UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the List 1 Chemicals

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

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## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: MGK 264

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMGK 264 FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................16

IV. Committee’s Assessment of Weight of Evidence..........................................20

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20

1. Tier 1 in vivo Assays.............................................................21

2. OSRI...............................................................................21

B. Estrogen Pathway...........................................................................23

C. Androgen Pathway.........................................................................24

D. Thyroid Pathway...........................................................................24

E. Conclusions.................................................................................24

V. EDSP Tier 2 Testing Recommendations....................................................24

VI. References.......................................................................................26

LIST OF TABLES: Table 1. Tier 1 Assays for MGK 264..................................................................8
Table 2. Estrogenic/Anti-Est rogenic Pathway for MGK 264....................................10
Table 3. Androgenic/Anti-A ndrogenic Pathway for MGK 264.................................14
Table 4. Thyroid Pathway for MGK 264...........................................................18

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................41MGK 264 FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationMGK 264 FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationMGK 264 FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)MGK 264 FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceMGK 264 FINAL

Page 1 of 49 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality, were considered in this weight of evidence (WoE) assessment.

In determining whether MGK 264 interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not the dose(s) at which endocri ne related responses occurred happened concurrently wi th general systemic toxicity.

On November 19, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of MGK 264 with the E, A,or T hormone path ways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For MGK 264, there was evidence of potential interaction with the estrogen pathway in
mammals but not in fish. In the in vitro Tier 1 assays, MGK 264 was negative for estrogen receptor (ER) binding, steroidogenesis and ER transactivation assay (ERTA). MGK 264 was positive for inhibition of aromatase activity. In the in vivo Tier 1 assays, MGK 264 was negative for estrogenic activity in the ut erotrophic assay. In the fish short-term reproduction assay (FSTRA), there were no treatment -related effects observed in fema les. In the female pubertal assay, delayed vaginal opening (VO) and delaye d mean age of first estrus, and decreased pituitary weight was observed at a dose of 800 mg/kg/day (near the limit dose). In a post-1998 two-generation reproduction toxicity study in rats (EDSP Tier 2 st udy), treatment-related effects seen also at a very high dose (952 mg/kg/day) included: decr eased absolute and relative ovary/oviduct weights in the parental (P) generati on; altered luteinization of the corpora lutea in
the P and F 1 offspring with an increased number of F 1 females in met-/diestrus cycle; a minor but significant decrease in implantations with conc omitant lower mean litter size at birth in F 1 pups; and an increase in VO by 4.2 days in the absence of alterations in body weights. There were no estrogenic/anti-estrogenic effects noted in th e other Part 158 mammalian toxicity studies.MGK 264 FINAL

Page 2 of 49 For the androgen pathway, there was no convincing evidence of potential interaction of MGK 264. It was negative in the in vitro [(androgen receptor (AR) bindi ng and steroidogenesis)] and in

vivo Tier 1 assays (Hershberger and male pubertal assays). In the FSTRA, while there was an effect on GSI, there were no other androgen-re lated effects observed. There were no androgenrelated effects observed in any of the Part 158 mammalian studies.

There was evidence for potential interaction of MGK 264 with the thyroid pathway in mammals but not in amphibians. In the pubertal assays, hi stopathology showed decreased colloid area with a concomitant increase in follicular cell height in both males and females. However, there were
no effects of MGK 264 noted on serum T 4 or thyroid stimulating hormone (TSH) levels or on thyroid weights in either assa ys. Minor but significant decr eases (p<0.05) in absolute ( ↓ 13) and relative ( ↓ 16%) pituitary weights were al so noted at the high dose in the female pubertal assay. In the post 1988 two-generation reproduction study in rats, histopathology revealed increased incidences of thyroid gland follicular cell hypertrophy in both generations and increased incidences of colloidal alteration in the P, F 1 males and F 1 females. In a chronic toxicity/ carcinogenicity study in the rat, increased inciden ces of thyroid follicular tumors were see in males. There were no thyroid-related effect s observed in the remaining Part 158 mammalian studies. In the amphibian metamorphosis assa y (AMA), while there was an increase in normalized hind limb (HLL) on day 21, there were no treatment-related effects on HLL on day 8, developmental stage, or thyroid histopathology (which wa s relatable to a thyr oid-related effect). The effect on day 21 limb length does not appear to be thyroid related.

In summary, there was evidence of potential interaction of MGK 264 with the estrogen and thyroid pathways in mammals but not in fish or amphibians. There wa s no evidence of potential interaction with the androgen path way either in mammals or fish.

In the EDSP Tier 1 male and female pubertal andOSRI, there was evidence for potential interaction with the thyr oid pathway in studies conducted with adult animals, but no such data exists for potential thyroid t oxicity in the young animals.

For MGK 264, the current Point of Departure (POD) of 6.1 mg/kg/day used for human health risk assessment is based on hepatocellular hypertrophy, the most sensitive endpoint, and decreased body weight. This POD was used for de riving the chronic Reference Dose (RfD) used for dietary risk assessment and calculating th e Margins of Exposure (MOEs) used for nondietary risk assessments. This POD is approximate ly 50 to 160-fold lower than the doses (range, 350 to 950 mg/kg/day) that caused estrogen- and thyroid-related effects in the pubertal assays.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the case of MGK 264, althoug h no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommendedMGK 264 FINAL

Page 3 of 49 since: 1) the POD is approximately 50 to 160-fold lower than the doses (range, 350 to 950 mg/kg/day) that caused hist opathological lesions of the thyroid glands in the Tier 1 pubertal assays and OSRI; 2) the POD is based on the more sensitive endpoint (hepatotoxicity) which mediates the thyroid toxic ity; 3) there were no effects of MGK 264 seen on serum T 4 or TSH levels or on thyroid weights in either assays; 4) the CTA woul d be tested at higher doses and would not be expected to yield a POD lower than the level currently used for risk assessment; and 5) the chronic RfD and MOEs are protective of the endocrine and/or thyroid related findings
in the Tier 1 in vivo assays and OSRI.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for MGK 264 since additional testi ng is not expected to impact EPA’s current regulatory point of departures a nd endpoints for human health risk assessments. As there was no evidence of potential interaction with the estr ogen, androgen, or thyroid pathway for wildlife (non-mammals), ESSP Tier 2 te sting is not recommended for wildlife (non-mammals).MGK 264 FINAL

Page 4 of 49 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality, were considered in this weight of evidence (WoE) assessment.

In determining whether MGK 264 interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not the dose(s) at which endocri ne related responses occurred happened concurrently wi th general systemic toxicity.

On November 19, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs and the Offi ce of Science Coordination and Policy conducted a weight-of-evidence (WoE) analys is of the potential in teraction of MGK 264 with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE eval uation in this report are presented by pathway (E, A and then T) be ginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for mammalian and wildlife studies ( 40 CFR Part 158 and literature).

MGK 264 has a water solubility of 13.7 mg/L (at 25° C) and a octanol:water partition coefficient (log K ow ) of 3.70. MGK 264 is stable to hydrolysis , direct aqueous photolysis, and soil photolysis. Aerobic and anaerobic soil metabolism are very slow (mean aerobic half-life was 341 days). Volatilization from soil or water is not expected to be important, based on the Henry’s Law constant ( 4.44 × 10 -7 atm·m 3 ·mol -1 ). MGK 264 was very mobile to moderately mobile in sand and sandy loam soils (K oc = 636 in sand) and essentially immobile in silt loam and clay loam soils (K oc = 3106 in clay loam).

The available information considered in determ ining the potential intera ction of MGK 264 with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.MGK 264 FINAL

Page 5 of 49 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 Screening assays were submitted as OSRI by the Test Order recipients. This included studies data submitted to support pesticide registration (e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the Re port of the Endocrine Disruptor Review Team on MGK 264 (USEPA, 2010). Since then, the Agency has also conducted a more recent search of
available scientific literature fo r any additional relevant informa tion. Summaries of the available OSRI are presented in Appendix 2.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and otherMGK 264 FINAL

Page 6 of 49 effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization?. Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic t oxicity?. The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in theMGK 264 FINAL

Page 7 of 49 presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g , lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.MGK 264 FINAL

Page 8 of 49 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1

. Table 1. Tier 1 Screening Assays for MGK 264

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirment Satisfied (MRID 48618003)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirment Satisfied (MRID 48618004)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirment Satisfied (MRID 48618001)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirment Satisfied (MRID 48672301)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirment Satisfied (MRID 48618002)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirment Satisfied (MRID 48618006)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirment Satisfied (MRID 48618005)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirment Satisfied (MRID 48686301)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirment Satisfied (MRID 48686301)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirment Satisfied (MRID 48923701)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirment Satisfied (MRID 48701701)MGK 264 FINAL

Page 9 of 49 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for MGK 264 to interact with the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

The Tier 1 in vitro ER binding, ERTA and steroidogenesis and in vivo uterotrophic assays were negative. MGK 264 was positive for aromatase inhibition with an estimated IC 50 of 10

− 4.7 M.

In the FSTRA, while it is noted that female fi sh (including controls) had findings of oocyte atresia (5-14%) sometimes associated with in flammation and presence of protozoal spores, fecundity was adequate in the assay, and there were no treatment-related effects observed (including fecundity) for the fema les up to 0.107 mg a.i./L, the hi ghest concentration tested.

In the female pubertal rat assay at 800 mg/ kg/day, initial and complete VO were delayed (p<0.001) by 3.1 and 3.4 days, respectively, and mean age of first estrus was delayed (p<0.05) by 2.5 days. Additionally, unadjusted and adjusted body weights at VO were increased (p<0.01) by 16-17% suggesting that the delay in VO was not a growth effect. In addition, a 13% decrease in ovarian and pituitary weight was seen at the high dose. Increased absolute, adjusted and relative liver weights was observed at both 400 and/or 800 mg/kg/day.

In the 2009 Part 158 two-generation reproduction t oxicity study in rats, effects observed in females at the high dose (952 mg/kg/day) included decreased absolute and relative ovary/oviduct weights (16-20%) in the P generati on, altered luteinization of the corpora lutea in the P (8/25)
and F 1 (11/25) females compared to 0/25 cont rols, with an increased number of F 1 females in met-/diestrus cycle at the high dos e (16/25 treated vs. 8/25 controls ). Also, there was a small but significant decrease in implantations with conc omitant lower mean litter size at birth in F 1 pups. At the high dose, the age at VO was increased by 4.2 days with body weights at VO unaffected by treatment. Additionally, increased absolute liver weights with corroborating gross and microscopic liver findings, increa sed kidney weights with increased storage of brownish pigment in the cortical tubules were observed at the mid and/or high dose in this study.

