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 

# &

Health Effects Divison

# ::~:E£'~:::e 

# f] g:ams d f 'v--

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

## ~~~ 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: ABAMECTIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYABAMECTIN FINAL

ii TABLE OF CONTENTS

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

I. Introduction.......................................................................................3

II. Sources of Scientific Data and Technical Information......................................4

A. EDSP Tier 1 Screening Assays............................................................4

B. Other Scientifically Rele vant Information (OSRI)......................................4

III. Weight of Evidence (WoE) Evaluation.......................................................4

A. EDSP Tier 1 Screening Assays.............................................................7

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................12

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

IV. Committee’s Assessment of Weight of Evidence..........................................18

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................18

1. Tier 1 in vivo Assays.............................................................19

2. OSRI...............................................................................20

B. Estrogen Pathway...........................................................................22

C. Androgen Pathway.........................................................................22

D. Thyroid Pathway...........................................................................23

E. Conclusions.................................................................................23

V. EDSP Tier 2 Testing Recommendations....................................................23

VI. References.......................................................................................24

LIST OF TABLES: Table 1. Tier 1 Assays for Abamectin................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Abamectin....................................9
Table 3. Androgenic/Anti-Androgenic Pathway for Abamectin.................................13
Table 4. Thyroid Pathway for Abamectin..........................................................17

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................27
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................39

APPENDIX 3. References Not Utilized in the Abamectin WoE Analysis...........................52ABAMECTIN FINAL

iii 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 eneration
F2 Second filial g eneration
Fcd Fecundit yABAMECTIN FINAL

iv Abbreviation Terminology

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 Concentration
MTD Maximum Tolerated Dose
N Ne g ativeABAMECTIN FINAL

v Abbreviation Terminology

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)

TP Testosterone Pro p ionate
T R Th y roid Rece p to rABAMECTIN FINAL

vi Abbreviation Terminology

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-EvidenceABAMECTIN FINAL

Page 1 of 53 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay batteryis designed to provide the necessary empirical data to evaluate the poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothala mic-pituitary thyroid (HPT) perturbations. In addition to the available Tier 1 data, othe r scientifically relevant information (OSRI), including general toxicity data a nd open literature studies of su fficient quality, were considered in this weight of evidence (WOE) assessment.

In determining whether abamectin 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 endocrine -related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On November 12, 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 abamectin with the E, A or T signaling pa thways. 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 abamectin, there was no evidence of potential interaction with the estrogen pathway in the
EDSP Tier 1 or OSRI in vitro assays with the exception of the steroidogenesis assay where estradiol production wa s decreased (p at 1 μ M abamectin (0.72-fold). Additionally, abamectin showed no estrogen-related effects in the Tier 1 in vivo mammalian assays, in the FSTRA or the
Part 158 mammalian or wildlif e toxicity studies.

There was no evidence of interaction of abamect in with the androgen pathway in the Tier 1 in

vitro ( i.e , AR binding and steriodogenesis a ssays were negative) or Tier 1 in vivo mammalian assays ( i.e ., Hershberger and male pubertal assays were negative) or in the FSTRA. There were no treatment-related effects on male reproductive parameters in the Part 158 mammalian or wildlife studies. While effects on the male repr oductive system (testicular damage, decreases in sperm count and motility, histopathological ch anges in testes and decreases in FSH concentrations) were observed in the OSRI literature study by Celik-Ozenki et al. (2011), no effects on gonadal histopathology were seen at simila r doses in the Tier 1 male pubertal assay or
in the Part 158 rat chroni c/carcinogenicity study. Overall, this is an isolated finding that is not corroborated by the Tier 1 or Part 158 data. Th erefore there is no convi ncing evidence for an interaction with the androgen pathway.ABAMECTIN FINAL

Page 2 of 53 There was no evidence of potential interaction of abamectin with the thyroid pathway. No
treatment-related effects were s een in thyroid, hormones, weight or histopathology in the male and female pubertal assays. In the AMA, no tr eatment-related finding were observed except for effects in growth parameters at the low concentr ation, with no effects on developmental stage or thyroid histopathology.

Based on weight of evidence considerations, mamm alian or wildlife EDSP Tier 2 testing is not
recommended for abamectin since there was no convi ncing evidence of potenti al interaction with the estrogen, androgen or thyroid pathways.ABAMECTIN FINAL

Page 3 of 53 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothala mic-pituitary thyroid (HPT) perturbations. In addition to the available Tier 1 data, othe r scientifically relevant information (OSRI), including general toxicity data a nd open literature studies of su fficient quality, were considered in this weight of evidence (WoE) assessment.

