the 

relative rates between species following the order of (fastest to slowest) rat>mouse>>human. 

The rate of metabolism of XDE-729 methyl in SGF was the slowest of all test media evaluated. 

The fastest rate of conversion occurred in SGF at pH 1.2 (representing the pH of human GI 

tract), with a half-life of approximately 100 hours; again, this was much slower than the rates of 

metabolism determined for liver S9 and whole blood. 

The in vitro-derived rates were incorporated as parameters into a PBPK model, and the model 

adapted to simulate 28-day dietary exposures of XDE-729 methyl to rats and humans. The 

predicted hydrolysis of XDE-729 methyl was most dependent on the rate of metabolism in liver 

tissue for both species, accounting for ~60% of total hydrolysis in rat and >95% in human. The 

model simulations indicate that XDE-729 methyl and XDE-729 acid achieve their steady-state 

concentrations soon after exposure is begun, the levels clear rapidly from blood and liver 

following the cessation of exposure, and that neither XDE-729 methyl nor XDE-729 acid would 

be expected to accumulate in either liver or blood following exposures to XDE-729 methyl. In a 

model-derived 28-day simulation at 10 mg/kg bw/day peak concentrations (and 24 h AUCs) of 

XDE-729 methyl in liver and blood are comparable between rats and human. The peak 

concentrations from a simulated 28-day exposure to XDE-729 methyl at 0.0001 mg/kg bw/day, a 

relevant exposure level based on residue data, resulted in predicted levels of XDE-729 methyl in 

humans that are 10000- to 15000-times lower than the limits of quantitation (LOQs: in blood 

~0.01 μg XDE-729 methyl/g; in liver ~0.04 μg XDE-729 methyl/g).

This in vitro mechanistic study is considered acceptable/nonguideline as a supplemental study. It 

provides information used to identify a mode of action for liver toxicity of halauxifen-methyl.

Discussion of MOA and Human Relevance Framework Analysis

XDE-729 methyl induces rodent liver effects via a proposed aryl hydrocarbon receptor-mediated 

mode of action (MOA) through the following key events (1) presystemic liver exposure to XDE729 methyl, (2) aryl hydrocarbon receptor (AhR) activation with associated liver weight increase 

and hepatocyte hypertrophy, leading to (3) hepatocellular proliferation. These key events have 

been evaluated in a series of guideline toxicity and MOA studies aimed at characterizing the 

MOA for XDE-729 methyl-mediated liver effects. This document (PMRA 2226486, MRID 

48557875) represents the weight of evidence approach used to evaluate the data based upon the 

Bradford-Hill criteria, followed by subsequent application in a Human Relevance Framework 

(HRF). 

Following dietary administration to rodents, XDE-729 methyl is rapidly hydrolyzed to the 

primary metabolite XDE-729. Although XDE-729 methyl is an AhR activator, XDE-729 has 

been shown through both in vitro and in vivo assays not to induce AhR activation and associated 
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liver responses. Therefore, this MOA/HRF analysis is focused on XDE-729 methyl induced 

AhR-mediated effects in the liver. Presystemic exposure of the rodent liver to XDE-729 methyl 

results in a dose responsive increase in Cyp1a1 transcript levels as a sensitive biomarker for AhR 

activation. In addition, corresponding increases in liver weight and associated hepatocellular 

hypertrophy were observed. Supporting events for an AhR-mediated MOA for hepatocellular 

effects include ligand binding to the receptor, and a series of in vitro assays, which have 

collectively indicated that XDE-729 methyl is a weak AhR agonist. Presystemic liver exposure 

to XDE-729 methyl, followed by activation of rat AhR, leads to liver weight increases with 

hepatocellular hypertrophy and increases in hepatocellular proliferation. Importantly, these 

effects (i.e., measureable molecular and apical end points) occur at doses ≥50 mg/kg bw/day 

XDE-729 methyl with a clear threshold for liver adverse effects at a no-observed-adverse-effect 

level (NOAEL) of 10 mg/kg bw/day, and for Cyp1a1 induction, of 3 mg/kg bw/day, based on 

mild induction (52-fold) at 10 mg/kg/day in the absence of other observable liver effects. The 

data from these evaluations provides a high level of confidence that XDE-729 methyl induces 

rodent liver effects through an AhR-mediated MOA. 

Comprehensive toxicokinetic analysis in the course of the studies evaluated in this MOA/HRF 

determined that XDE-729 methyl is rapidly metabolized and excreted in the urine following 

dietary administration. Low levels of XDE-729 methyl were present in the liver following 

dietary administration; however, after a four or 28 day recovery period, there was no XDE-729 

methyl present in the livers of rats. 

A physiologically-based pharmacokinetic (PBPK) model for XDE-729 methyl exposure 

predicted similar levels of XDE-729 methyl in the livers of humans and rats at the overall 

NOAEL from the toxicity studies of 10 mg/kg bw/day, as well as the conservative estimated 

human exposure scenario of 0.0001 mg/kg bw/day. Although PBPK model outputs indicate that 

human liver would be exposed to XDE-729 methyl, the concentrations modeled are well below 

those that would cause hepatic effects based on rodent toxicity studies (~50 mg/kg bw/day). 

Furthermore, there is a quantitative difference of ~10-fold for binding affinity of the prototypical 

AhR ligand dioxin to human versus rodents AhR, which would suggest that animal models 

would likely be more sensitive to AhR-mediated effects than humans (Connor and Aylward, 

2006). In addition, the doses causing hepatic toxicity in rats are considered to be well above 

those that would cause hepatic effects in humans based on quantitative differences in sensitivity 

to AhR activation and corresponding toxicity (discussed below). 

Several in vitro studies for AhR receptor activation were also completed as part of the 

assessment of human relevance. A conclusion from the rodent MOA data and in vitro 

mechanistic studies is that humans would be significantly less sensitive than rat to XDE-729 

methyl based on the following: 1) kinetic analysis of XDE-729 methyl indicated this molecule is 

rapidly hydrolyzed to XDE-729 by human liver S9 and does not bioaccumulate in rat liver 

tissues in which hydrolysis is substantially slower, 2) in vitro transactivation and binding assays 

with XDE-729 methyl indicated that this molecule was a relatively weak ligand for rodent AhR 

and had no transactivation activity for human AhR, 3) at concentrations of XDE-729 methyl that 

elicited substantial induction of Cyp1a1 transcript levels in the rat, primary human hepatocytes 

had minimal induction of CYP1A1 and CYP1A2. In addition, a clear threshold for adverse liver 

effects exists for XDE-729 methyl in rodents with comprehensive MOA and mechanistic data 
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indicating that molecular and apical endpoints (Cyp1a1 induction, liver enlargement with 

associated hypertrophy, and hepatocellular proliferation) do not occur below the dose level of 10 

mg/kg bw/day XDE-729 methyl in 28 day to 90 day repeat dose dietary studies. The data 

identify a dose (3 mg/kg bw/day) at which only slight Cyp1a1 induction is observed and which is 

well below the LOAEL for liver toxicity (52 mg/kg/day). This dose is considered protective for 

long-term exposure since the initial event for liver toxicity is not observed.

The data from these evaluations provide a high level of confidence that XDE-729 methyl induces 

rodent liver effects through an AhR-mediated MOA. Although XDE-729 methyl was able to 

activate the AhR, it is a weak agonist that is rapidly metabolized; therefore, XDE-729 methyl is 

not likely to result in sustained activation of the AhR signaling pathway. In addition, all hepatic 

molecular and apical end points were reversible after short recovery periods. Analysis of 

toxicity, toxicokinetic and molecular end points following XDE-729 methyl administration 

indicated a clear threshold for liver effects. In vitro transactivation studies and gene expression 

studies in human liver cells also demonstrated minimal activation of the human AhR with 

exposure to XDE-729 methyl, which is coupled by the fact that in vitro hydrolysis studies using 

human and rat liver S9 determined that hydrolysis of XDE-729 methyl is faster in the human 

compared to the rat. Although the human relevance of an AhR-mediated MOA conservatively

cannot be excluded, the mechanistic and PBPK data strongly support the presence of a threshold 

to the key events in rodents that would be protective of human health.

XDE-729 ACID

A.4.1 Subchronic Toxicity

870.3050 28-Day Oral Toxicity - Rat

In a subchronic oral toxicity study (PMRA 2226460, MRID 48557825), XR-729 acid (99.0% 

a.i., lot no. E2350-93, TSN030751-0002) was administered to 5 Fischer 344 rats/sex/group in the 

diet at dose levels formulated to supply 0, 10, 50, 250 or 1000/500 mg/kg/day for at least 28 days

(time-weighted average concentrations: 0, 10.5, 52.7, 270 or 734 mg/kg/day for males and 0, 10, 

50.3, 250 or 982 mg/kg/day for females, respectively). In addition to the standard toxicity 

parameters, a toxicokinetic analysis of the test material in blood (collected at three time points) 

and urine (collected overnight) was performed during the final week of the study.

There were no treatment-related effects on survival, ophthalmoscopy, hematology, clinical 

chemistry or gross pathology. 

Males at 734 mg/kg/day had body weight loss and decreased food consumption (↓9% from 

controls) during the first eleven days of the study due to reduced palatability of the diet; 

therefore, the targeted dose level for these males was reduced from 1000 to 500 mg/kg bw/day 

(targeted doses) for Days 11-29 with an achieved mean dose of 734 mg/kg bw/day for both 

targeted dose levels. After the reduction in the dietary concentration, the amount of food 

consumed increased to control levels and although animals began to gain weight, overall, the 

high-dose male body weights were still lower than the controls throughout the remainder of the 

study. Females at 982 mg/kg bw/day also had lower body weights and food consumption values. 
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One of the males that exhibited decreased body weights also appeared thin upon clinical 

examination. High-dose males and females had higher urine volumes (♂ ↓84%, ♀↓41% from 

controls) and lower specific gravity (♂ ↓0.028, ♀↓0.014 from controls) than the controls. These 

alterations were associated with treatment-related kidney lesions. In addition, high-dose females 

had cloudy urine and crystalline phosphate in the urine sediment. Final body weights were lower 

in males and females from the high-dose groups. This was accompanied by increased relative 

kidney weights (♂ ↓6% from controls). The kidneys of high-dose males and females had 

treatment-related histopathological tubular effects, which consisted of tubular degeneration 

(incidences ♂ 0, 0, 0, 0, 5, ♀ 1, 0, 1, 0, 4 in the respective dose groups), and hypertrophy (all 

animals affected vs. 0 in control and lower dose groups) and vacuolization of collecting duct 

epithelium (incidences 1♂, 4♀ vs. 0 controls). Splenic extramedullary hematopoiesis was also 

observed in 4 high-dose males and one high-dose female. 

The LOAEL is 734 mg/kg bw/day in males and 982 mg/kg bw/day in females, based on 

decreased body weight, body weight gains and food consumption, increased urinary 

volume, decreased urine specific gravity, increased relative kidney (males only) weights, 

renal histopathology (degeneration and hypertrophy of renal tubules and vacuolization of 

collecting duct epithelium) and splenic extramedullary hematopoiesis. Females also 

exhibited cloudy urine and phosphate crystals in urine sediment. The NOAEL is 270 mg/kg

bw/day in males and 250 mg/kg bw/day in females.

Absorption of the XR-729 acid test material from the diet appeared to be slightly saturated at the 

top three dose levels in both sexes. The ingested XR-729 acid was rapidly eliminated from 

plasma upon the removal of the fortified diet (fasting) and dropped below the limit of 

quantification after ~16 hours of fasting for all dose groups. On Day 23 (day of blood collection) 

the calculated intake were 11, 53, 286, 562 mg/kg bw/day for males and 10, 47, 233, 925 mg/kg

bw/day for females. The increase in the systemic dose (AUC24h) vs. administered dose 

demonstrated approximately linear proportionality. Total urinary elimination of XR-729 acid and 

metabolites at 24 hours post-dosing demonstrated a slight decrease in female rats at the highest 

two dose levels of 233 and 925 mg/kg bw/day compared to the two lower dose groups. In total, 

male rats eliminated in urine an average of 53-63% of what they consumed during a 24-hour 

period, while elimination by females averaged 48-69%. Overall, these pharmacokinetic data 

show that urine is a major elimination pathway in rats following dietary ingestion of XR-729 

acid after 28 days of continuous exposure and indicate that at 24 hours post-dosing decreased 

elimination in urine at high doses in females. In addition to parent XR-729 acid, four 

metabolites (glucuronide conjugate of O-demethyl XR-729 acid, sulfate conjugate of Odemethyl XR-729 acid, O-demethyl XR-729 acid, and acyl-glucuronide conjugate of parent XR729 acid) were tentatively identified in urine. This identified metabolites were comparable to 

those observed following single dose oral gavage administration of XR-729 acid at 1000 mg/kg 

bw. 

This subchronic toxicity study in the rats is acceptable/non-guideline and satisfies the guideline 

requirement (OECD 407) for a repeat-dose oral study in rats.

870.3100 90-Day Oral Toxicity - Rat
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In a subchronic oral toxicity study (MRID 48557828, PMRA 2226467), 10 F344/DuCrl 

rats/sex/group were given test diets formulated to supply XDE-729 acid (96.5% a.i., lot no. 