There were no estrogenic/anti-estrogenic effects not ed in the other Part 158 mammalian toxicity
studies.MGK 264FINALPage 10 of 49Table 2: Estrogenic/Anti-Estrogenic Pathway for MGK 264Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for MGK 2641Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48618003)NERTA(MRID 48618004)N4Aromatase(MRID 48618002)PSteroidogenesis(MRID 48672301)NUterotrophic(MRID 48618006)NNX5(H)Female Pubertal Rat(MRID 48686301)N↓13%(H)N↓13%(H)N↑3.4 dwt:↑17%(H)6liver wt.:↑26, 44%(L, H)NFSTRA(MRID 48923701)NENNNNNNOSRITwo-GenerationReproduction Toxicity(Rat; MRID 48084901)N↓16-20%(H)P7(H)NP8(H)↑4.2 d↑wt(H)P9(H)X(M, H)NTwo-GenerationReproduction Toxicity(Rat; MRID 42155701)NE NE N NE NNX(L, M, H)NDevelopmentalToxicity (Rat; MRID41660302)NX(H)X(H)MGK 264FINALPage 11 of 49Table 2: Estrogenic/Anti-Estrogenic Pathway for MGK 264Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for MGK 2641Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3DevelopmentalToxicity (Rabbit;MRID 40352301)NX(H)NChronic Toxicity/Carcinogenicity (Rat;MRID 43005301)NE N N NEX(M, H)X(H)Carcinogenicity(Mouse; MRID42093802)NE N N NEX(M, H)NChronic Toxicity (Dog;MRID 42148102)NE N N NX(H)NSubchronic OralToxicity (Rat; MRID42970601)NE N NE NEX(L, ML, M,MH)X (MH,H)Subchronic InhalationToxicity (Rat; MRID43309001)NENNENEX(L, ML,MH, H)NSubchronic DermalToxicity (Rabbit;MRID 42742201)NENENNEN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presented in this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. AMGK 264FINALPage 12 of 494. Although the responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected values, MGK 264 responseswere similar to those of the negative control corticosterone (0.7-3.9% of positive control (PC)) and not comparable to the responses of 17αmethyltestosterone, which was able to reach a maximum of 8.1-52.8% PC. MGK 264 was only able to reach a maximum of 1.2-5.2% PCwhen tested up tothe highest concentration possible based on cytotoxicity. The reference chemicals 17β-estradiol and 17α-estradiol (weak estrogen) responded as expectedin both runs. Because the RPCMax< PC10in both assay runs, MGK 264 was considered negative in this assay.5. One animal from the 1000 mg/kg/day group was euthanized moribund on Day 3, and terminal body weights were decreased (p<0.05)by 10% at 1000mg/kg/day. In addition, animals in the 1000 mg/kg/day group exhibited adverse clinical signs of toxicity (uncoordinated movement, decreased movement,hunched posture, twitching, piloerection, ungroomed appearance, a red nasal discharge, and/or diarrhea) following dosing.6. At 800 mg/kg/day, ages at initial and complete VO were delayed (p<0.001) by 3.1 and 3.4 days, respectively, and mean age offirst estrus was delayed7. In the female genital tract, altered luteinization of thecorpora lutea was observed at 6400 ppm in the P (8/25) and F1(11/25) animals compared to 0/25controls.8. There were an increased number of F1females in met-/diestrus cycle at 6400ppm (16/25 treated vs. 8/25 controls).9. At 6400 ppm, there was a small but significant decrease inimplantations with concomitant lower mean litter size at birth inF1pups. Although the meanlitter size is within the historical control range, a relationship to treatment cannot be ruled out due to the histopathological findings (altered luteinization) inthe parental females.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedMGK 264 FINAL

Page 13 of 49 2. Effects on Androgen Pathway

The potential for MGK 264 to interact with the androgen signaling pathway is summarized in

Table 3. The various targets of the androgen pathwa y across the relevant Tier 1 assays are
delineated so as to facilitate determination of potential for androgenic, anti-androgenic or HPG
axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of ov ert toxicity are discussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

In the in vitro Tier 1 assays, MGK 264 was negative in both the AR binding and steroidogenesis assays. In the in vivo Tier 1 assays, MGK 264 was negati ve in the Hershberger for both androgenic and anti-androgenic activity, and there were no treatment-related effects on androgen-related endpoints in the male rat pubertal assay. In the FSTRA, male GSI was increased (p<0.05) by 45% at the hi ghest concentration tested (0.107 mg a.i./L). It is noted that gonadal histopathology findings (e.g, increases in testicular degene ration) were reported in all treatments (including controls); however, they did not appear to be treatment-related and all other endpoints, except GSI, we re unaffected by treatment.

In the 2009 Part 158 two-generation reproductio n toxicity study in rats (MRID 48084901),
absolute and relative seminal ve sicle weights were increased (p <0.05) by 9-10% at the highest dose tested (650 mg/kg/day); however, the effect occu rred at a dose that resu lted in overt toxicity (>10% decrease in body weight). No other androge nic effects were noted in the study. There were no androgenic or anti-androgenic effects ob served in any of th e remaining Part 158
mammalian studies.MGK 264FINALPage 14 of 49Table 3: Androgenic/Anti-Androgenic Pathway for MGK 264Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for MGK 2641Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48618001)NSteroidogenesis(MRID 48672301)NHershberger(MRID 48618005)NENNNMale Pubertal Rat(MRID 48686301)NNNNNNNNX4liver wt.:↑23, 39%(L, H)NFSTRA(MRID 48923701)NE↑45%GSI(H)NNNNNOSRITwo-GenerationReproductionToxicity (Rat;MRID 48084901)NNNNNSV↑9-10%(H)NX(L, M, H)NTwo-GenerationReproductionToxicity (Rat;MRID 42155701)NENNENNENEX(L, M, H)X(H)Chronic Toxicity/Carcinogenicity(Rat; MRID43005301)NNNENNNEX(L, M, H)NMGK 264FINALPage 15 of 49Table 3: Androgenic/Anti-Androgenic Pathway for MGK 264Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for MGK 2641Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRID42093802)NNNENNENX(M, H)NChronic Toxicity(Dog; MRID42148102)NNNENENENEX(H)NSubchronic OralToxicity (Rat;MRID 42970601)NNENENENENEX(L, ML, M,MH)X(MH,H)SubchronicInhalation Toxicity(Rat; MRID43309001)NNNNNENEX(ML, MH,H)NSubchronic DermalToxicity (Rabbit;MRID 42742201)NNNENNENEN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.). Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate(DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. At 350 and 700 mg/kg/day, increased (p<0.05)absolute, adjusted, and relative liver weights (↑20-23% and↑39%, respectively). Additionally at 700mg/kg/day, increases (p<0.05) were noted in relative kidney weights (↑8%) and serum BUN, SDH, and total protein.P Positive findingsN Negative findings (in vitro)/No effect (in vivo);NE Not examinedMGK 264 FINAL

Page 16 of 49 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal male and female (rat) assays, and the AMA (frog).

The potential for MGK 264 to interact with the thyroid signaling pathway is summarized in

Table 4. The various targets of the thyroid pathway across the re levant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

In the EDSP Tier 1 pubertal assays, decreased colloid area with a concomitant increase in follicular cell height was observed microscopically at both dose levels in both the male (350 and 700 mg/kg/day) and female (400 and 800 mg/kg/day) pubertal assa ys. However, there were no
effects of MGK 264 noted on serum T 4 or TSH levels or on thyroid weights in either assays, and systemic toxicity, presenting primarily as increas ed liver weights, was noted at both doses in both assays. Small decrease s (p<0.05) in absolute ( ↓ 13) and relative ( ↓ 16%) pituitary weights were also noted at the high dose in the female pubertal assay.

In the AMA, nearly all exposed individuals were developed beyond NF stage 60 by Day 21 (referred to as ‘late-stage’). The only eff ect observed was an increase (p<0.05) of 28% in normalized hind limb length (HLL) at Day 21 at th e highest concentration (0.106 mg a.i./L). All other parameters were unaffected by treatment. Animals were stage matched to the greatest extent possible for thyroid histopathological evaluation. Histopathol ogical findings included mild follicular cell hypertrophy and hyperplasia in the negative control a nd all treatment groups. Specifically, for follicular cell hypertrophy, the numb er of mild diagnoses in the control group (7) was higher than the number for the mid a nd high treatment groups (6 and 5, respectively). Similarly for follicular cell hyperplasia diagnoses, th ere were more diagnoses of mild severity in the control group (5) than in the treatment groups (2, 1, and 0, respectively). Although a
decrease in the incidence of follicular ce ll hypertrophy or hyperplasia was observed in the treatment groups relative to the control, given that there we re no effects on developmental stage or effect on hind limb length on Day 7 (which may be more indicative of a potential interaction with the thyroid than on Day 21), the effects obs erved do not appear to be thyroid-related.

In a 2009 Part 158 two-generation reproduction study in rats, increased incidences of thyroid gland follicular cell hypertrophy were observed in both generations in the mid dose males and high dose females (159 and 952 mg/kg/day, respectiv ely). Increased incidences of colloidal
alteration were noted in the P males at the high dose (650 mg/kg/day), in the F 1 males at the mid and high dose, and in the F 1 females at the high dose. Overt and systemic toxicity, presentingMGK 264 FINAL

Page 17 of 49 primarily as decreased body weight s with increased liver weights, were also observed at these dose levels. In a chronic toxicity/ carcinogenicity study in the rat, an increase in thyroid follicular
tumors in males were observed in males. Th e incidence for adenomas alone were 6.7%, 8.3% and 10% at 50, 150, and 450 mg/kg/day, respectively, compared to 1.7-3.3% in the controls (n=60/group). The incidence for combined adenomas and carcinomas were 10%, 11.7% and 15% at 50, 150, and 450 mg/kg/day, respectively, co mpared to 1.7-6.7% for the controls (n=60/group). Again, overt and systemic toxi city, presenting primarily as decreased body weights were noted at 450 mg/ kg/day and increased liver weight s were observed at 50, 150, and 450 mg/kg/day. There were no thyroid-related ef fects observed in the remaining Part 158
mammalian studies.MGK 264 FINAL

Page 18 of 49 Table 4. Thyroid Pathway for MGK 264

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for MGK 264 1

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48686301) N P 4 (L, H) N N N X 5
liver: ↑ 23, 39%

(L, H) N

Female Pubertal Rat (MRID 48686301) N P 6 (L, H) N N ↓ 13% (H) P 7
liver: ↑ 26, 44%

(L, H) N

AMA (MRID 48701701) N HLL ↑ 28 % (H) 8 N N

OSRI

Two-Generation Reproduction Toxicity (Rat; MRID 48084901) N P 9 (M, H) N X (L, M, H) N

Chronic Toxicity/ Carcinogenicity (Rat; MRID 43005301) NE P 10 (M, H ♂ ) NE X (L, M, H) X ( ♀ H)

Carcinogenicity (Mouse; MRID 42093802) NE N NE X (M, H) N

Chronic Toxicity (Dog; MRID 42148102) N N N X (H) N

Subchronic Inhalation Toxicity (Rat; MRID 43309001) NE N NE X (ML, MH, H) N

Subchronic Dermal Toxicity (Rabbit; MRID 42742201) NE N NE N N

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment, Medium treatment (M), High treatment (H). Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV.MGK 264 FINAL

Page 19 of 49 4. Mean colloid area was decreased (p<0 .05) at 350 mg/kg/day; at 700 mg/kg/day, the incidence/grade of mean colloid area at 700 mg/kg/day was similar (NS) to that observed at 350 mg/kg/day. Follicular cell height was slightly increased (NS) at 350 and 700 mg/kg/day. Ther e was an increased incidence of minimal to moderate eosinophilic colloid in the thyroids of the 350 and 700 mg/kg/day males. 5. At 350 and 700 mg/kg/day, increased (p<0.05) absolute, adjusted, and relative liver weights ( ↑ 20-23% and ↑ 39%, respectively). Additionally at 700 mg/kg/day, increases (p<0.05) were noted in relative kidney weights ( ↑ 8%) and serum BUN, SDH, and total protein. 6. Mean colloid area was decreased (p<0.05) and mean fo llicular height was increased (p<0.05) in the 400 and 800 mg/kg/day groups, with an increased inciden ce of minimal to moderate eosinophilic colloid. 7. Absolute, adjusted, and relative liver weights were increased (p<0.001) by 21-26% and 40-44% at 400 and 800 mg/kg/day, respectively. 8. The increase (Jonckheere-Terpstra, p< 0.05) in normalized HLL (HLL) based on analysis using late stage only individuals (NF stage ≥ 60) as nearly all animals were ‘late-stage’ by Day 21. 9. In the thyroid glands, increased incidences of follicular cell hypertrophy were observed in both generations in the 1600 ppm males and 6400 ppm females. Increased incidences of colloidal alteration were noted in the P males at 6400 ppm, in the F 1 males at 1600 and 6400 ppm, and in the F 1 females at 6400 ppm. 10. Increased thyroid follicular tumors were seen in ma les with a response of 2 (3.3%), 1 (1.7%), 4 (6.7%), 5 (8.3%) and 6 (10%) for adenomas alone, and 4 (6.7%), 1 (1.7%), 6 (10%), 7 (11.7%) and 9 (15%) for combined adenomas and carcinomas for the two control, 50, 150 and 450 mg/kg/day dose groups respectively (n=60/dose group). P Positive findings N Negative findings NE Not examinedMGK 264 FINAL

Page 20 of 49 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of MGK 364 to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing.. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) is sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).MGK 264 FINAL

Page 21 of 49 1. Tier 1 in vivo Assays

Toxicity was not observed in the AMA at concentr ations up to 0.106 mg a.i./L, in the FSTRA at concentrations up to 0.107 mg a.i./L, or in the Hershberger assay at dose levels up to 850 mg/kg/day.