In determining whether abamectin 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 endocrine -related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On November 12, 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 weight-of-evidence analysis of the potential interaction of abamectin with the E, A or T signaling 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).

Abamectin has a vapor pressure of 1.5×10 -9 mm Hg at 20°C and it not anticipated to readily
volatilize. Abamectin has water solubil ity of <0.01 mg/L at 20°C and a log K OW of 5.0-5.5. The
available data indicate that abamectin has low mob ility in soil with half-liv es in soil of 2 weeks to 2 months. Abamectin also will be suscepti ble to photodegradation when exposed to sunlight on soil surfaces and the atmosphere. Bioc oncentration factors in fish are <100.

The available information considered in determin ing the potential interaction of abamectin with the E, A, T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity stud ies and/or other published articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies r ecommendations are presen ted in Section V.ABAMECTIN FINAL

Page 4 of 53 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 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 Guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on abamectin (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

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 wereABAMECTIN FINAL

Page 5 of 53 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 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 (complementarity) of the responses in the in dividual study is eval uated. 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 in teract 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 rele vant, 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 toxicity? 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.ABAMECTIN FINAL

Page 6 of 53 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at
the FIFRA SAP that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In Oc tober 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in e ndocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. second ary effects related to toxicity in non-endocrine 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.ABAMECTIN FINAL

Page 7 of 53 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 submitted in response to the agency’s test order for abamectin are shown
below in Table 1.

Table 1. Tier 1 Screening Assays for Abamectin

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID 48614405)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID 48614406)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID 48614403)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID 48614407)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48614404)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID 48614409)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID 48614408)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID 48926401)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID 48926402)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID 48675002)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID 48675001)ABAMECTIN FINAL

Page 8 of 53 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for abamectin to interact with th e E hormone pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 assays 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.

Abamectin was negative in the in vitro Tier 1 ER binding, ERTA, and aromatase assays. In the Tier 1 steroidogenesis assay, estradiol production was decreased in one of three runs at 0.1 μ M (0.92-fold) and in all three runs at 1 μ M (0.72-fold).

Abamectin was negative in the uterotrophic assay. In the female pubertal assay, the percent of females regularly cycling was decrease d (NS and p<0.05, respectively) at 0.75 and 1.5 mg/kg/day (38% and 50%, resp ectively, vs. 87.5% control). Also, a shift was noted in the cycle status at necropsy, with fe wer females in diestrus and more in estrus in treated groups compared to controls. Additionally, there was a s light and non-statistically significant increase (16%) in uterine weight at th e high-dose group. No histopathologi cal changes were noted in the ovaries. In the FSTRA, there were no effects in females up to the highest concentration tested (12 μg a.i./L).

In the Part 158 developmental neurotoxicity studie s with rats, minimal changes were seen in the age of attainment of VO (1.3 days to 1.9 days), which is considered to be within normal biological variability. Additionally, this eff ect was observed in the presence of significant (p ≤ 0.01) decreases in body weight (up to 10% at approximately the time of VO (PND 36). No estrogen-mediate effects were seen in the other Part 158 mammalian studies.