E2978-05-01, TSN030571-0005) at concentrations of 0, 10, 50, 250, or 750 mg/kilogram body 

weight/day (mg/kg bw/day) for 93-94 days. Average intake of XDE-729 acid corresponded to 

time-weighted average concentrations of 0, 10.5, 52.4, 262, or 782 mg/kg bw/day for males and 

0, 10.1, 50.3, 252, or 758 mg/kg bw/day for females, respectively. In addition to standard 

guideline parameters, toxicokinetic analyses were performed on blood (sampled at three time 

points) and urine (collected overnight) sampled during the last week of treatment.

There were no treatment-related effects on survival, clinical signs, food consumption, 

hematology, clinical chemistry, ophthalmoscopic parameters or gross pathology. 

Treatment-related decreases in body weight (6%) and body weight gains (9%) were observed in 

high-dose males. Males at 782 mg/kg bw/day also had increased urinary volumes (♂ 162%, ♀ 

89%), decreased urine specific gravity (♂ 0.03, ♀ 0.02), and increased renal histopathological 

lesions including tubular dilatation and degeneration (incidences: 0, 0, 0, 0, 8). The relative 

testes, thyroid, kidney and brain weights in high-dose males were higher than the concurrent 

control values (7-9% each) and were considered to be secondary to the lower mean final body 

weight. Both sexes of rats at the high-dose level exhibited renal necrosis (incidences: ♂ 0, 0, 0, 

0, 3; ♀ 0, 0, 0, 0, 1), increased mitotic figures (incidences: ♂ 0, 0, 0, 0, 4), hypertrophy of the 

collecting duct epithelial cells (incidences: ♂ 0, 0, 0, 0, 10; ♀ 0, 0, 0, 0, 5) and vacuolization of 

the collecting duct epithelium in the papilla of the kidney (incidences: ♂ 0, 0, 0, 0, 10; ♀ 0, 0, 0, 

0, 8). The incidence and severity was increased in males compared to females. The high-dose 

females exhibited amorphous crystals in urine sediment at the urinalysis.

The LOAEL in males is 782 mg/kg bw/day, based on decreased body weight and body 

weight gains, increased urinary volume, decreased urine specific gravity and renal 

histopathology (necrosis, increased mitotic figures, hypertrophy of collecting duct epithelial 

cells, vacuolization of collecting duct epithelium in the papilla of the kidney, tubular 

dilatation and degeneration). The LOAEL in females is 758 mg/kg bw/day, based on 

increased amorphous crystals in the urine sediment and renal histopathology (necrosis, 

increased mitotic figures, hypertrophy of collecting duct epithelial cells, vacuolization of 

collecting duct epithelium in the papilla of the kidney). The NOAEL is 262 mg/kg bw/day 

in males and 252 mg/kg bw/day in females. 

Based on the plasma concentrations of XDE-729 acid, the absorption and systemic 

bioavailability of XDE-729 acid were dose-proportional and comparable between both male and 

female rats. The amount of XDE-729 acid eliminated in the urine of male rats increased 

proportionally with dose; however, there was sublinearity in urinary excretion of test material in 

females between the 252 and 758 mg/kg bw/day.

This subchronic oral toxicity study in the rats is classified as acceptable/guideline and satisfies 

the guideline requirement for a subchronic toxicity study (OPPTS 870.3100; OECD 408) in rats. 

870.3050 28-Day Oral Toxicity – Mouse
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In a subchronic oral toxicity study, XR-729 acid (MRID 48557827; PMRA 2226464) (99.0% 

a.i., lot no. E2350-93, TSN030751-0002) was administered by diet to 5 Crl:CD-1 (ICR)

mice/sex/group at target concentrations of 0, 10, 50, 250 or 1000 mg/kg bw/day for 31 days 

(time-weighted average concentrations: 0, 10.2, 54.0, 269 or 1025 mg/kg bw/day for males and 

0, 9.9, 48.7, 241 or 958 mg/kg bw/day for females, respectively). Steady-state concentrations of 

the parent chemical in the blood were determined to characterize the relationship between the 

oral dose and internal systemic dose. Recovery of parent chemical in the urine was measured to 

characterize the relationship between the oral dose and the efficacy and dose proportionality of 

urinary elimination. Potential metabolites were not characterized in this study. 

There were no mortalities or clinical signs and no treatment-related effects on body weight, body 

weight gain, food consumption, or ophthalmology. In females there were no treatment-related 

changes in hematological parameters, organ weights, and no treatment-related gross or 

histopathological findings. In both sexes, the internal systemic dose in the blood plasma was 

proportional to the oral dose across all dose levels. Similarly, there was no overt effect of 

treatment dose level on the apparent fraction of the oral dose that was eliminated via the urine. 

Decreased urinary elimination of XR-729 acid at the high dose in males was considered an 

indication of a slight non-adverse effect on kidney function. There was no evidence of saturation 

effects in either the steady-state systemic exposure or the daily urinary elimination of the parent 

chemical.

In contrast to females, males dosed at 1000 mg/kg bw/day had non-adverse treatment-related 

decreases in red blood cell count (8%) and increases in the mean cell volume of red blood cells 

(RBC) (7%). The US EPA considered the RBC findings as a slight treatment-related effect but 

also noted that RBC findings were not observed in the 90-day mouse study and were not 

evaluated in the oncogenicity study. In both males and females there was a slight treatmentrelated decrease in serum ALT levels (♂ 33%, ♀ 26%) at the highest dose tested; this finding 

was considered to be treatment-related by Canada PMRA and Australia OCS. In males receiving 

1000 mg/kg bw/day, decreased kidney weight (absolute 12%, relative 12%) was concordant with 

an increased incidence of kidney tubule degeneration. The males at this dose level also exhibited 

increased incidences of multifocal karyomegaly (incidence of 1 vs. 0 in controls) and focal 

necrosis (incidence of 2 vs. 0 in controls) in the liver.

The LOAEL is 1025 mg/kg/day in males, based on decreased alanine aminotransferase 

activity (Canada PMRA/Australia OCS), decreased numbers and mean volume of red 

blood cells (US EPA), decreased weight and an increased incidence of tubule degeneration 

in the kidney (Canada PMRA/Australia OCS), and increased incidences of multifocal 

karyomegaly and focal necrosis in the liver. The LOAEL is 958 mg/kg bw/day in females, 

based decreased alanine aminotransferase activity. The NOAEL is 269 mg/kg bw/day in 

males and 241 mg/kg bw/day in females.

This subchronic toxicity study in the mice is classified as acceptable/guideline and satisfies the 

guideline requirement (OECD 407; OPPTS 870.3050) for a repeat-dose oral study in mice.

870.3100 90-Day Oral Toxicity – Mouse
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In a subchronic toxicity study (MRID 48557829; PMRA 2226468), ten male and ten female 

Crl:CD1(ICR) mice per group were given test diets formulated to supply 0, 50, 250, 500 1000 

milligrams XDE-729 acid (96.5% a.i., lot no. E2978-05-01/TSN030751-0005) per kilogram 

body weight per day (mg/kg bw/day) for at least 90 days which corresponded to time-weighted 

average doses of 0, 49.4, 247, 495, or 989 mg/kg bw/day for males and 0, 50.2, 258, 512, or 

1008 mg/kg bw/day for females, respectively. Parameters evaluated were daily cage-side 

observations, weekly detailed clinical observations, ophthalmic examinations, body weights, 

food consumption, blood and urine collection for toxicokinetics, hematology, clinical chemistry, 

organ weights, and gross and histopathological examinations.

There were no treatment-related effects on survival, clinical signs, body weight, food 

consumption, hematology, clinical chemistry, organ weights or gross pathology.

In high-dose males, treatment-related urinary bladder toxicity [acute inflammation (incidences: 

0, 0, 0, 0, 2), submucosal edema (incidences: 0, 0, 0, 0, 2), hyperplasia of transitional epithelium

(incidences: 0, 0, 0, 0, 1), multiple foci of ulceration (incidences: 0, 0, 0, 0, 1), crystals

(incidences: 0, 0, 0, 0, 1)] was observed and in one male, was associated with seminal vesicle 

acute unilateral inflammation. No urinary bladder effects were observed in females at any dose 

level. An increase in the proportion of neutrophils in the blood (↑31%) was observed in 989 

mg/kg bw/day males and was considered secondary to urinary bladder inflammation. 

The LOAEL is 989 mg/kg bw/day, based on urinary bladder histopathology and a 

secondary associated increase in neutrophils in males. The LOAEL in females was not 

established, as there were no adverse effects noted up to the limit dose. The male NOAEL is 

495 mg/kg bw/day and the female NOAEL is 1008 mg/kg bw/day.

Linear kinetics were observed in the plasma and urine after XDE-729 acid exposure, although 

high variability was noted in the female 512 mg/kg bw/day urine group. On average, 32-47% of 

the XDE-729 acid dose was excreted in the 24 h urine. There were no apparent sex differences in 

the toxicokinetic parameters examined.

This subchronic toxicity study in the mice is classified as acceptable/guideline and satisfies the 

guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in mice. 

Nonguideline Oral Rangefinding – Dog

In a palatability and range-finding toxicity study (MRID 48557871, PMRA 2226465), XR-729 

acid (99.0% a.i., lot no.E2350-93, TSN030751-0002) was administered to Beagle dogs in diet at 

dose levels of 0 or 7500 ppm for 3-day consecutive exposure periods during the palatability 

phase. Initial dose levels were subsequently escalated or de-escalated to assess palatability and 

determine dose levels for the 28-day phase. At 30000 ppm during the palatability phase, the 

females did not eat sufficient quantities, therefore, the females had a de-escalation to decrease 

the test material concentration to 22500 ppm. During the palatability phase, males that received 

30000 ppm and females that received ≥ 22500 ppm failed to maintain consistent body weight 

and food consumption during three day periods of exposure. The effects among females that 

received ≥ 22500 ppm were considered to be of sufficient severity to preclude the use of these 

concentrations during subsequent studies, whereas the effects among males that received 30000 
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ppm were not. Therefore, concentrations for the subsequent 28-day exposure phase were set at 

300, 3000, and 30000 ppm in males and 300, 3000, and 15000 ppm in females. Therefore, 

Beagle dogs were selected from the various palatability dosing groups and 2/sex/dose were 

administered XR-729 acid at 0, 300, 3000, 15000 (females only) or 30000 (males only) ppm (0, 

11, 80, 635 mg/kg bw/day in males; 0, 9, 85, 323 mg/kg bw/day in females) for 28 consecutive 

days for the main study. In addition, toxicokinetics were evaluated in blood and urine samples 

collected at 1, 2, 6 and 16 hours post-dose on Days 27 and 16 hours after food removal on Day 

29, and in the 30000 ppm males on Day 24.

There were no treatment-related effects on survival, ophthalmoscopic parameters, or gross 

pathology. 

Clinical signs of toxicity observed in both high-dose males were atypical muscle tone in one 

male and atypical palpebral closure in the other during week 4 of the study. The study authors 

considered the clinical signs to be non-adverse since they only occurred during Week 4; 

however, given the other treatment-related effects observed, the adversity of these observations 

cannot be excluded (Canada PMRA/US EPA). These clinical signs were not considered to be 

treatment-related by the US EPA, as there were only single findings in the 4th study week and 

they were not observed in the 90- or the 1-year dog studies.

The high dose males had lower body weights (↓29% overall) compared to controls and lower 

dose groups and both high dose males and females lost body weight over the duration of the 28-

day phase. Body weight loss was more marked in high dose males. The body weight loss was 

associated with decreased food consumption (↓29-100% from controls) in high dose males and 

females over the duration of the 28-day phase and the effect is considered to be associated with 

decreased palatability of the diet.

Both males at 635 mg/kg bw/day had increased total and HDL-cholesterol levels at termination. 

One of the high dose males (animal #113) had decreased red blood cell, hemoglobin, hematocrit 

and reticulocyte counts, increased LDL-cholesterol, increased bilirubin. The other high-dose 

male had increased AST levels. The above alterations may be secondary to the marked decrease 

in food consumption and associated body weight loss in high-dose males in the palatability phase 

of the study and during the 28-day phase.

At the high dose level, thymic atrophy (1/sex vs. 0 in controls) were observed. The thymic 

atrophy was attributed to stress by the study authors; however, no supporting information was 

submitted. In the absence of evidence to the contrary, this finding was considered to be 

treatment-related by Canada PMRA and Australia OCS; this observation was considered to be a 

secondary effect of treatment by US EPA. At ≥80 mg/kg bw/day in males and ≥323 mg/kg 

bw/day in females, treatment-related degeneration and regeneration of renal tubules was 

observed.

Canada PMRA/Australia OCS: The palatability phase of this study identified dose levels 

for the main study which used only 2 Beagle dogs/sex/dose with animals selected from the 

palatability study. Therefore, it is considered that no reliable identification of 

LOAEL/NOAEL values can be made from the main study, which itself is considered is a 

dose range finding study for a subsequent 90-day dog study [PMRA 2226469, MRID 
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48557831]. Consequently, a NOAEL/LOAEL has not been identified from this 

palatability/dose range-finding study which used the same animals in each phase.