In the pubertal rat assays, systemic toxicity, pres enting primarily as increa sed liver weights, was noted in both the male (350 and 700 mg/kg/day) and female (400 and 800 mg/kg/day) pubertal

assays.

In the uterotrophic assay, animals in the 1000 mg /kg/day group exhibited adverse clinical signs of toxicity (uncoordinated movement, d ecreased movement, hunched posture, twitching, piloerection, ungroomed appearance, a red nasal discharge, and/ or diarrhea) following dosing. Additionally, one animal from the 1000 mg/ kg/day group was euthanized moribund on Day 3. Body weights were decreased (p<0.05) by 10% on Day 4 in the 1000 mg/kg/day group compared to the vehicle controls, resulting in a mean overall body weight loss (p<0.05) of

− 8.9 g, compared to the vehicle c ontrol group (16.5 g gain).

2. OSRI

In a two-generation rat reproduction toxicity study, body weight decreases (p<0.05) during premating were noted at 1250 ( ↓ 5-10%), 2500 ( ↓ 5-7%), and 10,000 ( ↓ 9-15%) ppm in the F 1 dams and at 2500 ( ↓↓ 6-8%) and 10,000 ( ↓ 6-10%) ppm in the P dams (equivalent to 61/73, 125/151, and 649/759 mg/kg/day in ma les/females). In the F 1 males, body weighs were decreased (occasionally p<0.05) at 2500 ( ↓ 4-8%) and 10,000 ( ↓ 9-20%) ppm. Hepatocellular hypertrophy
was evident at all doses in the F 1 parental group and at higher le vels in the P parental group. Decreased body weight during lact ation was evident at all doses with there being decreases of about 5% to 14% (all p < 0.05) at 1,250 ppm, 5% to 13% at 2,500 ppm and 26% to 31% at 10,000 ppm. Following lactation, the pups gained weight comparable to the controls.

In a post-1998 two-generation rat reproduction toxicity study (ED SP Tier 2 study), systemic and overt toxicity were obser ved at 1600 and 6400 ppm (equiva lent to 159/225, and 650/952 mg/kg/day in males/fe males). The 6400 ppm F 1 group body weights and body weight gains were decreased (p<0.05) and relative (to body wei ght) food consumption was increased (p<0.05) during pre-mating. Body weights were decreased (p<0.05) in the 1600 ppm F 1 females at the end of the second gestation, and at 6400 ppm at the end of both ge stations and on post-natal day (PND) 4. Relative food consumption was dose- dependently increased in the 1600 and 6400 ppm

F 1 dams during the 2 nd mating. Absolute and relative liver weights were dose-dependently increased, with corroborating gross and micros copic liver finding in both sexes and/or generations. Microscopic findings were noted in the kidney at 1600 ppm and above in both sexes and/or generations. In the F 2a litters, small pups relative to ot her pups in the litter were observed with a higher frequency at 6400 ppm. A dditionally at 6400 ppm, decreased (p ≤ 0.05) offspringMGK 264 FINAL

Page 22 of 49 body weights were observed in both sexes in the F 1 and F 2b litters and in the F 2a females on PND 21 and 28 ( ↓ 11-17%), and body weights were decreased by 14% (p<0.05) on PND 14 in the F 2b females.

In a rat developmental toxicity study, increased salivation was not ed at 1000 mg/kg/day as a predose observation in four dams and post-dosing in 23 dams. Body weight gains were decreased by 25% (p<0.01) during GD 6-15 at 1000 mg/kg/da y. The pregnancy rate was decreased at 1000 mg/kg/day (68% treated vs. 80% controls); how ever, there was no corroborating evidence of effects on fertility.

In a rabbit developmental toxicity study, an increase in the inci dence of excessive salivation was seen at 100 mg/kg/day (44% of the does vs. 0% in the controls); this occurred on consecutive days in the middle of the dosing period and is considered a response to treatment.

In a rat chronic toxicity/carci nogenicity study, final body weights were decreased 8-9% in the males and 18-21% in the females at the high dos e (450 mg/kg/day). Decreases in body weight gains were also observed at this dose, reachi ng 58% in males and 50% in females during Weeks 52-78. Food consumption was also generally decreas ed in the 450 mg/kg/day females. Absolute liver weights were increased (p< 0.05) by 14-45% in the males at ≥ 50 mg/kg/day and by 9-22%
in females at ≥ 150 mg/kg/day. At 150 mg/kg/day and above, pathological changes were observed in the liver (hepatocyt e hypertrophy in both sexes and por tal bile duct proliferation and bile stasis, spongiosis hepatitis a nd cysts in females) and kidney (brown pigment in females). At 450 mg/kg/day, additional pathology was noted in the liver (bile duct prolifera tion, cysts and altered eosinophilic foci in males and spongiosis hepatitis in males) and kidney (brown pigment and cysts in males); and kidney, heart, and brain weights were increased.

In a mouse carcinogenicity study, at 400 mg/kg/da y and above, absolute and relative (to body) liver weights were dose-dependently increase d (p<0.01) in the males by 13-37% and 22-46%, respectively, compared to the controls. In the fe males, only relative (t o body) liver weight at these doses was significan tly (p<0.01) increased ( ↑ 11-16%), although absolute liver weights were slightly elevated at 400 ( ↑ 8%) and 800 mg/kg/day ( ↑ 6%). At 400 mg/kg/day there was increased calculi in the gall bladde r and intrahepatic bile stasis in males. At 800 mg/kg/day there was an increase in hepatocellular hypertrophy, ca lculus in bile duct in males and females and portal bile duct proliferation, portal mononuclear cell infiltrati on, spongiosis hepatis, cysts and vacuolar change in males and hyperplastic nodules in males and females. Increases in liver
adenomas were also observed in the 400 a nd 800 mg/kg/day males. Females at 400 and 800 mg/kg/day also had adenomas and a carcinoma whic h did not reach statistical significance, but
these liver tumors are rare in female CD-1 mice.MGK 264 FINAL

Page 23 of 49 In a one-year dog chronic toxic ity study, the only effects noted were brown pigment deposition in the liver of both sexes (3/4 males and fe males vs. 0/4 in controls) and hepatocellular hypertrophy in 2/4 males seen at 1000 ppm dos e level (equivalent to 34.9/32.5 mg/kg/day in males/females).

In a rat subchronic toxicity study, at 1000 mg/kg/day, body weight s were decreased (p<0.05) by
14% in the males and 17% in the females at Week 13. Body weight gains were also decreased at 500 and 1000 mg/kg/day in both sexes compared to controls (  10% and  21% in males and  18% and  33% in females). Food consumption was frequently decreased (p<0.05) at 500 and 1000 mg/kg/day in males and females throughout the study. Mean total protein values and globulin levels were increased at 250 mg/kg/ day and above, and cholesterol levels were increased at 500 mg/kg/day and above in the males; mean total protein values, globulin levels,
and cholesterol levels were increased at ≥ 500 mg/kg/day in the females. Absolute and relative (to body) liver weights were in creased in both sexes at ≥ 250 mg/kg/day. At necropsy, enlarged liver
was noted in males at 500 and 1000 mg/kg/day.

In a rat subchronic inhalation toxici ty in rats, clinical signs of t oxicity observed were red facial
stains and decreased activit y. Metaplasia/hyperplasia/hypertr ophy of the mucosal epithelium were observed in the larynx, ventra l diverticulum, and nasoturbinat es, and in the goblet cells in the respiratory epithelium and nasopharynx.

No systemic toxicity was observed in a rabbit subchronic derm al toxicity study.

B. Estrogen Pathway

There was evidence of potential interaction of MGK 264 with the estrogen pathway in mammals
but not in fish. In the in vitro Tier 1 assays, MGK 264 was negative for ER binding, steroidogenesis and ERTA. MGK 264 was positive fo r inhibition of aromat ase activity. In the in

vivo Tier 1 assays, MGK 264 was negative for estrogeni c activity in the uter otrophic assay. In the FSTRA, there were no treatmen t-related effects observed in females. In the female pubertal assay, delayed VO and delayed mean age of firs t estrus, and decreased pituitary weight was observed at a dose of 800 mg/ kg/day (near the limit dose).

In a two-generation reproduction toxicity study in rats conducted under the 1998 revised test guidelines, treatment-related eff ects seen also at a very high dose (952 mg/kg/day) included:
decreased absolute and relative ov ary/oviduct weights in the P genera tion; altered luteinization of the corpora lutea in the P and F 1 offspring with an in creased number of F 1 females in met- /diestrus cycle; a minor but significant decrease in implantations with concomitant lower mean
litter size at birth in F 1 pups; and an increase in VO by 4.2 days in the absence of alterations in body weights. There were no estrogenic/anti-es trogenic effects noted in the other Part 158 mammalian toxicity studies.MGK 264 FINAL

Page 24 of 49 C. Androgen Pathway

There was no convincing evidence of potential in teraction with the androgen pathway. MGK 264 was negative in the in vitro (AR binding and steroidogenesis) and in vivo Tier 1 assays (Hershberger and male pubertal assays). In the FSTRA, while there was an effect on GSI, there were no other androgen-related effects obser ved. There were no andr ogen-related effects observed in any of the Part 158 mammalian studies.

D. Thyroid Pathway

There was evidence for potential interaction of MGK 264 with the thyroid pathway in mammals but not in amphibians. In the AMA, while there was an increase in normalized hind limb (HLL) on day 21, there were no treatment-related effects on HLL on day 8, developmental stage, or thyroid histopathology (which was relatable to a thyroid-related effect).The effect on day 21 limb length does not appear to be thyroid related.

In the pubertal assays, histopathology showed decr eased colloid area with a concomitant increase in follicular cell height in both males and female s. However, there were no effects of MGK 264
noted on serum T 4 or TSH levels or on thyroid weights in either assays. Minor but significant decreases (p<0.05) in absolute ( ↓ 13) and relative ( ↓ 16%) pituitary weights were also noted at the high dose in the female pubertal assay. In the post 1988 two-generation reproduction study in rats, histopathology revealed incr eased incidences of thyroid gl and follicular cell hypertrophy in both generations and increased incidences of colloidal alteration in the P, F 1 males and F 1 females. In a chronic toxicity/ carcinogenicity study in the rat, increased incidences of thyroid
follicular tumors were see in males. There were no thyroid related effects observed in the remaining Part 158 mammalian studies.