No effects on reproduction were noted in the avia n reproduction study with mallard ducks (up to 12 mg a.i./kg-diet) or bobwhite quail (up to 20 mg a.i./kg-diet). Effects on survival and growth were observed in rainbow trout (at 2.2 or 0.96 μg a.i./L). Survival was affected at ≥ 0.67 μg a.i./L in sheepshead minnow fish.ABAMECTINFINALPage 9 of 53Table 2. Estrogenic/Anti-Estrogenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Abamectin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48614405)NERTA(MRID 48614406)NAromatase(MRID 48614404)NSteroidogenesis(MRID 48614407)PUterotrophic(MRID 48614409)NNX(H)Female Pubertal Rat(MRID 48926401)NNNNP4(H)NNNFSTRA(MRID 48675002)NENNNNNNOSRISubchronic OralToxicity/Neurotoxicity (Rat;MRID 46959201)NNN NENX(H)DevelopmentalToxicity (Rat; MRID00130819)NNNABAMECTINFINALPage 10 of 53Table 2. Estrogenic/Anti-Estrogenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Abamectin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3DevelopmentalToxicity (Rabbit;MRID 00130819)NX(H)NTwo-GenerationReproduction (Rat;MRID 00164151)NE NE N NENX(H)NChronic Toxicity/Carcinogenicity (Rat;MRID 40069601)NNNNX(H)X(H)Chronic Toxicity/Carcinogenicity(Mouse; MRID40069602)NNNNX(H)NChronic Toxicity(Dog; MRID00164022, 40375510)NE NE N NEX(H)NDevelopmentalNeurotoxicity (Rat;MRID 47116201)NNX(M, H)X(H)DevelopmentalNeurotoxicity (Rat;MRIDs 46727401-46727403)NNX,(M)X(H)Avian reproduction(Duck; MRID40318601)NN NABAMECTINFINALPage 11 of 53Table 2. Estrogenic/Anti-Estrogenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Abamectin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail; MRID49226708)NN N1.Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), Medium-high treatment (MH), High treatment (H). Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that this 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 X inthis 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. The percent of females regularly cycling was decreased (NSand p<0.05, respectively) at 0.75 and 1.5 mg/kg/day (38% and 50%, respectively, vs. 87.5%control). Also, a shift was noted in the cycle status at necropsy, with fewer females indiestrus and more in estrus in treatedgroups compared to controls.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedABAMECTIN FINAL

Page 12 of 53 2. Effects on Androgen Pathway

The potential for abamectin to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, 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 overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

Abamectin was negative (non-binde r) in the Tier 1 AR binding assa y and negative in the Tier 1 steroidogenesis assay. Abamectin was also negative in the Tier 1 in vivo Hershberger assay, and no androgen-related effects were observed in the Tier 1 male pube rtal rat assay or FSTRA.

No androgen-related effects were seen in any of the mammalian Part 158 studies. No effects on reproduction were noted in the avian reproduction study with mallard ducks (up to 12 mg a.i./kgdiet) or bobwhite quail study (up to 20 mg/kg-diet).

In a published, non-guideline study investigating the effect of abamectin exposure on testicular function in male rats, Celik-Ozenki et al. (2011) showed testicular damage and decreases in sperm count and motility following dosing w ith abamectin at 1 or 4 mg/kg/day. Histopathological changes in abamectin-treated rats included degenerative changes in the tubules and disrupted cellular or ganization. Sperm motility in the ep ididymis was decreased in a dosedependent manner in both abamectin treatment gr oups, and sperm motility also was decreased in
the in vitro motility test. Finally, decreases were noted in follicle stimulating hormone (FSH) concentrations, but luteinizing ho rmone (LH) and testosterone con centrations were not affected. Rather than a specific anti-andr ogenic effect, the study authors hypot hesized that the deleterious effects of abamectin on the male reproductive syst em (or capability) demonstrated in this study may be due to direct oxidative stress in the testes resulting in a cascade of poly(ADP-ribose) polymerase (PARP)-related cell damage.ABAMECTINFINALPage 13 of 53Table 3. Androgenic/Anti-Androgenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Abamectin1Study Type/Literature Citation AR Binding AR Activation 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding (MRID48614403)NSteroidogenesis(MRID 48614407)NHershberger (MRID48614408)N↓BWG(H)NMale Pubertal Rat(MRID 48926402)NNNNNNNNNNFSTRA(MRID 48675002)NENNNNNNOSRISubchronic OralToxicity/Neurotoxicity (Rat;MRID 46959201)NN N N NENX(H)Two-GenerationReproduction (Rat;MRID 00164151)NNNNNX(H)NChronic Toxicity/Carcinogenicity (Rat;MRID 40069601)N N NEN N NX(H)X(H)ABAMECTINFINALPage 14 of 53Table 3. Androgenic/Anti-Androgenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Abamectin1Study Type/Literature Citation AR Binding AR Activation 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic Toxicity/Carcinogenicity(Mouse; MRID40069602)N N NE N N NNX(H)Chronic Toxicity(Dog; MRID00164022, 40375510)NN NE NE NEX(M, H)X(H)DevelopmentalNeurotoxicity (Rat;MRID 47116201)NX(M, H)X(H)DevelopmentalNeurotoxicity (Rat;MRIDs 46727401-46727403)NX(H)NMechanistic Studies:Male ReproductiveEffects (Rat; CelikOzenskiet al., 2011)NN P4P5N NAvian reproduction(Duck; MRID40318601)NN NAvian reproduction(Quail; MRID49226708NN N1. Key to responses: Low treatment (L), Medium-Low treatment (ML), Medium treatment (M), Medium high treatment (MH), High treatment (H). Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changes inABAMECTINFINALPage 15 of 53weight 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 1 assays 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. Dose-dependent testicular damage including degenerative tubules with disrupted cellular organization was observed at both the low and high doses (1 and 4mg/kg/day).5. Following 6 weeks of dosing, sperm counts were decreased (p<0.01) by 17 and 21%, respectively, at doses of 1 and 4 mg/kg/day, respectively. Decreasedsperm motility was observed after 5 minutes of treatment with 0.42 μM abamectin in thein vitromotility test, with continued decreases through the end ofthe test period (p<0.01, 20 minutes).P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedABAMECTIN FINAL