US EPA: A NOAEL and LOAEL were selected for purposes of comparison to the definitive 

subchronic study. The LOAEL is 80 mg/kg bw/day in males, based on degeneration and 

regeneration of renal tubules and 323 mg/kg bw/day in females, based on body weight loss, 

reduced food consumption, thymic atrophy and degeneration and regeneration of renal 

tubules. The NOAEL in males is 11 mg/kg bw/day and 85 mg/kg bw/day in females.

The toxicokinetic analyses conducted after 24 or 27 days of dietary exposure indicated daily 

systemic doses (AUC24 h) of 10, 79, 331 mg/kg bw/day (male) and 9, 78, and 264 mg/kg bw/day 

(female) was 16, 185, and 5893 μg h mL

-1

in male and 26, 167, and 1900 μg h mL

-1

in female 

dogs, respectively. Large variation in the plasma concentrations was observed and was 

attributed to variability in the feeding pattern amongst the dogs. In male dogs, the increase in the 

systemic dose was dose-proportional up to the mid-dose level, but became more than 

dose-proportional at the high-dose level. The more than dose proportional increase in the 

systemic dose at the highest dose in male dogs is consistent with saturation of elimination of XR729 acid and was also observed at the highest dose in females to a lesser extent. In female dogs, 

the daily systemic dose increased proportional to dose up to the mid-dose level. These data were 

confounded by the high variability between the blood levels from the two female animals and 

thus, a definitive statement on the linearity of systemic doses across all dose groups cannot be 

made based on these data alone. 

Urinary elimination of XR-729 acid was 13, 49 and 53 % in male and 41, 70, and 44 % in female 

dogs at the low, mid and high doses, respectively. Female dogs were more efficient in 

eliminating XR-729 acid than male dogs. Based on the large variation in the plasma 

concentrations it is considered that definitive statements on the proportionality of urinary 

elimination with administered dose cannot be made from the study results.

This study in dogs is acceptable/nonguideline as a range-finding study. 

870.3150 90-Day Oral Toxicity – Dog

In an oral 90-day dog study (MRID 48557831; PMRA 2226469), XDE-729 acid (96.5% a.i. lot 

no. E2978-05-01, TSN) was administered to four Beagle dogs/sex/group in the diet at dose levels 

of 0, 500, 2500 or 12500 ppm (equal to 0, 16.75, 80.64 or 423.81 mg/kg bw/day in males and 0, 

18.71, 79.88 or 414.74 mg/kg bw/day in females, respectively). 

There were no treatment-related effects on survival, clinical signs, clinical chemistry or 

ophthalmoscopic parameters, or gross pathology. 

At 414.74 mg/kg bw/day, there was a treatment-related decrease in body weights (♂ ↓12-16%; ♀ 

8-10% from controls), body weight gains in males and to a lesser extent females (♂ ↓68%; ♀ 

61% from controls). In females, treatment-related reduced food consumption (↓20% from 

controls) was observed but was not completely attributable to the marked decrease in body

weight gain compared to controls. The decreased body weight gain in males and females at the 

high dose and decreased food consumption in females at this dose was considered to be adverse.
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In both sexes at the high-dose level, there were treatment-related decreases in erythrocytes (♂ 

↓20%, ♀ 23% from controls), hemoglobin (♂ ↓22%, ♀ 18% from controls) and/or hematocrit (♂ 

↓19%, ♀ 17% from controls) at Week 13 relative to concurrent controls and pre-test values. 

There were slight non-statistically significant increases in reticulocyte counts (♂ ↓32%, ♀ 35-

190% from controls) at Weeks 7 and/or 13 compared to concurrent controls and pre-test values 

in males and females. 

In females, these changes were associated with bone marrow hyperplasia and increased 

myelopoiesis identified in histopathological examinations. One of the high-dose females had 

increased band neutrophils and reactive lymphocytes, while the other high-dose female lacked 

mature neutrophils, had reduced band neutrophils, as well as increased reactive lymphocytes and 

monocytes indicative of ineffective myelopoiesis and maturation arrest at band neutrophil stage. 

Increased liver weights (♂ ↑38%, ♀ 51%) accompanied by extramedullary hematopoiesis in the 

liver and spleen was also observed in one of the high-dose females and was considered to be 

potentially adverse.

An additional target organ of toxicity was the kidney in both sexes. Increased incidences of 

degeneration and regeneration of renal tubules, degeneration of collecting ducts, and focal 

granulomas in the renal cortex or medulla were observed in both sexes at the high dose. 

Additionally, at the high-dose level, 3 males (#130/10A2918, #131/10A2919, #132/10A2920) 

and 2 females (#126/10A2934, #127/10A2935) had treatment-related decreased thymus weights 

compared to controls. This change in organ weight was accompanied with thymic atrophy upon 

histological examination in all cases except for male #130. These effects were attributed to stress 

by the study authors; however, no supporting information was submitted. In the absence of 

evidence to the contrary, these findings were considered to be treatment-related by Canada 

PMRA and Australia OCS; the US EPA considered this to be a secondary effect of treatment. 

The LOAEL is 423.81/414.74 mg/kg bw/day in males/females, based on decreased body 

weights (Canada PMRA/US EPA), decreased body weight gains, reduced food 

consumption (females), reduced red cell parameters (red blood cells, hemoglobin, 

hematocrit levels) and associated bone marrow hyperplasia characterised by an increase in 

myelopoiesis (females), decreased thymus weights and associated thymic atrophy, renal 

toxicity (including degeneration and regeneration of renal tubules) and increased liver 

weight and associated extramedullary hematopoiesis of liver and spleen (females). The 

NOAEL in males is 80.64 mg/kg bw/day and 79.88 mg/kg bw/day in females. 

Toxicokinetic analysis of XDE-729 acid in plasma during week 13 of dietary administration 

showed that the elimination half-life (t1⁄2) of XDE-729 was 2.37 hours in males and 4.89 hours in 

female dogs of the high-dose group. The elimination t1⁄2 could not be calculated for the mid- and 

low-dose groups because plasma concentrations at 27 hours were below the limit of quantitation 

in those groups. The daily systemic doses (AUC24h) for males in the low-, mid- and high-dose 

groups were 41, 183, and 2917 μg h/ml, respectively. The AUC24h for females in the low-, midand high-dose groups were 44, 212 and 3794 μg h/ml, respectively. When plotted against dose, 

the AUC24h values exhibited a linear relationship for the low- and mid-dose groups but appeared 

to become non-linear at the high dose with a more than dose proportional increase. This 

nonlinearity was proven statistically in females but not in males. 
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The total dose excreted in 24 hour urine as a percentage of the administered dose was 102.2, 72.6 

and 28.6% in males and 105.8, 47.0 and 32.1% in females for the low-, mid- and high-dose 

groups, respectively. Analysis of XDE-729 acid in excreted 24 hour urine across all dose levels 

showed the renal elimination of XDE-729 was sub-linear at the high dose in male dogs, as 

proven statistically. In female dogs, XDE-729 acid excreted in 24 hour urine showed a sublinear trend but, due to high variation among animals, non-linearity could not be proven by 

statistical analysis. 

The kinetics of XDE-729 acid in plasma and urine of male and female dogs during week 13 of 

dietary exposure appeared to be consistent with kinetics expected with the occurrence of 

saturation of elimination at the high dose. The supraproportional increase in systemic exposure 

and decrease in urinary elimination at high doses is consistent with observations in rats following 

a single oral dose of the salt and methyl forms [Hansen et al., 2010; PMRA 2226446] and in the 

28-day palatability and probe study in dogs [Popke, 2010; PMRA 2226465]. However, kinetic 

nonlinearity in the daily systemic doses (AUC24h) could be proven statistically only for females, 

and kinetic nonlinearity in the total dose excreted in 24 hour urine could be proven statistically 

only in males.

This subchronic toxicity study in the dogs is classified as acceptable/guideline, and satisfies the 

guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in dogs. 

870.3200 21/28-Day Dermal Toxicity – Rat

In a repeat-dose dermal toxicity study (MRID 48557834, PMRA 2226471), XDE-729 acid

(95.3% a.i.) suspended in 0.5% aqueous methylcellulose vehicle (dosing volume 4 mL/kg bw)

was applied to the shaved skin of 10 F344/DuCrl rats/sex/dose at dose levels of 0, 100, 300 or 

1000 mg/kg bw/day, 6 hours/day for 7 days/week during a 28-day period. The treatment site was 

semioccluded during exposure.

There were no treatment-related effects on survival, clinical signs, body weights, food 

consumption, ophthalmoscopic parameters, hematology, clinical chemistry, urinalysis, organ 

weights, gross pathology or histopathology. 

The LOAEL was not established, as no treatment-related effects were observed up to a 

limit dose. The NOAEL is 1000 mg/kg bw/day.

This 28 day dermal toxicity study in rats is classified as acceptable/guideline and satisfies the 

guideline requirement (OPPTS 870.3200, OECD 410) for a 21/28 day repeat-dose dermal 

toxicity study in rats.

870.3465 90-Day Inhalation – Rat

Study not conducted due to inability to generate acceptable test atmosphere

A.4.2 Prenatal Developmental Toxicity
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870.3700a Prenatal Developmental Toxicity Study - Rat

In an oral developmental toxicity study (PMRA 2226500, MRID 48557835), groups of 26 time-mated 

female Crl:CD(SD) rats were administered diets containing 0, 500, 2000, or 8000 ppm XDE-729 acid 

(95.3% a.i., lot no. E2837-52, TSN-030751-0006) on gestation day (GD) 6 through 21 (equal to 0, 

34.1, 140, or 526 mg/kg bw/day, respectively). In-life maternal study parameters included clinical 

observations, body weight, body weight gain and feed consumption. On GD 21, all rats were 

euthanized and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights 

were recorded, along with the number of corpora lutea, uterine implantations, resorptions and 

live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. 

Approximately one half of the fetuses were examined for visceral and craniofacial alterations, while 

skeletal examinations were conducted on the remaining fetuses.

There were no treatment-related effects on maternal liver weights or gross pathology. Two 

maternal animals at 526 mg/kg bw/day were sacrificed prematurely for humane reasons on 

gestation days 17 or 18 due to excessive body weight losses, reduced food consumption and 

decreased fecal output. These mortalities were considered to be treatment-related. Increased 

incidences of decreased fecal output (4 females) and perinasal red soiling (2 females) were noted 

at 526 mg/kg bw/day. Decreased body weight (GD 21; 6%), body weight gains (37%) and food 

consumption (6-18%) were observed throughout the study at the high dose level, but most 

notably during the last few days of gestation. Treatment-related decreases in gravid uterine 

weights (10%) and increased relative kidney weights (11%) were also observed at 526 mg/kg 

bw/day. 

The maternal LOAEL is 526 mg/kg bw/day based on increased mortality, decreased fecal 

output and increased perinasal red soiling, decreased body weight, body weight gains and 

food consumption, decreased gravid uterine weights, and increased relative kidney weights. 

The maternal NOAEL is 140 mg/kg bw/day.

No treatment-related external or visceral malformations or variations were observed. There was 

no evidence of treatment-related skeletal malformations in any dose group. In the 526 mg/kg/day 

group, treatment-related decreased fetal weights (3-5%), increased total resorptions (incidences: 

8, 13, 16, 22), resorptions per dam (0.9 vs. 0.3 in controls) and post-implantation loss (*2.2, 3.6, 

4.5, 6.2%), and increased incidences of delayed ossification of thoracic centra (designated a 

developmental variation; fetal(litter) incidences: 2(1), 1(1), 2(1), 7(7)) were observed. 

The developmental LOAEL is 526 mg/kg/day based on marginal effects including 

decreased fetal weights, increased total resorptions, resorptions per dam and postimplantation loss, and increased incidences of delayed ossification of thoracic centra 

(designated a developmental variation). The developmental NOAEL is 140 mg/kg/day.

The developmental toxicity study in the rat is as classified acceptable/guideline and satisfies the 

guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.

Nonguideline Prenatal Developmental Rangefinding Study – Rabbit
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In a developmental toxicity study (PMRA 2226504, MRID 48557837), groups of 26 time-mated 

female New Zealand White rabbits were administered diets containing 0, 4000, 10000, or 28292 

ppm XDE-729 acid (95.3%, lot no. E2837-52, TSN030751-0006) on gestation days (GD) 7-28 

which corresponded to time-weighted average doses of 0, 170, 434, or 1094 mg/kg body 

weight/day, respectively. In-life parameters evaluated for all groups included clinical 

observations, body weight, body weight gain, and food consumption. On GD 28, all surviving 

rabbits were euthanized and examined for gross pathological alterations. Liver, kidneys, and 

gravid uterine weights were recorded, along with the number of corpora lutea, uterine 

implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and 

examined for external and visceral alterations. The heads were also examined for craniofacial 

alterations by serial sectioning in approximately one half of the fetuses in each litter, while 

skeletal examinations were performed on all fetuses. Plasma concentrations of XDE-729 acid 

were measured in pregnant does and their respective litters on GD 28 to confirm systemic 

exposure and evaluate dose-proportionality.

Maternal toxicity: There were no treatment-related effects on maternal survival, clinical signs, 

gross pathology, terminal body weights, kidney, liver or gravid uterus weights, or Caesarean 

section parameters. Treatment-related decreases in body weight gains (14%) and food 

consumption (5-20%) were observed during gestation. 