E. Conclusions

There was evidence of potential interaction of MGK 264 with the estrogen and thyroid pathways in mammals but not in fish or amphibians. Th ere was no evidence of pot ential interaction with the androgen pathway either in mammals or wildlife (non-mammals).

V. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 male and female pubertal andOSRI, there was evidence for potential interaction with the thyr oid pathway in studies conducted with adult animals, but no such data exists for potential thyroid t oxicity in the young animals.

For MGK 264, the current Point of Departure (POD) of 6.1 mg/kg/day used for human health risk assessment is based on hepatocellular hypertrophy, the most sensitive endpoint, and decreased body weight. This POD was used for de riving the chronic Reference Dose (RfD) used for dietary risk assessment and calculating th e Margins of Exposure (MOEs) used for nonMGK 264 FINAL

Page 25 of 49 dietary risk assessments. This POD is approximate ly 50 to 160-fold lower than the doses (range, 350 to 950 mg/kg/day) that caused estrogen- and thyroid-related effects in the pubertal assays.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the case of MGK 264, althoug h no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the POD is approximately 50 to 160-fold lower than the doses (range, 350 to 950 mg/kg/day) that caused hist opathological lesions of the thyroid glands in the Tier 1 pubertal assays and OSRI; 2) the POD is based on the more sensitive endpoint (hepatotoxicity) which mediates the thyroid toxic ity; 3) there were no effects of MGK 264 seen on serum T 4 or TSH levels or on thyroid weights in either assays; 4) the CTA woul d be tested at higher doses and would not be expected to yield a POD lower than the level currently used for risk assessment; and 5) the chronic RfD and MOEs are protective of the endocrine and/or thyroid related findings
in the Tier 1 in vivo assays and OSRI.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for MGK 264 since additional testi ng is not expected to impact EPA’s current regulatory point of departures a nd endpoints for human health risk assessments. As there was no evidence of potential interaction with the estr ogen, androgen, or thyroid pathway for wildlife (non-mammals), ESSP Tier 2 te sting is not recommended for wildlife (non-mammals).MGK 264 FINAL

Page 26 of 49 VI. References

Blair, M. (1989) 13 week diet ary range-finding toxicity st udy in rats with MGK 264. International Research and Development Co., Mattawan, MI. Laborator y Project Id. 551-028, August 9, 1989. Unpublished. (MRID 42970601)

Blair, M. (1991) One year diet ary toxicity study in dogs with MGK 264. International Research and Development Co., Mattawan MI. Laboratory Study No. 551-031, December 16, 1991. Unpublished. (MRID 42148102)

Blair, M. (1991) Eighteen month dietary oncogenicity study in mice with MGK 264 insecticide synergist. Internationa l Research and Development Co., Ma ttawan, MI. Labor atory Study No.: 551-011, June 13, 1991. Unpublished. (MRID 42093802)

Eiben, R. (2009) MGK 264: two-generation reproduc tion study in Wistar rats by administration in the diet. Bayer Shering Pharma AG, W uppertal, Germany. Laboratory Report/Study No. AT05546/T1073335, September 30, 2009. Unpublished. (MRID 48084901)

Fort, D. (2012) MGK 264: 21-Day amphibian metamorphosis assay. Fort Environmental Laboratories, Inc., Stillwater, OK. Sponsored by McLaughlin Gormley King Co., Minneapolis, MN. Laboratory Report No. MGK01-00273, July 24, 2012. Unpublished. (MRID 48701701)

Fort, D. (2012) MGK 264: 21-Day fish reproduc tion assay. Fort Environmental Laboratories, Inc., Stillwater, OK; Golden P acific Laboratories, Inc., Fres no, CA; Experimental Pathology Laboratories, Inc., Sterling, VA. Sponso red by McLaughlin Gormley King Company, Minneapolis, MN. Laboratory Report N o. MGK01-00274, August 30, 2012. Unpublished. (MRID 48923701)

Goldenthal, E. (1993) 24-Month dietary chronic t oxicity and oncogenicity study in the rat with
MGK 264. International Research and Development Co., Mattawa n, MI. Laboratory Project No. 551-030, October 8, 1993. Unpublished. (MRID 43005301)

Lancaster, S., and Husband, R. (1993) MGK 264 s ynergist 13-week dermal toxicity study in the rabbit. Toxicol Laboratories Ltd., England. Laboratory Study No. MCA/17/92, March 19, 1993. Unpublished. (MRID 42742201)

Newton, P. (1993) A subchronic (3 -month) inhalation toxicity study of MGKR 264 insecticide synergist in the rat via whole body exposures . Unpublished study conducted by Pharmaco:LSR, East Millstone, NJ; Sponsored by McLaughlin Gormley and King Company. Laboratory Study No.: 91-8364. June 24, 1994. (MRID 43309001)

Schardein, J. (1987) Teratological study of MGK 264 insecticide syne rgist administered orally to
albino rabbits. International Research and De velopment Corporation, Mattawan, MI. SponsoredMGK 264 FINAL

Page 27 of 49 by McLaughlin Gormley King Company, Minnea polis, MN. Laboratory Project Id. 551-015, August 31, 1987 Unpublished. (MRID 40352301)

Schardein, J. (1990) Developmental toxicity study in rats. Intern ational Research and Development Corporation, Mattawan, MI. S ponsored by McLaughlin Gormley King Company, Minneapolis, MN. Laboratory Identificati on No. 551-026, June 8, 1990. Unpublished. (MRID 41660302)

Schardein, J. (1991) Two-generation reproduc tion study of MGK 264 in the albino rat. International Research and Development Corpor ation, Mattawan, MI. Laboratory Study No. 551- 027, December 18, 1991. Unpublished. (MRID 42155701)

USEPA (2010) MGK 264 - Report of the Endocrine Disruptor Review Team - Test Order#:
EDSP- 057001- 250. Office of Chemi cal Safety and Pollution Pr evention Science Coordination and Policy. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: evaluating results of EDSP Tier 1 screening to identify th e need for Tier 2 tes ting. Office of Chemical Safety and Pollution Prevention Science Coordina tion and Policy. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H. (2012) MGK 264: H295R steroidoge nesis assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9146V-100355STER, Janua ry 26, 2012. Unpublished. (MRID 48672301)

Wilga, P. (2012) MGK 264: human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9146V-100355AROM, January 11, 2012. Unpublished. (MRID 48618002)

Willoughby, J. (2012) MGK 264: androgen receptor bi nding (rat prostate cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Project Study No. 9146V- 100355ARB, January 11, 2012. Unpublished. (MRID 48618001)

Willoughby, J. (2012) MGK 264: estrogen receptor bi nding (rat uterine cy tosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Project Study No. 9146V- 100355ERB, January 11, 2012. Unpublished. (MRID 48618003)

Willoughby, J. (2012) MGK 264: estrogen receptor tr anscriptional activati on assay (human cell line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9146V-100355ERTA, January 11, 2012. Unpublished. (MRID 48618004)MGK 264 FINAL

Page 28 of 49 Zorrilla, L. (2012) The hershberger bioassay fo r MGK 264 Technical. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No. C202-200, January 11, 2012. Unpublished. (MRID 48618005)

Zorrilla, L. (2012) The uterotrophic assay for MGK 264 Technical. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No. C202-100, January 11, 2012. Unpublished. (MRID 48618006)

Zorrilla, L. (2012). Pubertal development and thyroid function with MGK 264 in juvenile/peripubertal female and male rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study No. C202-300, April 12, 2012. Unpublished. (MRID 48686301).

Weight of Evidence for MGK®-264: Assessment of Results of The Endocrine Disruptor Screen Program Tier I Battery. McLaughlin Gormle y King Company 8810 Tent h Avenue North Minneapolis, MN 55427. MRID No. 49202101MGK 264 FINAL

Page 29 of 49 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 48701701) of MGK 264 ( 95.4% purity) on amphibian metamorphosis
of Xenopus laevis was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to a negative
control and nominal concentr ations of 0.011, 0.033 and 0.100 mg a.i./L. The time-weighted average (TWA) concentrations were < 0.100 (<LOQ), 0.0171, 0.0390, and 0.106 mg a.i./L. The test system was maintained at 21.4 to 21.8 o C and a pH of 7.3 to 7.9.

The survival of tadpoles exposed to MGK 264 was not significantly affected (p>0.05) as survival
was 100% in the control and low and mid treatmen t groups with one mortality occurring in the high treatment group. There were no clinical signs of toxicity or observations of abnormal behavior at any concentration.

MGK 264 caused no significant acceleration or de lay (p>0.05) of median NF developmental stage at Day 7 or 21 at any treatment level re lative to the negative control. Further, no asynchronous development was observed. Nearly all individuals devel oped beyond NF stage 60 by Day 21, with 95% (57/60), 87% (52/ 60), 82% (49/60), and 83% (49/59) NF ≥ 61 individuals in the control, low, mid, and high treatment groups, respectively. The NF stage ranged from 61 to 64 for late stage individuals, with a mode of NF stage 61 for all te st levels, including the control. All other individuals (non-late-stag e) were at NF stage 60 by Day 21.

There was a significant increase in normalized (for snout-vent length) hind limb length (HLL) for Day 21 late-stage only indivi duals, at the high treatment level (Jonckheere-Terpstra, p<0.05),
relative to the control. There we re no other significant increases or decreases determined for Day 7 normalized HLL or for Day 7 and Day 21 snout-vent length (SVL) and wet weight in late-stage tadpoles. A separate analysis conducted on thos e individuals not reac hing stage 60 did not determine any significant effects; however the stat istical power from this analysis is low due to the decreased number of tadpoles.

Histopathological findings included mild fo llicular cell hypertrophy and hyperplasia in the negative control and all treatment groups. Speci fically, for follicular cell hypertrophy, the number of mild diagnoses in the control group (7) was higher th an the number for the mid and high treatment groups (6 and 5, respectively). Sim ilarly for follicular cell hyperplasia diagnoses, there were more diagnoses of mild severity in the cont rol group (5) than in the treatment groups (2, 1, and 0, respectively).MGK 264 FINAL

Page 30 of 49 Although a majority of the surviving frogs were ≥ NF stage 61 at test termination which may confound the ability to adequately evaluate potential thyroid-rela ted effects in the AMA, there was no significant acceleration or delay of median NF developm ental stage at any treatment concentration throughout the test nor effects on thyroid histopathol ogy compared to the control.

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48618001), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of MGK 264 (95.4% a.i., Batch #AB1412). DMSO was used as the solvent at a final assay concen tration of approximately 3.2%. A total of three independent runs were performed, and the assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radio-labeled reference androgen (R1881) pr ior to conducting AR competitive binding experiments. Saturation binding data were not or iginally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agenc y. The mean dissociation constant (K d ) for [ 3 H]-
R1881 was 0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. The mean and individual K d and B max values were below the expected ranges reported in the EPA validation program (K d : 0.685 to 1.57 nM; and B max : 7 to 16 fmol/100 μg protein).

In the competitive binding experiment, precipit ation of MGK 264 was visually observed at the highest concentration (10

− 3 M) tested in 2 of the 3 runs. In the first valid run, the mean specific binding was 48.7% at 10

− 3 M (without apparent precipitation), and 50.3% and 42.6% in the 2 runs with precipitation noted at 10

− 3 M. As data at 10

− 3 M were available from only one run, and were suspect because of precipitation at this c oncentration in the othe r two runs, the highest
concentration used for data evaluation was 10

− 4 M. The specific binding in the presence of
MGK 264 was >75% at concentrations ≤ 10

− 4 M in all three runs. An IC 50 and Relative Binding Affinity (RBA) could not be calculated for MGK 264 as it did not result in 50% displacement at

≤ 10

− 4 M.