Page 16 of 53 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 female and male (rat) assays, and the AMA (frog).

The potential for abamectin to interact with thyroid regulation is summarized in Table 4. The various targets of the th yroid pathway across the relevant 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 ar e not reported in the ta ble and not considered further in the WOE assessment.

There were no effects on the th yroid pathway in the Tier 1 in vivo male and female pubertal rat assays. In the AMA, abamectin did not dela y median Nieuwkoop-Faber (NF) developmental stage at Day 7 or 21 relative to the negative control, and did not affect normalized hind-limb length (HLL). In addition, no trea tment-related effects on thyroi d gland histopathology were observed. The only effects noted were an incr ease in Day 7 snout-to-vent length (SVL; ↑ 10%) and body weight ( ↑ 24%) at the low concentration, w ith no effects observed at Day 21.

No thyroid-related effects were seen in any of the Part 158 studies.ABAMECTIN FINAL

Page 17 of 53 Table 4. Thyroid Pathway for Abamectin

Lines of Evidence Indicating Potential Intera ction with the Thyroid Pathway for Abamectin 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic ToxicityObservd2Overt ToxicityObsercved3

EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48926401) N N N N N N N

Male Pubertal Rat (MRID 48926402) N N N N N N N

AMA (Frog; MRID 48675001) N N BW: ↑ 24% (L);

SVL: ↑ 10% (L) N

OSRI

Subchronic Oral Toxicity/ Neurotoxicity (Rat; MRID 46959201) NE N NE N X (H)

Two-Generation Reproduction (Rat; MRID 00164151) NE N NE X (H) N

Chronic Toxicity/ Carcinogenicity (Rat; MRID 40069601) N N N X (H) X (H)
Chronic Toxicity/ Carcinogenicity (Mouse; MRID 40069602) N N N X (H) X (H)

Chronic Toxicity (Dog; MRID 00164022, 40375510) NE N NE X (H) X (H) 1. Key to responses: Low treatment (L), Medium-Low treatment (ML), Medium treatment (M), Medium high treatment (MH), High treatment (H). Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. BW= Body weight; SVL= snout-to-vent length 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. A N Negative findings NE Not examinedABAMECTIN FINAL

Page 18 of 53 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 abamectin to interact with 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 analysis of those lines 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 evalua tion in helping elucidate 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 evalua tion of the results of the Ti er 1 assays stated that “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the 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).

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 and OSRI
studies are described below. Generally, one parameter (i.e ., death or >10% decrease in mammalian body weight) was 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).ABAMECTIN FINAL

Page 19 of 53 1. Tier 1 in vivo Assays

In the Hershberger assay androge n agonist phase, no treatment-rela ted clinical signs were noted in either of the abamectin treatment groups ( 1.5 and 5 mg/kg/day). Body weight and overall body weight gain for the low-dose group were compar able to control. High-dose animals had a decreased overall body weight gain ( ↓ 48%), and terminal body weight was decreased by 9%. Food consumption was generally similar to control; however, overall mean food consumption was decreased by 17% in the high-dose group. In the anti-androgen assay, one animal in the high-dose group was euthanized in extremis on Day 6 after decreases in body weight and food consumption. Moderate tremors and red materi al on the forelimbs and around the mouth were noted on Day 6. Although there were no macroscopic findings, the death was considered to be
related to treatment. All other treated rats su rvived until scheduled sacrifice, but the five surviving high-dose animals also exhibited red ma terial around the face, mouth, and/or forelimbs on Days 4-11, and one of these animals also ha d slight tremors appr oximately 4 hours postdosing on Day 6. The high-dose group had decreased overall body weight gain ( ↓ 27%), and terminal body weight was decreased by 6%. Food consumption in all treated groups was similar to control; however, overall average food cons umption was decreased by 11% in the high-dose

group.