The maternal LOAEL is 1094 mg/kg bw/day, based on decreased body weight gains and 

food consumption during gestation. The maternal NOAEL is 434 mg/kg bw/day. 

Developmental toxicity: There were no treatment-related effects on fetal survival or body 

weight, and no developmental malformations or variations upon external, visceral, craniofacial 

or skeletal examination. 

The developmental LOAEL was not established. The developmental NOAEL is 1094 mg/kg

bw/day. 

The mean terminal plasma concentrations of XDE-729 acid in both dam and fetal blood 

demonstrated systemic exposure. On average across all dose groups, fetal XDE-729 acid plasma 

concentrations were 3.0±0.9-fold lower than doe plasma concentrations (i.e. fetal plasma 

concentrations were 26–47% of doe plasma concentrations). Analysis of kinetic dose 

proportionality was not possible due to high variability between animals in each dose group, 

likely attributable to a combination of XDE-729 acid’s short plasma half-life in the rabbit (1.08 

h) and maternal feeding patterns at the end of pregnancy.

This study is classified as acceptable/guideline and satisfies the guideline requirement for a 

Prenatal Developmental Study – Rabbits; OPPTS 870.3700; OECD 414. 

A.4.3 Reproductive Toxicity

Nonguideline Dose Range-Finding Study – Rat
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In an oral reproductive toxicity probe dose range-finding study (MRID 48557880; PMRA 

2226495), groups of 12 male and 12 female Crl:CD(SD) rats were given test diets initially 

formulated to supply XDE-729 acid (96.5% a.i., lot no. E2978-05-01, TSN030751-0005) at 

doses of 0, 50, 250, 750, or 1000 mg/kg bw/day. The time-weighted doses were 0, 53.4, 266 or

754 mg/kg bw/day in males and 0, 53.9, 269 or 535 mg/kg bw/day, respectively. Males were fed 

the test diets for four weeks prior to breeding, continuing throughout breeding, and until 

necropsy on test day 47. The females were fed the test diets for four weeks prior to breeding, 

continuing through breeding (up to two weeks), through gestation (three weeks) and until 

necropsy on lactation day (LD) 14. Effects on gonadal function, mating behavior, conception, 

development of the conceptus, parturition and postnatal growth and survival were evaluated. In 

addition, a gross necropsy and histopathological examination of the liver and kidney of adults 

were conducted. Plasma concentrations of XDE-729 acid were measured in adult males on days

15 and 28, and in adult females and their culled pups on day 4 postpartum to confirm systemic 

exposure and evaluate dose-proportionality.

Parental toxicity: During the first week of treatment, males and females given a targeted dose of 

1000 mg/kg bw/day had lower food consumption, body weights, and fecal output relative to 

controls, with 4/12 males and 4/12 females losing weight. This level of toxicity exceeded the 

maximum tolerated dose and was considered excessive for use in subsequent studies. Therefore, 

the 1000 mg/kg bw/day group was terminated on day 8 with no further collection of data.

In the 754/535 mg/kg bw/day M/F mg/kg/day group, 1/12 males and 4/12 females lost weight 

between days 7-14. Therefore, the targeted dose level for both sexes was reduced from targeted 

750 to 500 mg/kg bw/day for the final two weeks of the pre-breeding period. One female 

continued to lose body weight and was removed from the study for humane reasons on day 20. 

Upon necropsy, this animal exhibited decreased ingesta in the gastrointestinal tract and a 

decreased amount of adipose tissue grossly and numerous adverse renal histological lesions; 

therefore, this mortality was considered to treatment-related. In the remaining females, gestation 

body weight and body weight gain was decreased, while there were no effects on food 

consumption, body weights, or body weight gains in males or lactating females. 

In parental animals, there were no treatment-related effects on kidney or liver weights. 

The primary target organ associated with dietary exposure to XDE-729 acid was the kidney in 

males and females given 754/535 mg/kg/day M/F. Treatment-related histopathological 

alterations consisted of 1) multifocal hypertrophy (incidences: ♂ 0, 0, 0, 6; ♀ 0, 0, 0, 10) and 

hyperplasia (incidences: ♂ 0, 0, 0, 3; ♀ 0, 0, 0, 9) of the epithelial cell lining of the kidney 

collecting ducts with accompanying nuclear karyomegally, 2) necrosis of individual collecting 

duct epithelial cells (incidences: ♂ 0, 0, 0, 2), 3) increased mitotic figures (mitotic alteration) 

within the collecting ducts (incidences: ♀ 0, 0, 0, 1), and 4) tubular dilatation (incidences: ♀ 0, 0, 

0, 5). Lesions were more prominent in females than males at the high-dose level. There was 

significant variability in the occurrence and severity of the treatment-related effects observed in 

individual animals at the high-dose level. 

Reproductive toxicity: No reproductive toxicity was observed.

Offspring toxicity: In offspring, there were no treatment-related effects on clinical signs, litter 

size or pup body weights. 
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Canada PMRA, Australia OCS: Maternal, reproductive and offspring NOAELs and 

LOAELs were not established in this range-finding study.

US EPA: NOAELs/LOAELs were identified for comparison with the definitive study. 

Parental systemic: The LOAEL is 754/535 mg/kg bw/day M/F, based on decreased 

gestation body weight and weight gain, kidney histopathology (multifocal 

hypertrophy/hyperplasia of collecting duct epithelial cell lining, nuclear karyomegally, 

necrosis of individual collecting duct epithelial cells, increased number of mitotic figures 

within collecting duct cells and tubule dilatation with inflammation). The NOAEL is 

255/269 mg/kg bw/day M/F. Reproductive: The LOAEL was not determined, as no dose 

related effects were seen. The NOAEL is 754/536 mg/kg bw/day M/F. Offspring: The

LOAEL was not determined, as no dose related effects were seen. The NOAEL is 754/536 

mg/kg bw/day M/F.

Blood samples collected from adult males (days 15 and 28) and females (LD 4) and PND 4 

culled pups indicated no deviation from linearity for systemic exposure of the test material. 

Following the reduction of the high-dose level, the mean plasma concentration was 

proportionately reduced and systemic linearity was maintained as demonstrated by the day 28 

plasma concentrations falling on the regression line. 

In PND 4 culled male and female pups, mean plasma XDE-729 concentrations were 9 and 11%, 

respectively, of the dam plasma levels. Interpretation of the relative pup plasma concentrations 

should be limited to a demonstration of exposure as the time elapsed between the last nursing 

event and pup blood collection is unknown (potentially several hours) and the molecule has a 

very short plasma half-life (t1⁄2α = 0.14 h).

This reproductive/developmental toxicity screen is classified as acceptable/non-guideline as a 

range-finding study; it was conducted as a dose range-finding study but does not meet the 

requirements for a Reproduction/Developmental Probe Study -- Rats (OPPTS 870.3550, OECD 

421).

870.3800 Reproduction and Fertility Effects – Rat

In an oral two-generation reproductive toxicity study (PMRA 2226496, MRID 48557841), groups of 

27 male and female Crl:CD(SD) rats were administered diets supplying XDE-729 acid (95.3% a.i., 

lot no. E2837-52, TSN030751-0006) at concentrations equivalent to target doses of supplying 0, 20, 

100, or 450 mg XDE-729 acid/kg bw/day for ten weeks prior to breeding, and through breeding, 

gestation and lactation for two generations. The time-weighted doses for the F0 generation were 0, 

20.7, 104 or 465 mg/kg bw/day in males and 0, 20.5, 103 or 465 mg/kg bw/day in females. The 

time-weighted doses for the F1 generation were 0, 20.3, 101 or 459 mg/kg bw/day in males and 0, 

19.5, 97.7 or 443 mg/kg bw/day in females. In-life parameters included clinical observations, food 

consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body 

weights, and puberty onset. In addition, post-mortem evaluations included gross pathology and 

organ weights in weanlings, and gross pathology, histopathology, organ weights, oocyte quantitation 

and sperm count, motility and morphology in adults.
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Parental toxicity: In parental animals, there were no treatment-related effects on survival, 

estrous cyclicity, sperm parameters, reproductive performance or ovarian follicle counts. At the 

high-dose level, treatment-related clinical signs including decreased quantity and abnormal 

colour of feces, periocular soiling, dehydrated skin, pale mucous membranes and a thin

appearance were observed in two females in late gestation and early lactation; these animals also 

had decreased body weight gains (7%) and food consumption (10%) from GD 14-21. Treatmentrelated effects at the high-dose level included increased kidney weights (absolute/relative 

10%/7%) in females and renal histopathology in both sexes. Renal toxicity was characterized by 

increased incidences of collecting duct epithelial cell hypertrophy (incidences: F0 ♂ 1, 0, 0, 10, 

♀ 0, 0, 0, 17; F1 ♂ 1, 0, 1, 16, ♀ 0, 0, 0, 19) and hyperplasia (incidences: F0 ♂ 0, 0, 0, 9, ♀ 0, 0, 

0, 9; F1 ♂ 1, 0, 1, 12, ♀ 0, 0, 0, 8), hyperplasia of the pelvic epithelium (incidences: F1 ♂ 0, 0, 0, 

6) and increased mitotic figures (incidences: F0 ♂ 0, 0, 0, 2, ♀ 0, 0, 0, 1; F1 ♂ 0, 0, 0, 6) in the 

collecting duct epithelium. Other adverse histological observations included increased 

incidences of mineralization of the aorta (incidences: F1 ♀ 1, 0, 0, 8), renal arteries (incidences: 

F0 ♀ 0, 0, 1, 2; F1 ♀ 0, 0, 0, 4), renal tubular basal lamina (incidences: F1 ♀ 0, 0, 0, 5) and

mammary gland artery (incidences: F1 ♀ 0, 0, 0, 2). 

The parental LOAEL is 465/465 mg/kg bw/day M/F, based on renal histopathological 

observations (collecting duct epithelial cell hypertrophy and hyperplasia, hyperplasia of 

pelvic epithelium, mitotic figures of the collecting duct epithelium) in both sexes; clinical 

signs, decreased body weight gains and food consumption during late gestation/early 

lactation, increased kidney weights, increased incidences of mineralization of the aorta, 

renal arteries, renal tubular basal lamina and mammary gland artery in females. The 

parental NOAEL is 104/103 mg/kg bw/day M/F.

Reproductive toxicity: There was no evidence of reproductive toxicity. 

Offspring toxicity: In offspring, there were no adverse effects on viability, clinical signs, litter 

parameters, body weights, sexual maturation, organ weights or gross pathology. 

The reproductive and offspring LOAELs were not established (>443 mg/kg bw/day). The 

reproductive and offspring NOAELs are 443 mg/kg bw/day.

This study is classified as acceptable/guideline and satisfies the guideline requirement for a 

Multi-generation Reproduction Study -- Rats (OPPTS 870.3800, OECD 416). 

A.4.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity – Rat

In a 2-year combined chronic/oncogenicity study (PMRA 2226483, MRID 48557842), groups 

of 60 male and 60 female F344/DuCrl rats were fed diets formulated to provide 0, 20, 100, 400, 

625 (males only) or 750 (females only) mg XDE-729 acid (95.3% a.i., lot no. E2837-52, 

TSN030751-0006)/kg body weight/day. These dietary doses corresponded to time-weighted 

average doses of 0, 20.2, 101, 404 and 633 mg/kg bw/day for males and 0, 20.3, 102 or 407 

mg/kg bw/day, respectively for females. Females given 750 mg/kg bw/day were terminated on 
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test day 518 due to excessive mortality (56%), indicative of exceeding a maximum tolerated 

dose (MTD); these animals were subsequently removed from the study and no histopathological 

assessments were conducted. Ten rats/sex/dose level were necropsied after one year (chronic 

toxicity group) and the remaining 50 rats/sex/dose level were maintained on their respective 

diets for up to two years. Animals were evaluated by cage-side examinations, clinical 

observations, detailed clinical observations, body weight, food consumption and food 

efficiency. Ophthalmic examinations were conducted on all rats, pre-study and all surviving 

rats prior to the scheduled necropsies. Clinical pathology examinations (hematology, 

coagulation, clinical chemistry, electrolytes, and urinalysis) were conducted at regular intervals 

throughout the study. Toxicokinetic analysis was conducted on blood and urine samples from 4 

rats/sex/dose at 6 and 12 months post-dosing. All rats had a complete necropsy with weights of 

selected organs recorded at the scheduled necropsies. Histopathological examination was 

performed on all control and high-dose level rats and all rats that died spontaneously or were 

euthanized due to their moribund condition. Histopathological examinations were not 

conducted on the female 633 mg/kg bw/day dose group due to early termination from the study. 

Histopathological examination of rats from the low- and intermediate-dose levels of the 12-

month chronic toxicity group was limited to the target organ (kidney) and relevant gross lesions. 

Histopathological examination of survivors from the low- and intermediate-dose levels of the 

24-month oncogenicity group was limited to the kidneys and relevant gross lesions for both 

sexes, and the urinary bladder, adrenal glands, and mesenteric tissues for males.