The estimated average log IC 50 was

− 9.0 M for R1881 and

− 4.6 M for the weak positive control (dexamethasone). Compared to R1881, the mean RBA for dexamethasone was 0.004%. All performance criteria were met for two of th e three runs. The Top % binding for MGK 264 and
dexamethasone were above the recommended toleran ce limit in the second run. However, as allMGK 264 FINAL

Page 31 of 49 criteria for the weak positive control were me t for the other two runs, it is unlikely that
information from an additional run would change the interpreta tion of this study.

Based on the results from the three runs, MGK 264 is classified as a Non-binder in the androgen receptor binding assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48618002), MGK 264 (95.4% a.i. Lot #AB1412) was incubated with human recombinant aromatase and tritiate d androstenedione ([1 β

- 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in 1% dimethyl sulfoxide (DMSO) at log
concentrations from 10

− 11 to 10

− 3 M for 15 minutes to determine the potential for MGK 264 to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritia ted water was quantified using liquid scintillation counting (LSC). For the three runs, each test concentration of MGK 264 was tested in triplicate. Each run also in cluded a full activity contro l, a background activity control, and a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN). Becau se the highest concentration of MGK 264 tested in Run 1 (10

− 3 M) showed precipitation during th e 15-minute incubation period, this concentration was not used for data interpretation. The highest successfully tested concentration
for all three runs that was used for data interpretation was 10

− 4 M.

Aromatase activity in the full act ivity controls was 0.611 ± 0.026 nmol

· mg-protein

− 1

· min

− 1 . The response of each full activity c ontrol within a run was between 90 to 110% of the average full activity. Activity in the backgr ound controls ranged from 0.25 to 0.37% and averaged 0.30% of the full activity control.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.27 M and the Hill slope was

− 0.94.

For MGK 264, aromatase activity averaged 0.603 ± 0.026 nmol

· mg-protein

− 1

· min

− 1 at the lowest tested concentration (10

− 11 M) and 0.062 ± 0.006 nmol

· mg-protein

− 1

· min

− 1 at the highest successfully tested concentration (10

− 4 M), corresponding to mean aromatase activity from 97.76% to 10.22% relative to the full activity control. The estimated log IC 50 for MGK 264 was

− 4.72 M and the estimated Hill slope was

− 1.0.MGK 264 FINAL

Page 32 of 49 MGK-264 showed >50% inhibition of aromatase act ivity at the concentra tions tested. Based on the data from the average response curve, MGK 264 is classified as an Inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48618003), uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct binding experiments. The competitive binding experiment measured the bindi ng of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (10

− 11 to 10

− 4 M) of MGK 264 (95.4% a.i., Batch #AB1412). DMSO was used as the solvent at a fina l concentration of 4%. A total of three runs were performed, and each run included 19- norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the na tural ligand reference
material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The protein concentrations used in the saturation binding runs varied between each run, and were approxima tely 3- to 6-fold greater than recommended (160 to 320 μg versus 50±10 μg). The K d for [ 3 H]-17 β -estradiol was 0.331±0.061 nM and the
estimated B max was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The K d for each run was within the expected Guideline range of 0.03 to 1.5 nM.

In the competitive binding experiment, the highest concentration tested in all three runs was

10

− 4 M based on the preliminary precipita tion assessment. The mean specific [ 3 H]-17 β -estradiol binding in the presence of MGK 264 was ≥ 85% at concentrations ≤ 10

− 4 M in all three runs. The estimated mean log IC 50 and Relative Binding Af finity (RBA) were not calculated for MGK 264 as the test chemical did not displace 50% of the radiolabeled ligand in any run.

The estimated mean log IC 50 was

− 9.0 M for the natural ligand (17 β -estradiol) and

− 5.5 M for the weak positive control (19-norethindrone). Compared to the natural ligand, the mean RBA was 0.030% for 19-norethindrone. All pe rformance criteria were met for 17 β -estradiol and 19- norethindrone; however, in two runs, octyltriet hoxysilane displaced greater than 25% of [ 3 H]-
17 β -estradiol from the ER at the highest concentration (10

− 3 M).

Based on the results from the three runs, MGK 264 is classified as Not Intera ctive in the estrogen receptor binding assay.MGK 264 FINAL

Page 33 of 49 ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48618004), hER α -HeLa-9903
cells cultured in vitro were exposed to MGK 264 (95.4% a.i ., Lot #AB1412) at logarithmically increasing concentrations from 10

− 11 to 10

− 4 M or 10

− 12 to 10

− 5 M in 0.1% DMSO for approximately 24 hours in two independent runs. The experiments were performed using 96-well plates and each MGK 264 concentration was tested in replicates of 6/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±2°C to induce report er (luciferase) gene products. Luciferase expression in response to ac tivation of the estrogen receptor was measured using a proprietary luciferase assay.

MGK 264 was tested up to the limit of cy totoxicity, which was observed at 10

− 4 M in the rangefinder and first run. The mean RPC Max for MGK 264 was 5.2% for the first run and 1.2% for the second run, and the associated PC Max was 10

− 7 M for the first run and 10

− 8 M for the second run.

In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, MGK 264 responses were similar to those of the negative control corticosterone (0.7-3.9% PC) and not comparable to the responses of 17 α -methyltestosterone,
which was able to reach a maximum of 8.1-52.8% PC. Cytotoxicity was observed at the highest
dose of 17 α -methyltestosterone tested (78.5% viability at 10

− 5 M). MGK 264 was only able to reach a maximum of 1.2-5.2% PC when tested up to the highest concentration possible based on cytotoxicity. The reference chemicals 17 β -estradiol and 17 α -estradiol (weak estrogen) responded as expected in both runs. Because the RPC Max < PC 10 in both assay runs, MGK 264 was considered negative for estrogen receptor tran scriptional activation in this test system.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (MRID 48923701) of MGK 264 with fathead minnows ( Pimephales promelas) was conducted under contin uous flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 4 groups/treatment; 4.5
- 6 months old) were exposed to MGK 264 (95.4% purity) at nominal concentrations of 0 (control), 0.011, 0.033, and 0.100 mg a.i./L; time-weighted av erage (TWA) concentrations were <LOQ (<0.001), 0.013, 0.036, and 0.107 mg a.i./L. The test system was maintained at 24.5 to 25.5 o C and a pH of 7.1 to 7.8.

Adult male and female survival were unaffect ed by treatment with MGK 264; overall survival
was 100% at all treatment levels. There were no nota ble observations with re gards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal napeMGK 264 FINAL

Page 34 of 49 pad in the control or treated groups or clinical signs of toxicity for any treatment group compared to the controls. Body weights and lengths in MGK 264-treated fish were similar to the control.

Spawning occurred at least two times in 7 days and fecundity in the negative control was 32.4 eggs/female/day/replicate (30.1 - 36.2); fertil ization success in the negative control group averaged 99.1% (99.0 - 99.2%). Male gonado-somatic index (GS I) was the only endpoint to exhibit a treatment-related effect; there was a significant 45% increase in male GSI at the high treatment level, relative to th e control (p<0.05; Dunnett’s test). There we re no statistically significant differences (p>0.05) in treated groups compared to negative control for any other endpoint (i.e., female GSI, and male and female vitellogenin and nuptial tubercle score).

Histopathology showed no treatment-related effects in males or females at any treatment level. However, it should be noted that female fish (i ncluding controls) had find ings of oocyte atresia (5-14%) sometimes associated with inflammati on (5-11%) and presence of protozoal spores. Male fish (including controls) had findings of increased proportion of spermatogonia (13-63%), and increases in testicular degeneration (38- 63%) sometimes associated with inflammation (13%) or interstitial cell (Le ydig) hypertrophy/hyperp lasia (25%) or a tes tis-ova (13%) or duct mineralization (25%). There were no statisticall y significant differences between gonadal stages in treated groups compared to the negative controls.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48618005) screen ing for androgenic activity, MGK 264 (95.4% purity, Batch/lot # AB1412) in corn oil was administered daily via or al gavage to groups of eight 61/62-day old, castrated male Sprague Dawley ra ts at dose levels of 0 (vehicle), 266, or 850 mg/kg/day. An androgenic positive control group consisted of eight castrated rats exposed to the vehicle plus 0.4 mg/kg/day of test osterone propionate (TP) by s ubcutaneous (s.c.) injection.

To screen for potential anti-andr ogenic activity, MGK 264 in corn oil was administered daily via oral gavage to groups of eight 61 /62-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 85, 266, or 850 mg/kg/day in conjunc tion with a daily dose of reference androgen, TP, at 0.4 mg/kg/day by s.c. injection. The an ti-androgenic positive control group consisted of eight castrated rats exposed to 3 mg/kg/day flutamide (FT, vi a oral gavage) and 0.4 mg/kg/day TP. The positive control group for the androgen a gonist assay was used as the anti-androgenic negative control group.MGK 264 FINAL

Page 35 of 49 For both components of the assay, body weights were determined da ily. The animals were dosed for 10 consecutive days and terminated a pproximately 24 hours after the final dose administration. At necropsy, the five androgen-de pendent tissues were co llected and weighed.

All animals survived until scheduled termination and no clinical signs of toxicity were observed in any animals 24 hours post dose. Body weight s and overall (Days 1-11) body weight gains in the MGK 264 treatment groups were similar to cont rols in both the androgen agonist and antiandrogen assays.

In the androgen agonist assa y, body weights on Day 11 in the pos itive control (vehicle + TP) group were increased (p<0.05) by 8%, and overall body weight gains were increased (p<0.05) by 63% compared to the vehicle controls. There we re no treatment-related increases in any of the accessory sex organ weights of MGK 264-dosed an imals. Animals in the positive control (vehicle + TP) group had accessory sex organ wei ght increases (p<0.05) as follows: 1120% in seminal vesicles; 1123% in ventral prostate; 220% in levator ani-bulbocavernosus muscle complex (LABC); 565% in Cowper’s glands; and 64% in glans penis.

In the anti-androgen assay, body weights and over all body weight gains in the positive control (FT + TP) group were comparable to the negative controls. There were no treatment-related decreases in accessory sex organ weights for all treatment groups in the anti-androgen assay. Animals in the positive control (FT + TP) gr oup had accessory sex organ weight decreases (p<0.05) as follows: 84% in seminal vesicles; 78% in ventral prostate; 55% in LABC; 60% in Cowper’s glands; and 25% in glans penis.

The dosing for this study (high dose of 850 mg/kg/day) was consid ered adequate based on data from an earlier study showing clinical signs of toxicity and decreased body weights at 1000 mg/kg/day. The performance cr iteria indicated that the assa y performed as expected.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissues. MGK 264 was negative for androgenicity and anti-androgenicity in the Hershberger

assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal assay (MRID 48686301), 16 Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily via oral gavage (5 mL/kg) with MGK 264 (95.4% a.i., Lot #: AB1412) in corn oil at doses of 0, 400, or 800 mg/kg/day from post-natal day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and ag e and weight at dayMGK 264 FINAL

Page 36 of 49 of attainment were recorded. Following s acrifice on PND 42 or 43, blood was collected for clinical chemistry and hormone analyses. Total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined using radioimmunoa ssays. Adrenals, liver, thyroid, pituitary, and urogenital organs were weighed and examined m acroscopically. The uterus, left ovary, left kidney, and thyroid were ex amined microscopically.