There were no effects of treatment for eith er abamectin dose (0.75 or 1.5 mg/kg/day) on mortality, clinical signs, in itial or final body weights, overall body weight gain, and food consumption in the female and male pubertal rat studies.

In the uterotrophic Assay, all animals survived until scheduled termination. No treatment-related effect was observed at the low dose (2.5 mg/kg/da y), but all six females at the high dose (7.5 mg/kg/day) had slight to moderate tremors on Days 1 or 2, and the tremors persisted for three females until sacrifice on Day 4. Additional fi ndings included pale body and red material around the nose, mouth and forelimbs. Body weights at the high dose were decreased by 14% and 21% on Days 3 and 4, respectively, and there wa s an overall negative body weight gain (

− 37.3 g) for Days 1-4, indicating that the high dose was overtly toxic. Overall mean food consumption at the high dose also was decreased by 60%.

In the 21-day FSTRA study, although Replicate A of the medium-treatment group had high mortality (33% survival, combined sexes), this was not considered treatment-related and was excluded from analysis of the all endpoints. Abamectin exposure was not associated with increases or decreases in body wei ght and/or length in either sex at any treatment concentration. A few observations of lethargy, partial loss of equilibrium and dark coloration were observed among females (1-2) in each of th e three treatment groups, but no s econdary sex characteristic or clinical signs were observed in males.

Abamectin treatment had no effect on the surviv al of tadpoles in the 21-day AMA study. Body weight was increased at Day 7 in the low-concentration group only ( ↑ 24%), with no furtherABAMECTIN FINAL

Page 20 of 53 affect at Day 21. Spinal curvature was observed in the control and all treatment groups, and did not appear to be treatment-related (similar in cidence across control a nd treatment groups, except for a lower incidence in the high-concentration group).

2. OSRI

In a subchronic combined oral toxicity and ne urotoxicity study, there we re no treatment-related mortalities in the low- and medium-dose groups (0.4 and 1.6 mg/kg/day) during the study. Overt toxicity occurred at the high dose (4.0 mg/kg/day) as all rats were sacrifi ced after Week 7 due to adverse clinical signs and decr eased body weights. Clinical signs , observed in high-dose males and females included: piloerection, mouth-staini ng, irregular breathing, si des pinched in, upward curvature of the spine, and tip-t oe gait. Decreased body weight ga in was observed in both the high-dose males and females in the first four weeks of treatment, although the effects were minimal in the males ( ↓ 11%) and greater in females ( ↓ 40%). The high-dose males had minimally decreased body weight ( ↓ 5%) at Week 4, with weight similar to control by Week 6. Body weights in the high-dose females were decreas ed by 4-7% through Week 7, and the overall (Week 1-7) body weight gain was decreased by 26 %. No clinical signs, or decreases in body weight or body weight gain, were observed at the lower doses.

In a developmental toxicity study in rats, th ere were no mortalities during the study, and no clinical signs were noted. No decreases in body weight or body weight gain were observed; body weight gain was increased among all treatment groups, but the extent was not reported. In a developmental toxicity study in rabbits, decreases in maternal body weights were seen in the dose-range finding study at the medium-high dose only (2.0 mg/kg/day). This dose was selected as the high dose for the rabbit developmental st udy in rabbits, but furt her effects on body weight from this study were not reported.

In a two-generation reproduction study in rats, nine mortalities of F 0 parental rats and five
mortalities of F 1b parental rats during dosing occurred, but were not considered to be related to treatment. No signs of clinical toxicity were noted for the parental generations during the study. Body weight gain increases were reported for th e medium- and high-dose F 0 males and females during the growth phase. Body weight gain incr eases also were reported for the medium- and high-dose F 0 females during the F 1a gestation period, with no difference noted during the F 1b gestation period. Body weight gains in F 0 females during the F 1b lactation period were decreased compared to control. High-dose F 1b pups (both sexes) and medium-dose females selected for the parental generation had lower mean body weights compared to control at dose initiation. Body weight in the high-dose F 1b males continued to be decrease d up to Day 28, similar to control through Day 168, and increased between Days 175- 272. Body weight gain in high-dose females was decreased during the growth phase, with no differences in overall body weight gains during
the F 2a and F 2b gestation periods. Decreased body weight gains in F 1b females also were noted during the F 2a and F 2b lactation periods.ABAMECTIN FINAL