The cumulative mortality of males given 633 mg/kg bw/day at 24 months was higher and 

statistically significant as compared to controls. The mortality rates at the end of the study were 

32, 44, 44, 34 and 62% in males and 16, 10, 28 and 30% in females in the specified dose groups, 

respectively. The higher mortality was attributed to treatment-related toxic effects in the 

kidneys. The increased mortality was not due to tumours, which indicates that this dose level 

had exceeded an MTD. An MTD was achieved for females given 407 mg/kg bw/day, based on 

treatment-related degeneration with regeneration of renal tubules and necrosis of individual 

epithelial cells lining the collecting ducts was observed in females at this dose level. There were 

no treatment-related effects on survival in the remaining dose groups. 

At 633/766 mg/kg bw/day M/F, in addition to decreased survival, treatment-related decreases in 

body weight and/or body weight gains, as well as a roughened kidney surface (24 months) were 

observed in both sexes. In males, increased incidences of mineralization of the pelvic epithelium 

in kidney, increases in the incidence of very slight necrosis of individual epithelial cells of the 

tubules and atrophy of mesenteric adipose tissue (secondary to decreased body weights) were 

also observed. Treatment-related increased blood urea nitrogen levels, necrosis of collecting duct 

epithelial cells of the kidney and vacuolization of the zona glomerulosa of the adrenal glands

were noted at doses greater than the MTD in females.

There were no adverse effects on clinical observations, food consumption, food efficiency, 

ophthalmological observations or hematology parameters in any of the dose groups.

At 404/407 and 633/766 mg/kg bw/day M/F in males and females, treatment-related renal effects 

consisted of increases in urinary volume (53-270%), decreased urine specific gravity (0.017-

0.045), increased kidney weights (absolute 8%, relative 12-14%), and increased incidences of 

renal histopathology [hypertrophy of renal collecting duct epithelium (incidences: ♂ 27, 29, 30, 
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48, 47, ♀ 8, 5, 11, 49), increased numbers of mitotic figures in collecting duct epithelial cells 

(males, 12 months only; incidences: 0, 0, 0, 3, 1), chronic interstitial inflammation of medulla

(incidences: ♂ 14, 18, 17, 37, 38, ♀ 9, 3, 14, 40), hyperplasia of pelvic epithelium (females, 12 

months, incidences: 0, 0, 1, 6, 9), vacuolization of collecting duct epithelium in the papilla (12 

months, incidences: ♂ 0, 0, 0, 4, 4; ♀ 0, 0, 0, 9, 10), slight necrosis of collecting duct epithelium 

and individual tubular epithelial cells (24 months; incidences: ♂ 0, 1, 0, 1, 21; ♀ 0, 0, 1, 6). In 

males at 404 and 633 mg/kg bw/day, decreased terminal body weights (5-7%), and increased 

incidences of hypertrophy of the zona glomerulosa of the adrenal glands (incidences: 0, 0, 0, 2, 

26), presence of calculi in the renal pelvis of the kidney (incidences: 0, 0, 0, 1, 2), degeneration 

and regeneration of renal tubules (incidences: 0, 1, 0, 4, 26), bladder effects [extensive 

hyperplasia of the transitional epithelium (incidences: 0, 1, 0, 3, 7), subacute to chronic 

inflammation in the submucosa beneath the hyperplastic epithelium (incidences: 0, 0, 0, 3, 7), 

microscopic calculi in the lumen of the urinary bladder (incidences: 0, 0, 0, 1, 2)] were also 

observed. In females, increases in chronic progressive glomerulonephropathy (12 months only; 

incidences: 2, 2, 4, 6, 10) and chronic interstitial inflammation (incidences: 0, 0, 0, 3, 7) were 

noted in the kidneys. 

At 102 mg/kg bw/day, the only treatment-related effect was a statistically identified increase in 

hyperplasia of pelvic epithelium of the kidney (incidences: 8, 5, 22, 43) in females. 

The LOAEL in males is 404 mg/kg bw/day, based on increased urinary volume, decreased 

urine specific gravity, decreased terminal body weights and decreased body weight gain, 

increased kidney weights, adverse renal histopathology findings, hypertrophy of the zona 

glomerulosa of the adrenal glands, presence of calculi in the renal pelvis of the kidney, 

degeneration and regeneration of renal tubules and bladder histopathology (including 

calculi). The LOAEL in females is 102 mg/kg bw/day, based on hyperplasia of pelvic 

epithelium of the kidney. The male NOAEL is 101 mg/kg bw/day and the female NOAEL is 

20.3 mg/kg bw/day.

There was no evidence of oncogenicity. The MTD was achieved at 404/407 mg/kg bw/day M/F 

based on treatment-related renal toxicity. 

Plasma levels of XDE-729 acid (AUC24h) at 6 and 12 months of exposure, when plotted against 

dose, were linear, with the exception of males at 6 months. Plasma levels in males at 6 months 

were sublinear at the high dose (749 mg/kg/day). Urine levels of XDE-729 acid at 6 and 12 

months were also linear, when plotted against dose. Because terminal plasma levels (samples 

taken at the time of necropsy) were either very low or non-quantifiable (below the lower limit of 

quantitation), t1/2 values could not be calculated for most animals. However, the average t1/2

from those that could be calculated was 3.3 hours. At 6 months of exposure, most (approximately 

72%, on average) of the administered dose of XDE-729 acid was excreted in the urine. At 6 

months in females there appeared to be some saturation of urinary elimination at the high dose 

with 85, 96, 57 and 55% of the administered dose excreted in 24 hour urine at 20, 97, 425 and 

832 mg/kg bw/day, respectively. However, this trend was not apparent in males. At 12 months, 

there was no apparent trend and the fraction of the administered dose excreted in the urine at 24 

hours in of males and females had declined to approximately 41%, on average. 
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This study is classified as acceptable/guideline and satisfies the guideline requirement for a 

Combined chronic/carcinogenicity [feeding] F344/DuCrl rats (OPPTS 870.4300; OECD 453).

870.4100b Chronic Toxicity – Dog

In a 1-year dog study (PMRA 2226470, MRID 48557832), XDE-729 (95.3% a.i., lot no. 2857-32) 

was administered to 4 Beagle dogs/sex/group in the diet at dose levels of 0, 500, 2500 or 10000 

ppm. Due to the body weight losses of females in the high dose group, the dietary concentration 

was reduced to 5000 ppm on Day 47 in the females only. The test material intake averaged 13.90, 

82.42, and 354.75 mg/kg/day in males and 16.72, 89.85 and 219.80 mg/kg/day in females at the 

low-, mid- and high-dose respectively. 

There were no treatment-related effects on survival, clinical signs, food consumption, food 

efficiency, hematology, clinical chemistry, urinalysis or ophthalmoscopic parameters, organ 

weights or gross pathology.

Following 6 weeks of treatment, the mean body weight in high-dose females was 9% lower in 

comparison with the Week -1 mean body weight value and body weight losses were observed. 

The changes in female body weight and body weight gains were considered to be treatmentrelated but of limited toxicological significance, since recovery was observed after the 

adjustment to the dietary concentration on Day 47 and overall body weight and body weight 

gains were comparable to controls. 

Treatment-related renal toxicity (graded very slight) consisting of degeneration with regeneration 

of the renal proximal and distal tubules (incidences ♂ 0, 0, 0, 1, ♀ 0, 0, 1, 1), degeneration of 

collecting ducts (incidences ♂ 0, 0, 0, 1, ♀ 0, 0, 1, 0) and multifocal glomerulosclerosis

(incidences ♂ 0, 0, 0, 2, ♀ 0, 0, 1, 0) was observed in mid- and high-dose females and in the 

high-dose males upon histological examination. In males, treatment-related granulomas 

(incidences ♂ 0, 0, 0, 1) and multifocal fibrosis (incidences ♂ 0, 0, 0, 1) were observed in the 

renal cortex of the kidneys at the high dose-level. 

The LOAEL is 89.85 mg/kg bw/day, based on degeneration with regeneration of the renal 

proximal and distal tubules, degeneration of collecting ducts and multifocal 

glomerulosclerosis in females. The NOAEL in females is 16.72 mg/kg bw/day. The LOAEL 

is 354.75 mg/kg bw/day in males, based on degeneration with regeneration of the renal 

proximal and distal tubules, degeneration of collecting ducts, multifocal glomerulosclerosis, 

and granulomas and fibrosis in the renal cortex. The NOAEL in males is 82.42 mg/kg 

bw/day. 

XDE-729 was present in approximately 50% of plasma, and all urine samples from treated 

animals, at levels above the analytical lower limit of quantitation. Based on the AUC24h values, 

XDE-729 exhibited supralinear kinetics at the high dose (10,000 ppm) in male dogs after 13 

weeks of exposure. The AUC24h values also appeared to be supralinear in male and female dogs 

of the high dose group at 26 weeks of exposure, but this nonlinearity was not statistically 

significant. At 52 weeks of exposure, there were too few plasma samples with quantifiable 

levels of XDE-729 to assess kinetic linearity across doses. Terminal plasma levels (after 16 
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hours of fasting) were below the lower limit of quantitation, consistent with a relatively short 

elimination half-life.

On average, males excreted 40% of the dose intact in the urine and females excreted 55% of the 

dose intact in urine. The total amount of XDE-729 excreted in the urine over 24 hours exhibited 

a linear relationship between all three doses in females and between the 500 and 2500 ppm doses 

in males. Urine levels appeared to be sublinear at 10,000 ppm in males at 13, 26 and 52 weeks 

of exposure, though this nonlinearity was not found to be statistically significant.

This study in dogs is classified as acceptable/guideline and satisfies the guideline requirement for 

a 1-year oral toxicity study in dogs (OPPTS 870.3150; OECD 409). 

A.4.5 Carcinogenicity

870.4200a Carcinogenicity Study - rat

See 870.4100

870.4200b Carcinogenicity (feeding) - Mouse

In an oncogenicity study (MRID 48557843, PMRA 2226485), groups of 50 male Crl:CD1(ICR) 

mice were fed diets formulated to provide 0, 50, 250, or 750 mg/kg body weight/day 

(mg/kg/day) and groups of 50 female Crl:CD1(ICR) mice were fed diets formulated to provide 0, 

50, 250, or 1000 mg/kg bw/day XDE-729 acid (95.3% a.i., lot no. E2837-52, TSN030751-0006) 

for up to 18 months to evaluate the potential for oncogenicity. These dietary concentrations 

corresponded to time-weighted average doses of 0, 50, 251, or 751 mg/kg bw/day for males, and 

0, 50.1, 251, or 1004 mg/kg bw/day for females, respectively. Animals were evaluated by cage 

side observations, clinical observations, detailed clinical examinations, body weights, and feed 

consumption. Ophthalmic examinations were conducted pre-exposure and prior to necropsy. 

Toxicokinetic analysis was conducted on blood and urine samples from 4 mice/sex/dose at 6 and 

12 months. All mice had a complete necropsy examination with total white blood cell (WBC) 

and differential WBC counts and weights of selected organs recorded at the scheduled necropsy. 

An extensive set of organs was examined histopathologically from all control and high-dose 

group mice, as well as all mice that died or were euthanized in a moribund condition. 

Histopathological examination was also conducted on target organs, selected major organs and 

all gross lesions of mice from the low- and intermediate-dose groups from the terminal necropsy.

There were no adverse effects on clinical observations (including detailed ranked assessments),

food consumption, food efficiency, ophthalmological observations, hematology parameters

(white blood cell count, leukocyte differential count), or incidence of palpable masses in any of 

the dose groups.

At 251 mg/kg bw/day and above, treatment-related moderate subacute to chronic inflammation

(incidence: 0, 4, 3, 27) and gross and microscopic calculi in the lumen (incidence: 0, 0, 2, 10) 

were observed in the kidneys of males. In females, the only treatment-related effects were 
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decreased body weight gains (8-9%) and increased hypertrophy of the intercalated cells in 

kidneys (incidence: 1, 1, 8, 36). 

The highest dose tested in male mice (751 mg/kg bw/day) was considered to be in excess of the 

maximum tolerated dose. The mortality rate at study termination was 46% at the highest dose 

tested and was statistically identified and attributed to the high prevalence of treatment-related 

renal and urinary bladder toxicity in deceased males. There were no treatment-related effects on

survival in the remaining dose groups. 

At 751 mg/kg bw/day in males, treatment-related increases in mortality due to renal and urinary 

bladder lesions were observed along with decreased kidney weight (absolute 8%, relative 7%), 

urinary bladder toxicity [thickened bladder wall (incidences: 0, 2, 0, 7), hyperplasia of the 

transitional epithelium lining of the mucosa (incidences 0, 1, 0, 7), increased numbers of mitotic 

figures within the epithelium (incidences: 0, 0, 0, 10), ulceration of the epithelium (incidences: 0, 

0, 0, 2), necrosis of individual transitional epithelial cells (incidences: 0, 1, 0, 11); decreased 

aggregates of mononuclear cells secondary to inflammation (incidences: 31, 31, 32, 11)] and 

renal toxicity [degeneration with regeneration of cortical tubular epithelium (incidences: 0, 0, 0, 

12), tubular dilatation (incidences: 1, 1, 3, 14), hypertrophy of the intercalated cells of the 

collecting ducts (incidences: 2, 0, 0, 39)]. There were no treatment-related effects in females at 

1004 mg/kg bw/day.