No adverse, treatment-related related effects were observed on surviv al, clinical signs, body weight, body weight gain, clini cal chemistry, gross pathology, or uterine, ovarian, or renal histology.

At 800 mg/kg/day, ages at initial and complete VO were delayed (p<0.001) by 3.1 and 3.4 days, respectively, and mean age of first estrus was delayed (p<0.05) by 2.5 days. Additionally, unadjusted and adjusted body weights at VO we re increased (p<0.01) by 16-17%. VO occurred in all animals, and mean cycle length, % cycling, % re gularly cycling, and the cycle status at necropsy were similar to controls at both doses.

Serum T 4 and TSH levels in the treated groups were similar to the controls. The following clinical chemistry parameters differed (p ≤ 0.05) in the treated groups co mpared to controls: (i) potassium decreased by 8-11% at both doses; (ii) chloride increased by 2% each at both doses; (iii) calcium decreased by 4% each at both doses ; (iv) creatinine decreased by 20% each at both doses; (v) SDH decreased by 37% at 400 mg/kg/ day; (vi) ALP decreased by 28% at 800 mg/kg/day; and (vii) total prot ein increased by 5% at 800 mg/kg/ day. These differences were minor and all clinical chemistry values were within the reported normal range, with the exceptions of phosphorus, AST, ALT, ALP, and SDH.

Significant decreases in mean colloid area and si gnificant increases in mean follicular height were observed in the thyroid gland in animal s administered 400 and 800 mg/kg/day MGK 264. There was an increased incidence of hyper-eosinoph illic colloid (minimal to moderate severity) in the thyroids of females treated with 400 and 800 mg/kg/day MGK 264. However, serum T4 and TSH levels were not affected, and there were no treatment-related e ffects on thyroid weight.

Absolute, adjusted, and relative liver weights were increased (p<0.001) by 21-26% and 40-44% at 400 and 800 mg/kg/day, respectively. Additio nally at 800 mg/kg/day, adjusted and relative pituitary weights were decreased (p<0.05) by 13 and 16%, respectively; absolute and adjusted ovary weights were decreased (p<0.05) by 13% e ach; and relative kidney weights were increased (p<0.05) by 5%. Absolute pituit ary weight at 800 mg /kg/day was also decreased by 13%, but this decrease was not statistically significant.

Although there were no adverse clin ical signs or significant decr eases in body weights observed at the high dose to indicate the high dose was at an adequate concentration, there was a positiveMGK 264 FINAL

Page 37 of 49 response (delayed VO and day of first estrus) observed at 800 mg/kg/day. Additionally, the findings observed at 1000 mg/kg/day in the ra nge-finding and uterotr ophic assays (mortality, abnormal behavior, lethargy, la bored breathing, piloerection, hunched posture, and mild twitching) indicate that th e high-dose of 800 mg/kg/ day was appropriate.

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal assay (MRID 48686301), 16 male Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily via oral gava ge (5 mL/kg) with MGK 264 (95.4% purity, Lot #AB1412) in corn oil at doses of 0, 350, or 700 mg/kg/day from post-natal day (PND) 23 to 53/54. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment were recorded. Following sacrifice on PND 53 or 54,
blood was collected for clinical chemistry and hormone analyses. Total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (T SH) levels were determined using radioimmunoassays. Adrenals, liv er, thyroid, pituitary, and urogeni tal organs were weighed and examined macroscopically. The thyroid, left kidn ey, left epididymides, and left testis were examined microscopically.

No treatment-related related effects were observed on survival, clinical si gns, body weights, final body weight, body weight gains, gross pathology, or testicular, epididymal, or renal histology.
No treatment-related related effects were observe d on body weight or age at PPS, age at partial PPS, proportion/incidence of un-separated preputia , androgen-dependent tissu e organ weights, or
serum testosterone levels.

The following clinical chemistry parameters differ ed (p<0.05) in the treated groups compared to controls: (i) potassium decrease d by 5-8% at both doses; (ii) ALP decreased by 17-19% at both doses; (iii) BUN increased by 17% at 700 mg/ kg/day; (iv) SDH in creased by 50% at 700 mg/kg/day; and (v) total prot ein increased by 7% at 700 mg/kg/day. Creatinine increased by 25% at both doses, but the incr ease was statistically significant only at 350 mg/kg/day. These differences were minor and, with the exception of ALP, were within the reported normal range. ALP levels at all doses, includ ing controls, exceeded the report ed normal range by 2.4-fold or more. The increase of ALP was slight, without clear dose-dependence, and was considered
incidental.

Absolute, adjusted, and relative liver weights were increased (p<0.001) by 20-23% and 39% at 350 and 700 mg/kg/day, respectively. Gross pathology and clinical chemistry did not indicate hepatotoxicity. Additionally at 700 mg/kg/day, re lative kidney weights were increased (p<0.01) by 8%, a minor effect. The adjusted kidney we ights showed a linear trend of increased (p ≤ 0.05) weight with increasing dose; howev er, no statistically significant effect was detected between theMGK 264 FINAL

Page 38 of 49 treated groups and control using Dunnett’s test. Therefore, th is linear trend was considered
incidental.

A significant decrease (p<0.05) in mean coll oid area was observed at 350 mg/kg/day. The incidence/grade of colloid area at 700 mg/ kg/day was similar to those observed at 350 mg/kg/day, but the finding did not attain statistical significance. Follicular cell height was slightly increased at 350 and 700 mg/kg/day, but did not attain statistical signi ficance. There was an increased incidence of hyper-eosinophillic co lloid (minimal to mode rate severity) in the thyroids of males treated with 350 and 700 mg /kg/day MGK 264. However, as serum T4 and TSH levels and thyroid weights were unaffected, the biological relevance of these histological changes is uncertain.

Although the animals could have tolerated a hi gher dose, the high dose (700 mg/kg/day) was considered adequate, based on the information provided from the 14-day range finding study and the Hershberger assay. The inve stigators assumed that the decreased final body weights noted at 1000 mg/kg/day after 14 days of administration ( ↓ 16%) and at 850 mg/kg/day after 10 days of administration ( ↓ 5%) may have been more severe after 31 days of administration in the male pubertal assay. Additionally, 700 mg/kg/day approached the limit dose of 1000 mg/kg/day, considering the lack of a steep dose-response curve. No clearly adverse effect was noted in this study, but a treatment-related ( possibly adaptive response) wa s observed on liver weight.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48672301), H295R cells cultured in vitro in 24-well plates
were incubated with MGK 264 (95.4% purity, Lot # AB1412) at log concentrations from 0.0001 to 100 μM for 48 hours in triplicate for four independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration in the assay of 0.05%. Because precipitation and cytotoxicity was observed at 100 μM of MGK 264, this concentration was not evaluated statistically. In addition, Run 1 was not analy zed because one of the forskolin concentrations on the quality c ontrol plate was incorrect (3.3 μ M was used instead of 1 μ M).

Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards. A Quality Control (QC) plate was run concurrently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to pos itive control agents at two concentration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including adequate production of testosterone and estrad iol, acceptable reproducibility (low %CV), and appropriate induction and inhibi tion with positiv e controls.MGK 264 FINAL

Page 39 of 49 Statistically significant inhibition of testosterone was noted in Run 4 at 10 μM of MGK 264, and
induction of estradiol was observed in Run 3 at 0.1 and 1 μM. However, these effects were not reproducible in this assay in th e other runs, and the response was similar to the solvent control (SC) when normalized for cell viability. Additio nally, the apparent effect on estradiol was not concentration-dependent. Based on these findi ngs, MGK 264 did not inhibit or induce hormone production under the conditions for this assay.

Based on hormone responses in at least thr ee independent runs, MGK 264 treatment did not result in statistically signifi cant and reproducible alterations in testosterone or estradiol production

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48618006) conducted to screen for potential estrogenic activity, MGK 264 (95.4% purity, Batch/lot # AB1412) in corn oil was administered daily via oral gavage to groups of eight ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 320, or 1000 mg/kg/day on post-natal days (PND) 56-58. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.05 mg/kg/day by or al gavage. Body weights were determined daily. All animal s were terminated and necropsied on PND 59 approximately 24 hours after the final dose admini stration to determine wet and blotted uterine weights.

One animal from the 1000 mg/kg/day group wa s euthanized moribund on Day 3; all other
animals survived until scheduled termina tion. Animals in the high dose MGK 264 group exhibited adverse clinical signs of toxicity (uncoordinated movement, decreased movement, hunched posture, twitching, piloerect ion, ungroomed appearance, a red nasal discharge, and/or diarrhea) following dosing.

Body weights were decreased (p<0.05) by 10% on Day 4 in the 1000 mg/kg/day group compared to the vehicle controls, resulting in a mean ove rall body weight loss (p<0. 05) of -8.9 g, compared to the vehicle control group (16.5 g gain). Fi nal body weights and overa ll body weight gains in the 320 mg/kg/day group were comparable to c ontrols throughout the duration of the assay.

Uterine weights in the MGK 264 treated groups we re comparable to the vehicle controls.

The body weights and overall body we ight gains for the positive control (EE) group were not statistically evaluated; however, there was a mean overall body weight loss of

− 2.4 g over the duration of the study. Absolute wet and blotted uterus weights for the positive control (EE) group were each increased (p<0.05) by 99%, as expected.MGK 264 FINAL

Page 40 of 49 Dosing for the assay was adequate, as animals we re dosed up to the limit dose (1000 mg/kg/day), which resulted in decreased body wei ght and adverse clinical signs.

No statistically si gnificant changes were s een in uterine weight in this assay. MGK 264 is negative in the uterotrophic assay.MGK 264 FINAL

Page 41 of 49 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of MGK 264 with the endocrine system are summarized below. Given the breadth of toxicity data available on MGK 264, the only Part 158 studies discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects (i.e., mu ltiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 48084901 ), groups of 25 Wistar rats/sex
were administered MGK 264 Insectic ide Synergist (94.2% pu rity) in the diet at dietary levels of 0, 400, 1600, or 6400 ppm (equivalent to 0/0, 36/52, 159/225, and 650/952 mg/kg/day in males/females) for two consecutive generations . The P generation animals were fed the diets beginning 10 weeks prior to mating to produce the F 1 litters. F 1 offspring (25/sex/dose group) selected to be parents of the next generation recei ved the same doses as th eir parents. One set of litters (F 1 ) was produced by the P parents, and the F 1 adults produced two litters (F 2a and F 2b ).

At 6400 ppm, F 1 male and female body weights were d ecreased (p<0.05) by 6% at Week 17, and Week 5-17 body weight gains were decreased (p<0.05) by 5%. Relative (to body weight) food consumption was increased (p<0.05) by 11% during Weeks 5-16. In the 1600 ppm F 1 females during the 2 nd mating, body weights were decreased (p<0.05) by 5% at the end of gestation. At 6400 ppm, body weights were decr eased (p<0.05) by 8-10% at the end of both gestations and on post-natal day (PND) 4. Relative food consumption was dose-dependently increased in the 1600 and 6400 ppm F 1 dams during the 2 nd mating.

There were no effects of treatmen t on uterus, testes, epididymides , pituitary, or thyroid weights or microscopic pathology. Absolute and relative liver weights were dose-dependently increased in both generations in both sexes. With the exception of the 1600 ppm F 1 females, these increases were statistically si gnificant (p<0.05) in both sexes fro m both generations at 1600 and 6400 ppm. At 400 ppm, the only in creases were in the P and F 1 males. Liver enlargement was
found in 10 P females and 12 F 1 females at 6400 ppm compared to 0 controls. In the P rats,
increased incidences of distinct lobulation of the liver were ob served at 1600 ppm (9 males, 4 females) and 6400 ppm (10 males, 8 females) compar ed to 0 controls. Incidences of centrilobular hypertrophy and eosinophilic centrilobular cy toplasmic changes in the liver were dosedependently increased at 1600 and 6400 ppm in bot h sexes from both genera tions. Centrilobular hypertrophy was found at these doses in the male s (16-25 treated) and fe males (9-24 treated), and eosinophilic centrilobular cytoplasmic change s were noted in the males (9-25) and females (9-24), all compared to 0 cont rols. Additionally at 6400 ppm, periportal hypertrophy was noted in 21 P females. Deposition of brownish pigmen t in/around bile ducts w ith prominent bile duct epithelium, and periductular cellular infiltration and/or degeneration of bi le duct epithelium wereMGK 264 FINAL

Page 42 of 49 reported at 6400 ppm in both sexes of both gene rations. Presence of hydropic foci was noted in both generations of males at 6400 ppm. There was an increased amount of periportal/centrilobular fat storag e in both generations of male s at 1600 ppm and above and in females at 6400 ppm.

In the kidney, increased storage of brownish pigment in the cortical tubules was observed at 1600 ppm and above in both sexes and generations. Increased severity in basophilic cortical tubules hyaline droplets were noted in P males at 6400 ppm. In the thyroid glands, increased incidences of follicular cell hypertrophy were observed in both generations in the 1600 ppm males and 6400 ppm females. Increased incidences of colloidal alteration were noted in the P males at 6400 ppm, in the F 1 males at 1600 and 6400 ppm, and in the F 1 females at 6400 ppm.

Altered luteinization of the corpora lutea wa s observed at 6400 ppm in the P (8/25) and F 1 (11/25) females compared to 0/25 controls . There were an increased number of F 1 females in met-/diestrus cycle at 6400 ppm (16/25 treated vs 8/25 controls). Additionally at 6400 ppm in the P generation: absolute and rela tive ovary/oviduct weights were significantly (p<0.01) decreased by 16-20%, and absolute and relative seminal vesi cle/coagulating gland we ights were increased by 9-10% (p<0.05). However, because there were no corroborating microscopic findings and no effect was found on sperm parameters, insemi nation, or fertility, the increased seminal vesicle/coagulating gland weights were not considered adverse.

The parental LOAEL is 1600 ppm (equivalent to 159/225 mg/kg/day) based on decreased body weights in females and adverse effects on the li ver, kidneys, and thyroid in both sexes. The NOAEL is 400 ppm (equivalent to 36/52 mg/kg/day in males/females).

Live birth index, sex ratio, number of pups born, prenatal losses, and viability and lactation indices were unaffected by treatment.

In the F 2a litters, small pups relative to other pups in the litter were observed with a higher frequency at 6400 ppm compared to contro ls. At 6400 ppm in both sexes of the F 2b litters, a nonstatistically significant reducti on in body weight was observed fo r PND 1-7. Except for the F 2a males on PND 21, significantly (p ≤ 0.05) decreased offspring body weights were observed in
both sexes in the F 1 , F 2a , and F 2b litters on PND 21 and 28. Additionally, in the F 2b females, body weights were decreased by 14% (p<0.05) on PND 14.

Body weight at the time of balano-preputial se paration was significantly lower (p<0.01) at 6400 ppm; however, the time to sexual maturation was unaffected by treatment, indicating that the decreased growth did not affect sexual maturation. Furthermore, the mean body weight of the 6400 ppm males at the time of sexual maturation was within the hi storical control range. In contrast, vaginal opening was dela yed (p<0.01) by 4.2 days in the F 1 generation. Although not statistically significant, the body weights at vaginal opening we re dose-dependently increased, indicating that sexual ma turation in the females was not secondary to growth (body weight).MGK 264 FINAL

Page 43 of 49 Therefore, a direct effect on sexual maturati on cannot be discounted. There was no effect on anogenital distance in either males or females in the study.

The offspring LOAEL is 6400 ppm (equivalent to 650/952 in males/females) based on decreased pup body weights and delayed vaginal openi ng. The NOAEL is 1600 ppm (equivalent to 159/225 mg/kg/day in males/females).

There were no treatment-related e ffects on estrus cycle, sperm pa rameters, insemination, fertility, pre-coital interval, gestation, rearing, gestation duration, or number of litters born. At 6400 ppm, there was a small but significan t decrease in implantations with concomitant lower mean litter
size at birth in F 1 pups. Although the mean litter size is with in the historical control range, a relationship to treatment cannot be ruled out due to the hi stopathological findings (altered luteinization) in the parental females.

The reproductive LOAEL is 6400 ppm (equivalent to 650/952 in males/females) based on decreased number of implanta tions and litter size, possibl y related to effects on the ovaries/corpora lutea. The NOAEL is 1600 ppm (equivalent to 159/225 mg/kg/day in males/females).

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a multi-generation reproduction toxicity study ( MRID 42155701 ), groups of 26 SpragueDawley rats/sex were administ ered MGK 264 (100% purity) in th e diet at dose levels of 0, 2500, 10,000 or 20,000 ppm. However, high mortality was observed in the 20,000 ppm group, so this group was terminated, and a second control group and a 1250 ppm group were initiated approximately two months after study init iation. These dose levels (0, 1250, 2500 and 10,000 ppm) correspond to mean doses of approxima tely 0/0, 61/73, 125/151, and 649/759 mg/kg/day in
males/females. The diets were administered to the P generation for at least 80 days prior to mating for the F 1a litters, and to the F 1 parents for at least 102 da ys prior to mating for the F 2a litters.

There were no treatment-related mortalities or clinical signs. Organ we ights were not reported. There were no effects of treatment reported for microscopic findings in the ovaries, testes, or epididymides.

Body weight decreases (p<0.05) duri ng pre-mating were noted at 1250 ( ↓ 5-10%), 2500 ( ↓ 5-7%), and 10,000 ( ↓ 9-15%) ppm in the F 1 dams and at 2500 ( ↓↓ 6-8%) and 10,000 ( ↓ 6-10%) ppm in the
P dams. In the F 1 males, body weighs were decreased (occasionally p<0.05) at 2500 ( ↓ 4-8%) and 10,000 ( ↓ 9-20%) ppm. Hepatocellular hypertrophy wa s evident at all doses in the F 1 parental group and at higher levels in the P parental gr oup. The parental LOAEL is 1250 ppm (equivalent to 61/73 mg/kg/day in males/females), based on decreased body weight and hepatocellular hypertrophy. The parental NOA EL was not established.MGK 264 FINAL

Page 44 of 49 Decreased body weight during lact ation was evident at all doses with there being decreases of about 5% to 14% (all p < 0.05) at 1,250 ppm, 5% to 13% at 2,500 ppm and 26% to 31% at 10,000 ppm. Following lactation, the pups gained wei ght comparable to the controls. Because of a variety of factors including lack of a true dose response between 1250 and 2500 ppm, the fact that the pups are taking in large quantities of milk and feed and because the difference was relatively small and reversible, the dose level of 1250 ppm is cons idered to be near the NOAEL and additional testing is not considered necessary. The developmental LOAEL is 2500 ppm (equivalent to 61/73 mg/kg/day in males/females) , based on slight decreases in body weight. The developmental NOAEL was not established.

There were no adverse effects on reproductive performance. The reproductive performance LOAEL was not observed. The reproductive NOAEL is 10,000 ppm (equivalent to 649/759 mg/kg/day in males/females).

Developmental Toxicity (Rat, OCSPP 870.3700)

In a developmental toxicity study ( MRID 41660302 ), groups of 25 presumed pregnant

COBS ® CD ® rats were administered MGK 264 (98% purity) in 0.5% methylcellulose (dose volume 10 mL/kg) by daily oral gavage at dos e levels of 0, 100, 300, or 1000 mg/kg/day during gestation days (GD) 6-15. Dams were euthanized on GD 20 by carbon dioxide inhalation, and the fetuses removed by cesarean section. Fetuses were weighed, sexed, and examined for external, visceral, and skeletal alterations.

There was one death in the 100 mg/kg/day group a nd one death in the 1000 mg/kg/day group; all
other rats survived to scheduled euthanasia. Increased salivati on was noted at 1000 mg/kg/day as a pre-dose observation in four dams and pos t-dosing in 23 dams. Body weight gains were decreased by 25% (p<0.01) during GD 6-15 at 1000 mg/kg/day. The maternal LOAEL was 1000 mg/kg/day, based decreased body weight gain. The matern al NOAEL is 300 mg/kg/day.

The pregnancy rate was decreased at 1000 mg/kg/ day (68% treated vs. 80 % controls); however, there was no corroborating eviden ce of effects on fertility. Th ere were no treatment-related external, visceral, or skeletal alterations observed. The develo pmental LOAEL was not observed. The developmental NOAEL is 1000 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a developmental toxicity study ( MRID 40352301 ), groups of 16 New Z ealand white rabbits were administered MGK 264 (98% purity) in 0.5% methylcellulose by daily oral gavage at dose levels of 0, 10, 30, or 100 mg/ kg/day during GD 7-19. Does were euthanized on GD 29, and the fetuses removed by cesarean section. Fetuses were weighed, sexed, and examined for external, visceral, and skeletal alterations.MGK 264 FINAL

Page 45 of 49 There were no treatment-related effects on morta lity, body weight gains, parental gross necropsy or cesarean parameters. One doe died on study in both the 10 and 30 mg/kg/day groups. There was one abortion each in the control, 10, and 100 mg/kg/day groups. There were three abortions in the 30 mg/kg/day group including an abortion in the animals that died (this animal was said to have aborted and then died). An increase in the incidence of excessive salivation was seen at 100 mg/kg/day (44% of the does vs. 0% in the cont rols); this occurred on consecutive days in the middle of the dosing period and is considered a response to trea tment. The maternal LOAEL for maternal toxicity is 100 mg/kg/day based on excessive salivation. The maternal NOAEL is 30 mg/kg/day.

There were no effects of treatment on fertility or on external, viscer al, or skeletal alterations. The developmental LOAEL was not observed. Th e developmental NOAEL was 100 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a chronic toxicity /carcinogenicity study ( MRID 43005301 ), groups of 60 CD rats/sex were administered MGK 264 (100% purity) in the diet at doses of 0 (two se parate groups), 50, 150, or 450 mg/kg/day for 104 weeks.

There were no effects of treatment on mortality, clinical signs, ophthalmoscopic examinations, hematology, clinical chemistry, or urinalysis. No tr eatment-related changes we re seen in absolute or relative weights of the testes, ovaries or adrenal glands, and no treatment-related histopathological le sions were seen in the test es, epididymides, or ovaries.

At 450 mg/kg/day, body weights were decreased (p<0.05) after Week 18 in males and Week 10 in females, with final body weights decreased 8- 9% in males and 18-21% in females, compared to the two control groups. Decreases in body wei ght gains were also observed at this dose, reaching 58% in males and 50% in females during Weeks 52-78. Food consumption was also generally decreased in fe males at 450 mg/kg/day.