The LOAEL in both sexes is 251 mg/kg bw/day, based treatment-related moderate 

subacute to chronic inflammation and gross and microscopic calculi in the lumen of the 

kidneys of males and decreased body weight gains and increased hypertrophy of the 

intercalated cells in kidneys of females. The NOAEL in males and females is 50 mg/kg

bw/day.

There was no evidence of oncogenicity. The MTD was exceeded in males at 751 mg/kg bw/day 

M/F based on treatment-related mortalities attributed to renal and urinary bladder toxicity. A 

limit dose (1004 mg/kg bw/day) was tested in females. 

The plasma concentrations of XDE-729 acid were dose proportional (linear) in females at 6 and 

12 months and in males at 12 months. Less than dose-proportional plasma concentrations of 

XDE-729 acid were observed (sublinear) at the high dose in males at 6 months. XDE-729 

concentrations in urine were dose-proportional in both male and female mice at 6 and 12 months 

of dietary exposure. These data are consistent with data from previous studies in mouse and rat, 

in which plasma and urine concentrations of XDE-729 acid were usually dose-proportional, but 

occasionally sublinear at high doses. Though not statistically significant, visually there appears 

to be a trend toward sublinearity in plasma concentrations at high doses in the current and 

previous studies. These data suggest that the high dose of ~750 mg/kg bw/day may be 

approaching a dose at which nonlinear kinetics would be consistently observed.

This carcinogenicity study in mice is classified as acceptable/guideline and satisfies the guideline 

requirement for a carcinogenicity study (OPPTS 870.4200; OECD 451) in mice.

A.4.6 Mutagenicity
Halauxifen-methyl Human Health Risk Assessment DP No. D406145

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Gene Mutation

Guideline 870.5100, bacterial reverse 

mutation test (preincubation assay)

MRID 48557844

Acceptable/guideline

0, 50, 10, 500, 1500, or 5000 μg/plate +/- S9

Negative in Salmonella typhimurium strains TA 1535, TA1537, TA98, 

and TA100 and Escherichia coli Wp2 uvrA up to the limit concentration 

with no evidence of compound insolubility or cytotoxicity.

Guideline 870.5300, in vitro 

mammalian cell forward gene mutation, 

Chinese hamster ovary cells, HGPRT 

locus

MRID 48557848

Acceptable/guideline

0, 187.5, 375, 750, 1500, 2000, 2500, 3000 or 3400 μg/plate +/- S9

Negative for induced mutant colonies at the HGPRT locus up to 

precipitating and cytotoxic concentrations.

Cytogenetics

Guideline 870.5375, in vitro 

mammalian cell chromosomal 

aberration assay, primary rat 

lymphocytes, SD rat

MRID 48557852

Acceptable/guideline

0, 800, 1500 or 2000 μg/mL in the absence of mammalian metabolic 

activation (S9) ; 0, 400, 800 or 1500 μg/mL in the absence of 

mammalian metabolic activation

Negative for induction of chromosomal aberrations up to cytotoxic 

concentrations.

Guideline 870.5395, in vivo mammalian 

micronucleus assay, CD1 mouse

MRID 48557856

Acceptable/guideline

0, 500, 1000 or 2000 mg/kg

Negative for increased frequency of micronucleated reticulocytes in 

peripheral blood up to a dose causing systemic toxicity.

A.4.7 Neurotoxicity

870.6100 Delayed Neurotoxicity Study - Hen

Study not required

870.6200 Acute Neurotoxicity Screening Battery

In an acute oral neurotoxicity study (MRID 48557857; PMRA 2226505), ten male and ten female 

F344/DuCrl rats per group were given a single, oral gavage dose of 0, 250, 750, or 2000 mg XDE729 technical/kg body weight (acid form, 95.3% a.i., Lot no. E2837-52, TSN030751-0006). Body 

weights were recorded and a functional observational battery (FOB) and an automated test of motor 

activity were conducted pre-exposure (baseline), the day of dosing (day 1), day 8, and day 15. The 

FOB included hand-held and open-field observations as well as measurements of grip performance, 

landing foot splay and rectal temperature. Clinical observations were conducted on days 2, 3, and 4, 

and body weights were also recorded on day 2. At the end of the study, on day 16, five rats/sex/dose 

level were perfused in situ for histopathologic evaluation of the central and peripheral nervous 

systems, which was conducted on the control and high-dose groups. Positive control data 

demonstrating the proficiency of the laboratory personnel in conducting neurobehavioral 

assessments were provided.
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Treatment with XDE-729 did not affect clinical observations (days 2-4), rectal temperature, grip 

performance, landing foot splay, or motor activity in males or females, at any dose level tested. 

There was a slight, treatment-related decrease in the body weight of males and females given 750 

or 2000 mg/kg bw on days 2, 8 and/or 15 when compared to controls, with the greatest decrease 

seen in males at 2000 mg/kg bw on Day 2 (-7.8%). This effect was not considered adverse due to 

the small magnitude of the change at all time points (<10%). At the end of the study, body weight 

gains of males given 750 or 2000 mg/kg bw/day were lower (-9%/-11%) when compared to 

controls. Body weight gains in females were not affected by treatment. There was a slight decrease 

in the level of activity during the open-field evaluation of the FOB on day 1 in male rats only given 

750 or 2000 mg/kg bw (2.3 at both doses versus 2.9 in controls), which was considered an 

equivocal effect due to the lack of an effect on other related neurobehavioral endpoints including 

motor activity and absence of any treatment related neuropathological findings. 

The results of the neuropathologic evaluation indicated that XDE-729 had no effect on the 

central or peripheral nervous system. 

Canada PMRA, and Australia OCS: Based on the decrease in body weight in males on Day 

2, the NOAEL for F344/DuCrl male rats dosed with XDE-729 was 750 mg/kg bw. In 

females, no adverse effects were observed up to the highest dose tested, therefore the 

NOAEL was set at 2000 mg/kg bw. There was no evidence of neurotoxicity up to the 

highest dose tested.

US EPA: The neurotoxicity and systemic toxicity NOAELs are 2000 mg/kg bw (HDT and 

limit dose) for both males and females. LOAELs for neurotoxicity and systemic toxicity 

were not identified (>2000 mg/kg bw). 

This study is classified as acceptable/guideline and satisfies the guideline requirement for an 

acute neurobehavioral screening study in the rat (OPPTS 870.6200; OECD 424).

870.6200 Subchronic Neurotoxicity Screening Battery

In a subchronic neurotoxicity study (MRID 48557858; PMRA 2226506), XDE-729 (acid form, 

95.3% a.i., lot no. E2837-52, TSN030751-0006) was administered in the diet at levels equivalent 

to target doses of 0, 50, 250, and 750 mg/kg body weight/day in male and female F344/DuCrl 

rats for at least 13 weeks. The time-weighed average dosages were: 0, 52.5/51.2, 260/256, or 

774/772 mg/kg body weight/day in M/F. Ten rats/sex/dose were evaluated pre-exposure and at 2, 

4, 8, and 13 weeks of exposure using a functional observational battery (FOB), determinations of 

grip performance, rectal temperature, landing foot splay, and an automated test of motor activity. 

Weekly clinical observations, body weights and food consumption were also evaluated, and 

ophthalmic examinations were conducted pre- and post-exposure. At the end of the study, five 

rats/sex/dose level were perfused for histopathologic evaluation of the central and peripheral 

nervous systems, which was conducted on the control and high-dose groups. Positive control 

data were provided that demonstrated proficiency of the testing laboratory personnel.

Treatment with XDE-729 did not affect FOB observations, grip performance, rectal 

temperature, landing foot splay, motor activity, ophthalmic examinations, or clinical 
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observations in males or females at any dose level tested. At the end of the study, there was a 

treatment-related decrease in the body weight (7%) and body weight gain (12%) of males given 

750 mg/kg bw/day when compared to controls, which correlated with a slight, treatment-related 

decrease in feed consumption across the study in this group. There were no treatment-related 

gross or histopathologic observations in the central or peripheral nervous systems of rats 

administered XDE-729.

Canada PMRA: The no-observed-adverse-effect level (NOAEL) for systemic toxicity was 250 

mg/kg bw/day based on the decrease in body weight of males at 750 mg/kg bw/day. The 

NOAEL for females was 750 mg/kg bw/day; a LOAEL was not identified. There was no 

evidence of neurotoxicity.

US EPA and Australia OCS: The no-observed-adverse-effect level (NOAEL) for both 

neurotoxicity and systemic toxicity was 750 mg/kg bw/day (HDT) in males and females. 

The decrease in body weight of males at 750 mg/kg bw/day was not considered biologically 

significant (<10%). LOAELs for neurotoxicity and systemic toxicity were not identified in 

this study.

This study is classified as Acceptable/guideline and satisfies the guideline requirement for a 

subchronic oral neurobehavioral screening study in the rat (OPPTS 870.6200; OECD 424).

870.6300 Developmental Neurotoxicity Study

Study not required

A.4.8 Metabolism

Nonguideline Metabolism Probe Study – Rat and Mouse

In a metabolism probe study (MRID 48557859, PMRA 2226444), absorption, distribution, 

metabolism and elimination (ADME) of uniformly labeled 14C-XR-729 acid (radiochemical 

purity 96.6%, lot no. INV 030887-0001) was determined in Fischer 344 rats (2/sex) and CD1 

mice (4/sex) after administration of a single oral dose at 100 mg/kg bw. Time-course blood

(plasma and RBC) and excreta were collected from rats and time-course excreta only were 

collected from mice for up to 72 hours post-dosing. The urine samples collected during the first 

12 hours post-dosing were analyzed for parent compound and metabolite(s). Additionally, two 

rats (1/sex) and four mice (2/sex) were orally dosed with 14C-XR-729 acid at 100 mg/kg bw and 

plasma was collected at the peak plasma concentration (tmax; 0.5 hour post-dosing estimated 

from rats) and analyzed for parent and metabolite(s). 

The orally administered 14C-XR-729 acid was rapidly absorbed from the GI tract both by the 

rats and mice with a tmax of approximately 0.5 hours and eliminated mainly in the urine. The rate 

of absorption of 14C-XR-729-derived radioactivity from the GI tract was same for both male and 

female rats. Total urinary elimination accounted for 65-75% of the dose in rats and 65-66% of 

the dose in mice, most of which occurred within the first 12 hours after dosing. Fecal 

elimination accounted for 12-21% in rats and 19-20% in mice. Elimination of radioactivity from 
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plasma was bi-exponential in the rat with an initial half-life (t1⁄2) of 0.14 hours indicating rapid 

distribution to tissues and terminal elimination half-life (t1⁄2) ranging from 3.37 (male) to 3.67 

(female) hours. Elimination of 14C-XR-729-derived radioactivity from red blood cells (RBC)

was mono-exponential with a half-life (t1⁄2) of 0.84 (male) and 0.77 (female) hours. 

In the 12-hour urine samples, eight peaks were detected, but only one peak was found 

representing >5% of the dose and was identified as XR-729 acid (parent). Four metabolites were 

tentatively identified: the glucuronide conjugate of O-demethyl XR-729, the sulfate conjugate of 

O-demethyl XR-729, O-demethyl XR-729, and acyl-glucuronide conjugate of parent XR-729. 

The metabolic profile looked very similar between sexes and species with only slight changes in 

the percentages of the peaks. Unchanged XR-729 acid was found as the major component in all 

Cmax plasma extracts and the acyl glucuronide conjugate of XR-729 acid was also found in the 

male mouse plasma extract, representing approximately 7.5% of the recovered radioactivity. 

In summary, XR-729 acid is rapidly absorbed from the GI tract of rats and quickly eliminated from 

the male and female rat and mouse mainly in urine within the first 12 hours after dosing. The vast 

majority of the radioactivity in the urine and plasma of rats and mice was unchanged XR-729 acid.

This study is classified as acceptable/nonguideline. It was performed as a probe for definitive 

general metabolism study and was not conducted to satisfy the guideline requirement for a 

metabolism study (OPPTS 870.7485, OECD 417) in the rat or mouse.

Nonguideline Metabolism – Dog

In a non-guideline metabolism study (MRID 48557870; PMRA 2226445), three male and three 

female beagle dogs were administered 50 mg/kg bw 14C-XDE-729 (96.8%; Lot# DE3-102069, 

INV# INV-031412-0003), formulated as the sodium salt, via oral gavage. Time-course plasma and 

excreta were collected up to 168 hours (7 days) post-dosing to determine absorption, metabolism, 

and elimination (AME) of this test material. Urine and fecal samples collected at 168 hours postdosing and plasma samples collected at Cmax and 1⁄2Cmax were analyzed to characterize and quantify 

XDE-729 acid (parent) and metabolites.

Plasma Cmax was achieved slightly earlier in females than male dogs (mean tmax: 0.7 vs. 1.3 h) 

and was also slightly higher in female dogs (143 vs. 118 μg-Na eq./g). The vast majority of the 

14C-radioactivity cleared from dog plasma by 6 hours post-dosing. Elimination of radioactivity 

from the plasma was biphasic, with initial half-life (t1⁄2α) of 1 h indicating rapid distribution to 

tissues and terminal elimination half-life (t1⁄2β) of 9-12 h. XDE-729 acid represented the major 

radiolabeled component in plasma determined by LC/MS-MS (analysis of non-radiolabel) and 

liquid scintillation counting. These data are consistent with metabolite profile results obtained by 

HPLC-RAM analysis of selected Cmax plasma samples indicating XDE-729 acid as the major 

component in the plasma of dogs. Comparison of pharmacokinetic parameters with rats showed 

that the dose corrected plasma AUC24 in dogs was 2 to 4-fold higher than rats administered 10 

and 100 mg/kg bw of 14C-XDE-729 sodium salt indicating higher systemic exposure to 14CXDE-729 acid in dogs following a single oral dose.

Analysis of excreta showed that approximately 80% of the dose was excreted in the urine in 168 

h, with the largest fraction excreted within 12 h. Fecal elimination accounted for ~10% (males) 
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or ~14% (females) of the dose, most of which were excreted within 48 hours post-dosing. In 

total, 95-98% of the dose was recovered from excreta and cage wash within 168 hours postdosing demonstrating adequate mass balance.

XDE-729 acid was the major radio-labeled component in urine/rinse (77-79% of the dose) and 

was present in feces at levels higher than or similar to those of other identified metabolites. The 

primary metabolites identified in urine, feces, or plasma included O-demethyl XDE-729 and the 

sulfate and glucose conjugates of O-demethyl XDE-729, hydroxy XDE-729, and an acyl 

glucuronide conjugate of XDE-729.

In summary, animals dosed by oral gavage with XDE-729 sodium salt were systemically 

exposed to XDE-729 acid (and associated metabolites), as assessed by radioactivity and HPLC in 

plasma samples. Absorption of the dose appeared to be fairly rapid (tmax of 0.5 to 1 hour postdosing) and complete (~80% of the dose was excreted in urine and only 10-14% in feces at 168 

hours post-dosing). Elimination from plasma was biphasic and fairly rapid (t1⁄2α of 1 h and t1⁄2β of 

9-12h). Excretion of the dose was fairly rapid; the largest fraction of the dose was recovered 

from urine collected at 12 hours post-dosing and most of the dose excreted in feces was 

recovered by 48 h after oral gavage. Parent compound generally represented the major 

radioactive component analyzed in plasma and excreta samples. 

This study is classified as Acceptable/non-guideline. It was conducted to provide additional 

information on the metabolism of XDE-729, namely to evaluate the potential for renal saturation 

of elimination. The study was not submitted to satisfy the guideline requirement for a general 

metabolism study (OPPTS 870.7485; OECD 417).

 

870.7485 Metabolism - Rat

In a rat metabolism study (MRID 48557860, PMRA 2226446), groups of four male and female 

F344/DuCrl rats were administered 14C-XDE-729 (14C-(UL-phenyl)-XDE-729 [Lot# XS9-

100040-68, 97.2%] or 14C-(2,6-pyridine)-XDE-729 [Lot# XS9-100040-69, 97.3%]), as the 

sodium salt, following oral (single or multiple dose of 10 mg/kg bw; single dose of 750 mg/kg 

bw) and intravenous (IV) exposure (single 10 mg/kg bw). In addition, absorption, distribution, 

metabolism, and excretion (ADME) of 14C-XDE-729 methyl (X11393728, 97.4%) (the methyl 

ester form of XDE-729; 10 mg/kg bw) was also determined in male and female F344/DuCrl rats 

(n=4/sex) to determine the bioequivalence between the acid (as sodium salt) and methyl ester 

forms of XDE-729. Animals were monitored and excreta collected for 168 hours (7 days) postdosing to determine ADME of the test compounds.

Orally administered XDE-729, as the sodium salt, was rapidly absorbed without any apparent lag 

time (tmax 0.1-0.2 hours and 0.3-1.8 hours following low oral single doses and a high oral single 

dose, respectively). The tmax for the low oral XDE-729 methyl group was slightly longer (0.44-

0.50 hours) compared to the low oral XDE-729, as the sodium salt. The percent absorption of the 

orally administered dose sodium salt in all low oral groups (single 10 mg/kg bw, multiple 10 

mg/kg bw, single 10 mg/kg bw pyridine-ring label) was at least 77-93%, based on recovery in 

urine and non-GI tissues and was comparable for low oral XDE-729 methyl dose group (82%). 

The percent absorption estimated from urine and non-GI tissue was slightly lower following the 
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high single oral dose of 14C-XDE-729 sodium salt (69-73%). Total recovery of radioactivity 

from all the animals averaged 102 ± 3 and 99 ± 3% in the oral and IV dose groups, respectively.

Absolute oral bioavailability, calculated from comparison of the plasma AUC data for the low 

oral and IV dose groups (14C-(UL-phenyl)-XDE-729 sodium salt), was ~100% for both female 

and male rats.

The orally absorbed dose was rapidly excreted in urine (68-92% of the administered dose for all 

five oral dose groups) with a higher percentage excreted in the urine of female rats. The majority 

of the urinary elimination (90-99%) occurred within the first 24 hours post-dosing. A smaller 

percent (11-29% for all five oral dose groups) of the administered oral dose was eliminated in 

feces. The amount and rates of urinary and fecal elimination were comparable between XDE729, administered as sodium salt (both labels) and XDE-729 methyl as a low oral dose. Rats 

orally administered 750 mg 14C-XDE-729 sodium salt/kg bw demonstrated decreased urinary 

elimination compared to rats administered 10 mg 14C-XDE-729 sodium salt/kg bw. There was no 

excretion of radioactivity via exhaled CO2.

The IV-administered XDE-729 sodium salt was also rapidly and extensively excreted in urine 

(85-88% of administered dose). The majority of the urinary elimination (98-99%) occurred in 

the first 24 hours post-dosing. A smaller percent (11-15%) of the IV dose was eliminated in 

feces. 

At 168 hours post-dosing, levels in the tissues ranged from non-quantifiable to 0.3% of the orally 

administered dose from either XDE-729 (low single, high single, multiple dose, and low dose 

pyridine-ring label) or 14C-XDE-729 methyl (low single). Less than 0.03% of the IV 

administered 14C-XDE-729 remained in the tissues at 168 hours post-dosing. 

In the rats orally administered as a single dose at 10 mg 14C-XDE-729 sodium salt/kg bw (both 

labels) or 10 mg 14C-XDE-729 methyl/kg bw, the Cmax was 1.6 to 3.6 fold lower for the methyl 

group. Elimination of the radioactivity from plasma was rapid during the initial distribution 

phase (t1⁄2α = 0.5-0.9 hours) followed by a slower elimination during the terminal phase (t = 5-

16 hours). Rats orally administered 750 mg 14C-XDE-729 sodium salt/kg bw displayed a slower 

initial distribution phase (t1⁄2α = 2.7- = 5.6-8.7 hours) 

was within the range of the 10 mg/kg bw group. Clearance of the IV-administered XDE-729 

sodium salt was ~ 0.5 mL/h/kg. The plasma AUC was 2.0-2.6 times higher at the high dose than 

would be expected for dose proportionality demonstrating a supraproportional increase in 

systemic exposure at the high dose. The AUC non-linearity is consistent with the longer initial 

distribution phase of the 750 mg/kg bw group and decreased urinary elimination at 168 h postdose, likely due to a slower redistribution at the high dose and saturation of elimination. The 

time-course of radioactivity in RBC was less than the plasma time-course concentration profiles 

with lower concentrations. At 10 mg/kg bw oral dose (both labels, sodium salt and methyl forms) 

when compared to the concentrations in plasma, the maximum concentrations in RBC were 8- to 

33-fold less. At the high dose of 750 mg/kg bw, the Cmax was 2- to 3-fold less than in plasma. 

The time to peak, maximum concentration and absorption half-lives in RBC were comparable to 

plasma numbers. The time for distribution, as reflected by t , and elimination (t ) was less 

than those in plasma for all five treatment groups, with the exception of the high dose group 

which demonstrated a longer elimination (t ) in RBC compared to plasma. The AUC numbers 
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were substantially less than those in plasma for all five treatment groups. Overall, similar 

kinetics were observed across sexes for the sodium salt and methyl test materials. The IVadministered XDE-729 sodium salt demonstrated higher clearance values for RBC (5- to 8-fold) 

when compared to plasma. As seen in plasma in RBC’s there was a supraproportional increase in 

AUC from the low to the high dose, however, in contrast to plasma the AUC non-linearity is 

consistent with the longer terminal elimination phase of the 750 mg/kg bw group.

Analysis of blood samples from the methyl dose group afforded primarily XDE-729 and no 

parent ester, indicating rapid hydrolysis of the ester in vivo. Although XDE-729 methyl showed 

increased tmax and decreased Cmax when compared to the same dose of XDE-729 sodium salt/kg 

bw, the administration of both test compounds resulted in similar AUC values. 

The major radiolabeled component present in the urine and fecal extracts from animals 

administered 14C-XDE-729 sodium salt, was XDE-729 acid. The major radiolabeled component 

present in urine and fecal extracts of animals dosed with XDE-729 methyl was the hydrolysis 

product, XDE-729 acid. Minor metabolites of both test materials consisted of the acylglucuronide conjugate of parent XDE-729 (present in selected samples at greater than 5% of the 

administered dose) and O-demethylated XDE-729 and the corresponding sulfate and glucuronide 

conjugates.

In summary, administered XDE-729 sodium salt or methyl was rapidly and highly absorbed, 

poorly metabolized and readily excreted mainly in urine from the rat, with lesser amounts 

excreted in feces and very low tissue residues following single oral and IV low, high oral doses 

and low oral repeat doses. The similarity in absorption, tissue distribution, elimination kinetics 

and excretion, and metabolite profiles between XDE-729 sodium salt (low single, phenyl- and 

pyridine-ring labels) and XDE-729 methyl (low single phenyl-ring label) and blood analyses 

showing that the methyl ester is rapidly hydrolyzed to XDE-729 acid in vivo at 10 mg/kg bw, 

demonstrates bioequivalence between these two test materials at low doses.

This study is classified as acceptable/guideline and satisfies the guideline requirement for a 

general metabolism study in the rat.

870.7600 Dermal Absorption - Rat

Study not required (see Executive Summaries for Halauxifen-methyl)

A.4.9 Immunotoxicity

870.7800 Immunotoxicity

Study conducted on halauxifen-methyl – see Executive Summaries for halauxifen-methyl

A.4.9 Special/Other Studies

See Executive Summaries for halauxifen-methyl

METABOLITE X11449757 (GENOTOXICITY ONLY)
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A.4.6 Mutagenicity

Gene Mutation

Guideline 870.5100, Bacterial Reverse 

Mutation Test (Pre-Incubation Assay)

MRID 48557847

Acceptable/guideline

0, 156.25, 312.5, 625, 1250, 2500 or 5000 μg/plate - S9 activation 

and

0, 51.2, 128, 320, 800, 2000 or 5000 μg/plate + S9 activation

Negative in Salmonella typhimurium strains TA98, TA100, TA102, 

TA1535, TA1537 for induced mutant colonies over background at the 

HGPRT locus up to precipitating and cytotoxic concentrations.

Guideline 870.5300, in vitro 

mammalian cell forward gene mutation 

CHO cells, HGPRT locus

MRID 48557850

Acceptable/guideline

0, 100, 200, 400, 800, 1600 or 3210 μg/mL +/- S9 activation

Negative for induced mutant colonies over background at the HGPRT 

locus up to precipitating and cytotoxic concentrations.

Cytogenetics

Guideline 870.5375, in vitro 

mammalian cell chromosomal 

aberration assay, human peripheral 

lymphocytes

MRID 00141673

Acceptable/guideline

0, 25.3, 50.6, 101.3, 202.5, 405 or 810 μg/mL +/- S9 activation

Negative for increased chromosomal aberrations over background up to 

cytotoxic and precipitating concentrations.
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Appendix B. Metabolism Summary Tables

Table B.1. Tabular Summary of Metabolites and Degradates

Chemical 

Name

Matrix Percent TRR (PPM) 1

Matrices - Major Residue 

(>10%TRR)

Matrices - Minor Residue (<10%TRR)

Parent

HalauxifenMethyl

Wheat Forage: PH 5.0 (0.010)

Wheat Forage: PY 8.3 (0.012)

Wheat Hay: PH 1.0 (0.002)

Wheat Hay: PY 5.4 (0.022)

Wheat Straw: PH 1.2 (0.002)

Wheat Straw: PY 1.9 (0.001)

Turnip Tops: PH 13.5 (0.011)

Turnip Tops: PY 12.2 (0.010)

Rotational Crops - -

Ruminant Milk PY- day 3: 0.4 (<0.001)

Milk PY- day 4: 0.5 (<0.001)

Kidney PY: 0.6 (<0.001)

Poultry Skin with Fat PH: 0.5 (<0.001)

Rat <5% of the AD in excreta

Water ND - 97%

HalauxifenMethyl 

Glucose 

Conjugate

Turnip Tops: PH 26.4 (0.021)

Turnip Tops: PY 28.7 (0.024)

Rotational Crops - -

Ruminant - -

Poultry - -

Rat - -

Water - -

HalauxifenMethyl

N-Glucuronic 

Acid 

Conjugate

Rotational Crops - -

Ruminant Milk PY- day 3: 3.5 (<0.001)

Milk PY- day 4: 4.2 (<0.001)

Poultry - -

Rat - -

Water - -

XDE-729 Acid Wheat Forage: PH 1.6 (0.003)