Both absolute and relative liver weights were dose-dependently increased in males and females. Compared to the two control groups, both absolute and relative liver weights were increased in females at 150 mg/kg/day by 9-20% (NS) and 22- 24% (NS), respectively, and at 450 mg/kg/day by 22-34% (p<0.05) and 61-63% (p<0.01), respectivel y. In males, absolute and relative liver weights were also increased at 50 mg/kg/day by 9-14% (p<0.05) and 9-12% (NS), respectively, at 150 mg/kg/day by 10-16% (p<0.05) and 18-21% (p<0.05), respectively, and at 450 mg/kg/day by 39-45% (p<0.01) and 55-59% (p<0.01) , respectively. At dose levels ≥ 150 mg/kg/day, pathological changes were observed in the liver (hepatocyte hypertrophy in both sexes and portal bile duct proliferation an d bile stasis, spongiosis hepatitis and cysts in females) and kidney (brown pigment in females). At 450 mg/kg, add itional pathology was noted in the liver (bile duct proliferation and cysts in both se xes, and altered eosinophilic foci and spongiosis hepatitis in males) and kidney (brown pigment and cysts in males); and kidney, hear t, and brain weightsMGK 264 FINAL

Page 46 of 49 were increased. The systemic LOAEL is 150 mg/kg/day based on increased liver weight supported by liver pathology (hepatocyte hypert rophy and other liver histopathology). The NOAEL is 50 mg/kg/day.

An increase in thyroid follicular tumors in male s was noted. A response of 2 (3.3%), 1 (1.7%), 4 (6.7%), 5 (8.3%) and 6 (10%) for adenomas alone , and 4 (6.7%), 1 (1.7%), 6 (10%), 7 (11.7%) and 9 (15%) for combined adenomas and car cinomas for the two control, 50, 150 and 450 mg/kg/day dose groups resp ectively (N=60/dose group).

Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study ( MRID 42093802 ), groups of 50 CD-1 mice/sex were administered MGK 264 (98% purity) in the di et at 0 (two separate groups ), 50, 400, or 800 mg/kg/day for 18
months.

There were no treatment-related effects observe d on mortality, clinical signs of toxicity, body weights, food consumption, ophthalmological parameters, or hematology parameters at any dose
in either sex.

No treatment-related changes were se en in the absolute or relative (to body) weights of the testes, ovaries, or adrenal glands. At 400 mg/kg/day an d above, absolute and relative (to body) liver weights were increased (p<0.01) in the males by 13-37% and 22-46%, respectively, compared to the controls. In the females, only relative (to body ) liver weight at these doses was significantly (p<0.01) increased ( ↑ 11-16%), although absolute liver wei ghts were slightly elevated at 400 ( ↑ 8%) and 800 mg/kg/day ( ↑ 6%).

No treatment-related histopatholog ical findings were seen in th e testes, epididymides, seminal vesicle, prostate, ovaries, uterus, mamma ry, thyroid, adrenal or pituitary glands.

At 400 mg/kg/day there was increase d calculi in the gall bladder and intrahepatic bile stasis in males. At 800 mg/kg/day there was an increase in hepatocellular hypert rophy, calculus in bile duct in males and females and portal bile duct pr oliferation, portal mononuclear cell infiltration, spongiosis hepatis, cysts and vacuolar change in males and hyperplastic nodules in males and
females.

The 400 and 800 mg/kg/day doses were associated with increases in liver adenomas in males. In females, the 400 and 800 mg/kg/day doses had ad enomas and a carcinoma which did not reach statistical significance, but these liver tu mors are rare in female CD-1 mice.

The systemic LOAEL was 400 mg/kg/day based on in creased absolute and relative liver weight and liver pathology findings in males. The systemic NOAEL was 50 mg/kg/day.MGK 264 FINAL

Page 47 of 49 Chronic Toxicity (Dog, OCSPP 870.4100)

In a one-year chroni c toxicity study ( MRID 42148102 ), groups of 4 beagle dogs/sex were administered MGK 264 (92% purity) in the diet at concentrations of 0, 65, 250, or 1000 ppm (equivalent to 0/0, 2.1/2.0, 7.5/ 7.4, and 34.9/32.5 mg/kg/day in male s/females) for one year.

There were no effects of treatment on mortalit y, clinical signs, body weight gains, food consumption, ophthalmoscopic examinations, hema tology, clinical chemistry, urinalysis, or gross pathology. There were no tr eatment-related effects on organ weights or microscopic pathology in the ovary, testes, p ituitary, adrenal, or thyroid.

The only effects noted were seen at the 1000 ppm dose level and consisted of brown pigment deposition in the liver of both sexes (3/4 males and females vs. 0/4 in controls or lower doses) and hepatocellular hypertrophy in 2/4 males. The LOAEL is 1000 ppm (equivalent to 34.9/32.5 mg/kg/day in males/females), based on liver effects. The NOAEL is 250 ppm (equivalent to 7.5/7.4 mg/kg/day in males/females).

Subchronic Oral Toxicity (Rat, OCSPP 870.3100)

In a subchronic toxicity study ( MRID 42970601 ), groups of 10 Sprague-Dawley rats/sex were administered MGK 264 (98% purity) in the di et at nominal dose levels of 0, 125, 250, 500, 1000, or 2000 mg/kg/day for 13 weeks. Actual achieved doses were 0, 140, 279, 564, 1124 and 1842 mg/kg/day in males and 0, 130, 260, 522, 1034 and 1933 mg/kg/day in females.

Clinical signs of toxicity were observed in high-dose male and females, including decreased defecation in all animals, discol ored urine in all males and 8 fe males, anogenital staining in 4 males and one females, and labored breathing in 2 males and 6 females. In addition, high levels of mortality were observed at the high dose, w ith three males and six females dying on study or being sacrificed in extremis . Because of this excessive mortality, the surviving animals in this group were removed from test-article containing diet and placed on basal diet prior to the end of the study. Mortality in this group was likely due to decreased food consumption (50% decrease in food consumption in males and 35% in females at week one) resulting from an unpalatable
diet. Once removed from the test diet the surviving animals in th is group rapidly gained weight and appeared healthy. There were no premature deaths or clinical signs in either sex of any other
test group.

There were no effects of treatment on hematology, testes, ovary, or adrenal weights, or on gross pathology of the testes, epididymides, ovary, or adrenal. None of the collected tissues were examined for histologically.

Terminal body weights (Week 13) were decreased by 7 and 9% (NS) in males and females at 500 mg/kg/day, respectively, and by 14 and 17% (p<0.05) in males and females at 1000MGK 264 FINAL

Page 48 of 49 mg/kg/day. Body weight gains we re also decreased at 500 and 1000 mg/kg/day in both sexes compared to controls ( ↓ 10% and ↓ 21% in males, and ↓ 18% and ↓ 33% in females). Food consumption was frequently decreased (p<0.05) at 500 and 1000 mg/kg/day in males and females throughout the study.

In males, dose-dependent increases were observed in mean total protein values ( ↑ 7-11%) and globulin levels ( ↑ 14-24%) at 250 mg/kg/day and above , and in cholesterol levels ( ↑ 22-67%) at 500 mg/kg/day and above. In females, dose-dependent increases were also observed in mean total protein values ( ↑ 8-18%), globulin levels ( ↑ 25-38%), and cholesterol levels ( ↑ 26-89%) at 500 mg/kg/day and above.

Dose-dependent increases were observed in ab solute and relative (to body) liver weights in males and females at doses from 125 to 1000 mg/ kg/day, with the changes becoming significant (p<0.05) at ≥ 250 mg/kg/day. Absolute liver weights we re increased (p<0.05) by 6% (NS) to 56% in males and by 8% (NS) to 61% in females, and relative (to body) liver weights were increased (p<0.05) by 8 to 104% in male s and by 6 (NS) to 84% in females.

The only findings observed at gross necropsy th at were likely related to compound exposure were findings of enlarged liver in males (0% in controls compared to 30% at 500 and 20% at 1000 mg/kg/day) .

The NOAEL is 125 mg/kg/day. The LOAEL is 250 mg/kg/day based on evidence of liver alterations (increased serum globulins and total pr oteins in males, and increased absolute and relative liver weights in both sexes).

Subchronic Inhalation Toxicity (Rat; OCSPP 870.3465)

In a subchronic inhalati on toxicity in rats ( MRID 43309001 ), groups of 15 Sprague-Dawley rats/sex were exposed to atmospheres cont aining MGK 264 at concentrations of 0, 0.010, 0.040, 0.135 or 0.400 mg/L for three months (5 days/week, 6 hours/day). An additional 15 rats/sex were exposed to MGK 264 at concentrations of 0 or 0.400 mg/L as above and maintained unexposed for three additional months in a recovery phase.

There were no effects of treatment on body we ights, body weight gains, food consumption, ophthalmoscopic examinations, hematology, clinical chemistry, absolute or relative (to body) organ weights, or gross or microscopic pathol ogy with the exceptions of the nasoturbinates, nasopharynx, and larynx. The ovaries, testes, and epididymides weights and testes and epididymides pathology were unaffected by treatment.

There were no treatment-related deaths. One cont rol male died on Day 46; a probable cause of death was chronic pyelitis affecting the kidneys. One high-dose male died during the recovery period; a cause of death was not determined. Clinical signs of t oxicity observed were red facialMGK 264 FINAL

Page 49 of 49 stains in all treated females and in males at ≥ 0.040 mg/L, and decrease d activity in the 0.400 mg/L group.

In the larynx, metaplasia /hyperplasia and metaplastic keratini zed epithelium of the mucosa were present in all dosed groups, and the severity of these lesions increased with the dose level. Stratified squamous epithelium (normal) hyperplasi a and hyperkeratosis we re present in the midhigh and high dose groups. Vent ral diverticulum: pseudostratified cuboidal/columnar epitheliumsquamous/squamoid metaplasia/hyperplasia was also reported to be pres ent only in the mid-high dose (3 males and 4 females) and high-dose (6/s ex) groups. Mucosal metaplasia/hyperplasia was minimally present in 12 males and 8 females in the high-dose group after the recovery period. The keratinized tissue was still present in tw o high dose males only (minimal degree) also implying a slow rate of recovery. The normal hype rplasia and hyperkeratosis were also present to minimum degree in one or two of the high dose group further indicat ing a slow recovery.

In the nasoturbinates, the mid-high and high dos e groups were associated with increases in frequency (for females) and inte nsity (for both sexes) of eosinoph ilic material in the epithelium. The severity of hypertrophy/hyperp lasia of goblet cells in th e respiratory epithelium was increased in the high dose group. The hypertrophy/hype rplasia incidence and severity scores for the control and 0.400 mg/L groups su ggested that the condition is re versed in males but persisted in females. The eosinophilic material incidence a nd severity scores indicated reversal after the 3 month recovery period.

In the nasopharynx hypertrophy/hyper plasia of the goblet cells in the epithelium was increased in frequency in the 0.400 mg/L group. Recovery was observed.

Subchronic Dermal Toxicity (Rabbit, OCSPP 870.3250)

In a subchronic dermal toxicity study ( MRID 42742201 ), groups of 10 New Zealand white
rabbits/sex were administered MGK 264 (92% purity) in corn oil (1 mL/kg) by dermal application to the shaved, intact skin at con centrations of 0, 10, 30, or 100 mg/kg for 6 hours/day, 7 days/week for 13 weeks. No treatment-related effects were observed on mortality, clinical signs, body weights, food consumption, ophthalmosc opic parameters, hematology and clinical chemistry parameters, organ weights (liver, test es, or kidneys), or gross and histopathological findings (including testes, epididymides, and ovaries). Signs of epidermal hyperplasia, hyperkeratosis, scab formation, chronic inflamma tion, and hemorrhage were noted in all groups without evidence of compound -related effects, and were considered to be related to the corn oil vehicle and/or bandaging.

The systemic LOAEL was not observed. Th e systemic NOAEL was 100 mg/kg/day.