Wheat Forage: PY 1.9 (0.003)

Wheat Hay: PH 1.7 (0.004)

Wheat Hay: PY 1.8 (0.007)

Wheat Straw: PH 1.2 (0.002)

Wheat Straw: PY 1.8 (0.001)

Turnip Tops: PH 3.1 (0.002)
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Turnip Tops: PY 3.5 (0.003)

Rotational Crops - -

Ruminant Kidney PY: 34.4 (0.014)

Renal Fat PH: 44.8 (0.007)

Liver PH: 12.1 (0.009)

Kidney PH: 36.7 (0.044)

Milk PY- day 3: 2.0 (<0.001)

Milk PY- day 4: 3.6 (<0.001)

Liver PY: 5.8 (0.002)

Poultry Skin w/ Fat PH: 26.0 (0.044) Liver PH: 6.3 (0.003)

Liver PY: 5.5 (0.002)

Rat ND ND

Water ND - 99%

XDE-729 Acid 

Glucose 

Conjugate

Turnip Tops: PH 12.5 (0.010)

Turnip Tops: PY 12.6 (0.010)

Rotational Crops - -

Ruminant - -

Poultry - -

Rat - <5% of the AD in excreta

Water - -

X11406790 Wheat Forage: PH 2.6 (0.005)

Wheat Forage: PY 3.8 (0.006)

Wheat Hay: PH 0.5 (0.001)

Wheat Hay: PY 0.8 (0.003)

Wheat Straw: PH 0.9 (0.002)

Wheat Straw: PY 0.8 (0.001)

Turnip Tops: PH 2.2 (0.002)

Turnip Tops: PY 1.5 (0.001)

Rotational Crops - -

Ruminant Milk PY- day 3: 2.5 (<0.001)

Milk PY- day 4: 0.3 (<0.001)

Kidney PH: 1.2 (0.002)

Kidney PY: 0.3 (<0.001)

Liver PY: 8.8 (0.003)

Poultry Skin with Fat PH: 4.3 (0.001)

Liver PH: 5.7 (0.003)

Rat - -

Water 0.5% - 33%

X11406790

Glucose 

Conjugate

Wheat Forage: PH 2.6 (0.005)

Wheat Forage: PY 1.4 (0.002)

Wheat Hay: PH 4.4 (0.010)

Wheat Hay: PY 4.9 (0.020)

Wheat Straw: PH 3.2 (0.006)

Wheat Straw: PY 3.0 (0.002)
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Rotational Crops - -

Ruminant - -

Poultry - -

Rat - -

Water - -

X11406790

Glucose

Malonyl 

Conjugate

Wheat Forage: PH 10.3 (0.021)

Wheat Forage: PY 5.9 (0.009)

Wheat Hay: PH 5.0 (0.012)

Wheat Hay: PY 4.8 (0.020)

Wheat Straw: PH 5.2 (0.010)

Wheat Straw: PY 3.7 (0.002)

Turnip Tops: PH 5.9 (0.005)

Turnip Tops: PY 7.2 (0.006)

Rotational Crops - -

Ruminant - -

Poultry - -

Rat - -

Water - -

X11406790

Glucuronic 

Acid

Conjugate

Rotational Crops - -

Ruminant Milk PY- day 3: 40.7 (0.005)

Milk PY- day 4: 24.5 (0.002)

Kidney PH: 5.9 (0.007)

Poultry - -

Rat - -

Water - -

X11406790

Sulfate

Conjugate

Rotational Crops - -

Ruminant Milk PY- day 3: 21.8 (0.003)

Milk PY- day 4: 12.5 (0.001)

Kidney PH: 0.8 (0.001)

Poultry Skin with Fat PH: 12.3 (0.002)

Liver PH: 14.5 (0.007)

Liver PY: 8.3 (0.004)

Rat - -

Water - -

X11449757 Wheat Forage: PH 5.1 (0.010)

Wheat Forage: PY 4.7 (0.007)

Wheat Hay: PH 1.9 (0.005)

Wheat Hay: PY 1.9 (0.008)

Wheat Straw: PH 2.0 (0.004)

Wheat Straw: PY 1.7 (0.001)

Rotational Crops - -

Ruminant Renal Fat PH: 17.4 (0.003)

Liver PH: 62.0 (0.048)
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Liver PY: 31.9 (0.010)

Kidney PH: 14.7 (0.018)

Kidney PY: 14.4 (0.006)

Milk PY- day 3: 10.1 (0.001)

Milk PY- day 4: 21.9 (0.002)

Poultry Liver PH: 32.3 (0.015) Skin with Fat PH: 2.9 (<0.001)

Rat <5% of the AD in excreta

Water 3% - 77%

X11449757 

Sulfate 

Conjugate

Rotational Crops - -

Ruminant Kidney PH: 18.3 (0.022)

Kidney PY: 10.4 (0.004)

Renal Fat PH: 9.5 (0.002)

Poultry Skin with Fat PH: 0.4 (<0.001)

Liver PH: 0.9 (<0.001)

Rat <5% of the AD in excreta

Water - -

X11449757 

Glucuronide 

Conjugate

Rat - <5% of the AD in excreta

Degradate 1 Water 20% - 41%

Degradate 2 Water 8% - 13% 

Degradate 4 Water 0.7% - 15%

Degradate 10 Water ND – 13%

Degradate 11 Water ND – 16%

Degradate 14 Water ND – 12%

The wheat metabolism studies included use of a safener.

The Turnip samples were taken at 14 days after treatment

No metabolites were identified in the rotational crop study. TRR in all commodities was 0.001 ppm or less

<5% of the AD: <5% of the administered dose 

Table B.2. Structures of Halauxifen-Methyl Metabolites and Degradates

Code Name Chemical Abstracts Name Structure Where Found

Halauxifenmethyl

methyl 4-amino-3-chloro-6-(4-

chloro-2-fluoro-3-

methoxyphenyl)pyridine-2-

carboxylate

Metabolism: 

wheat, turnips, 

poultry, 

drinking water

Env fate: 

hydrolysis, 

soil photolysis
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Table B.2. Structures of Halauxifen-Methyl Metabolites and Degradates

Code Name Chemical Abstracts Name Structure Where Found

Halauxifenmethyl

N-glucose 

conjugate 

(anomers)

methyl 3-chloro-6-(4-chloro-2-

fluoro-3-methoxy-phenyl)-4-

[[3,4,5-trihydroxy-6-

(hydroxymethyl)tetrahydropyra

n-2-yl]amino]pyridine-2-

carboxylate (anomers = α- and 

β-conjugated glucose)

Metabolism: 

turnips

Halauxifenmethyl

N-glucuronic 

acid

conjugate 

6-[[3-chloro-6-(4-chloro-2-fluoro3-methoxy-phenyl)-2-

methoxycarbonyl-4-

pyridyl]amino]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid

or isomer

Metabolism: 

ruminants

XDE-729 

Acid

4-amino-3-chloro-6-(4-chloro-2-

fluoro-3-methoxy-phenyl)-

pyridine-2-carboxylic acid

Rat metabolism DER: XR-729 

Parent

Metabolism: 

wheat, turnips, 

ruminants, 

poultry, rats, 

drinking water

Env fate: 

hydrolysis, 

soil photolysis, 

anaerobic soil 

metabolism
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Table B.2. Structures of Halauxifen-Methyl Metabolites and Degradates

Code Name Chemical Abstracts Name Structure Where Found

XDE-729 

Acid

glucose 

conjugate

[3,4,5-trihydroxy-6-

(hydroxymethyl)tetrahydropyra

n-2-yl] 4-amino-3-chloro-6-(4-

chloro-2-fluoro-3-methoxyphenyl)pyridine-2-carboxylate

 

 or

3-chloro-6-(4-chloro-2-fluoro-3-

methoxy-phenyl)-4-[[3,4,5-

trihydroxy-6-

(hydroxymethyl)tetrahydropyra

n-2-yl]amino]pyridine-2-

carboxylic acid

or

Metabolism: 

turnips

X11406790 methyl 4-amino-3-chloro-6-(4-

chloro-2-fluoro-3-

hydroxyphenyl)pyridine-2-

carboxylate

Metabolism: 

wheat, turnips, 

ruminants, 

poultry, 

drinking water

X11406790 

glucose 

conjugate

Metabolism: 

wheat

X11406790 

glucosemalonyl 

conjugate

Metabolism: 

wheat

N

Cl

NH2

Cl

O

HO O

F
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Table B.2. Structures of Halauxifen-Methyl Metabolites and Degradates

Code Name Chemical Abstracts Name Structure Where Found

X11406790 

N- or Oglucuronic 

acid 

conjugate

6-[[3-chloro-6-(4-chloro-2-fluoro-3-

hydroxy-phenyl)-2-methoxycarbonyl4-pyridyl]amino]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid or

6-[3-(4-amino-5-chloro-6-

methoxycarbonyl-2-pyridyl)-6-chloro2-fluoro-phenoxy]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid

or

Metabolism: 

ruminants

X11449757 4-amino-3-chloro-6-(4-chloro-2-

fluoro-3-hydroxyphenyl)-pyridine2-carboxylic acid

Rat metabolism DER: Odemethyl- XR-729

Metabolism: 

wheat, 

ruminants, 

poultry, rats

drinking water

Env. fate: 

anaerobic soil 

metab., 

aerobic 

aquatic metab.

X11449757 

Sulfate 

Conjugate

4-amino-3-chloro-6-(4-chloro-2-

fluoro-3-sulfoxy-phenyl)pyridine2-carboxylic acid

Rat metabolism DER: sulfate 

conjugate of O-demethyl-XR-729

Metabolism: 

ruminants, 

poultry, rats

X11449757 

Glucuronide 

Conjugate

Rat metabolism DER: glucuronide 

conjugate of O-demethyl-XR-729

Metabolism: 

rats

X11449757 

Acyl 

Glucuronide 

Conjugate

Rat metabolism DER: acylglucuronide conjugate of Odemethyl-XR-729

Metabolism: 

rats

N

F

HO

Cl

O

OH

Cl

NH2

O

O

S

O

N

F

Cl

NH2

Cl

OH

O

HO
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Table B.2. Structures of Halauxifen-Methyl Metabolites and Degradates

Code Name Chemical Abstracts Name Structure Where Found

Degradate 1 Unknown

Unknown

Metabolism: 

drinking water

Env. fate: 

aqueous 

photolysis

Degradate 2 Unknown

Unknown

Metabolism: 

drinking water

Env. fate: 

aqueous 

photolysis

 

Degradate 4 Unknown

Unknown

Metabolism: 

drinking water

Env. fate: 

aqueous 

photolysis

Degradate 10 Methyl 4-amino-6-(4-chloro-2-

fluoro-3-methoxy-phenyl)-3-

hydroxy-pyridine-2-carboxylate

Metabolism: 

drinking water

Env. fate: 

aqueous 

photolysis

Degradate 11 Methyl (2E)-2-(4-chloro-2-fluoro3-methoxy-benzoyl)iminoacetateMetabolism: 

drinking water

Env. fate: 

aqueous 

photolysis

 

Degradate 14

2-[(E)-(4-chloro-2-fluoro-3-

methoxy-phenyl)methyleneamino]

acetaldehyde

Metabolism: 

drinking water

Env. fate: 

aqueous 

photolysis

 
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Appendix C. Physical/Chemical Properties

Table C-1. Physicochemical Properties of the Technical Grade Test Compound

Parameter

Value for

Halauxifen-methyl

Value for

XDE-729 Acid

(X11393729)

Reference

Water solubility

pH 5

pH 7

pH 9

unbuffered

mg/L at 20°C

1.66 

1.67 

1.69 

1.83 

at 20°C

102 mg/L

3.07 g/L

46.1 g/L

84.0 mg/L

Tolerance 

Petition 

2E8086

Octanol/water partition 

coefficient at 20°C

Log KOW Log KOW

pH 5 buffer solution 3.75 0.42

pH 7 buffer solution 3.76 -0.83

pH 9 buffer solution 3.92 -1.01

Vapor Pressure 5.9 x 10-9 Pa at 20°C

(4.4 x 10-11 mmHg)

1.5 x 10-8 Pa at 25°C

(1.1 x 10-10 mmHg)

2 x 10-5 Pa at 20°C

(1.5 x 10-7 mmHg)

3 x 10-5 Pa at 25°C

(2.3 x 10-7 mmHg)

Organic solvent solubility g/L at 20°C g/L at 20°C

methanol 38.1 4.2

acetone >250 4.4

ethyl acetate 129 1.2

n-octanol 9.83 0.43

Melting point 146°C not available

pH 6.24 not available

Dissociation constant (pKa) 2.84 @ 20°C 5.11 @ 21.9°C
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Appendix D. Review of Human Research

This risk assessment relies in part on data from studies in which adult human subjects were 

intentionally exposed to a pesticide or other chemical. These data, which include studies from 

PHED 1.1 and the AHETF database are (1) subject to ethics review pursuant to 40 CFR 26, (2) 

have received that review, and (3) are compliant with applicable ethics requirements. For certain 

studies, the ethics review may have included review by the Human Studies Review Board. 

Descriptions of data sources, as well as guidance on their use, can be found at the Agency 

website1.

 

1 http://www.epa.gov/pesticides/science/handler-exposure-data.html and 

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