Page 21 of 53 In a chronic toxicity/carcinoge nicity study in rats, treatmen t-related whole body tremors and overall unthrifty appearance were observed in some rats in the high- dose group. In some cases,
the tremors correlated with the increase in dosage from 2.0 to 2.5 mg/kg/day for the high-dose group; however, the tremors persisted intermittently until the time of sacrifice, despite the reduction back to 2.0 mg/kg/day. The tremors were c onsidered to be relate d to treatment because there were no pathological lesions to correlate with the tremors. There were no treatment-related effects on mortality or survival. Body weights were increased for both male and female rats in all treated groups compared to cont rol; overall body weight gains for female rats were not affected,
but were increased for male rats in all treated groups.

In a chronic toxicity/oncogenici ty study in mice, tremors observed in two females in the highdose group late in the study (Weeks 89 and 91). Tremor observed in a medium-dose female the day prior to death were considered secondary to a metastatic lymphoma of the pituitary, and
tremors observed in moribund male rats in the co ntrol and high-dose groups were considered secondary and not related to treat ment. There was increased mortal ity in male mice in the highdose group, which was considered to be possibly related to treatment, but there were no treatment-related effects on mortality in female mice. Overall body weight gain was decreased ( ↓ 23-25%) in high-dose females compared to the tw o control groups at termination of the study. Similar decreases were reported to have b een observed throughout the study. Decreased body weight gain in the high-dose fe males was considered to be trea tment-related; no changes were seen at the lower doses. In male mice, body wei ght gains in the mid- and high-dose groups were decreased ( ↓ 4-5%) compared to control group I and increased ( ↑ 3-4%) compared to control group II.

In a chronic toxicity study in dogs, there was a high incidence of mydriasis in male and female dogs at the medium and high doses (occurrences of 3 and 15%, respectively, in the 0.5 and 1.0 mg/kg/day dose groups). In addition, three high- dose dogs exhibited sali vation which may have
been treatment-related as no othe r treated dogs exhibited this si gn. Three male dogs in the highdose group died or were sacrificed prior the end of the study; the deaths were attributed to abamectin treatment. Overall body weight gain in dogs in the high-dose group were decreased ( ↓ 23%) compared to control.

In a developmental neurotoxicity study, there were no effects of treat ment on mortality or clinical signs in maternal rats at doses up to 0.4 mg/kg/day. Body weights ( ↑ 1-5%) and body weight gains ( ↑ 10-18%) were increased in all treated dams during gestation, with body weights and body weight gains generally comparable to control during lactation. However, body weight decreases ( ↓ 10-36%), clinical signs of toxicity and mortality were observed in high-dose pups, with all pups being deceased by post-natal day (PND) 38. In an earlier developmental neurotoxicity study, there also we re no effects of treatment on mo rtality or clinical sign in maternal rats at the same doses. Body weights ( ↑ 3-7%) and body weight gains ( ↑ 10 -16%) were
increased in all treated animal s during gestation (body weight ga in and food consumption were comparable to control in the low-dose group). Increased body weights we re still present atABAMECTIN FINAL

Page 22 of 53 lactation day (LDs) 1-3, with values similar to control by LD 29, but overall body weight gains were decreased. Pup body weights were decrease d by 4-5% and 7-10% in the mid and high-dose

groups.

In an avian reproduction study, a single, non-treatm ent-related mortality occurred with a mallard duck hen in the medium-concentration group. In the avian bobwhite quail reproduction study, a reduction in food consumption was observed at ≥ 5.1 mg/kg-diet, but no clini cal signs of toxicity were observed. In a fish early life-cycle toxicity study with rainbow trout (MRID 40069609), fry survival at 60 days and standard length at 36 days were decreased at the high concentration (2.2 μg a.i./L), and wet weight at 60 days was decreased at the medium-high concentration (0.96 μg a.i./L). In a sheepshead minnow early life-cycle study, 33-day larval survival was decreased at 0.67 μg/L; there were no eff ects on hatching or growth.

In a published, non-Guide line study (Celik-Ozenki et al. , 2011), no mortality or signs of toxicity
were observed in male rats treated with abamect in at 1 or 4 mg/kg/day for up to 6 weeks. In addition, no changes in body wei ghts or investigated tissue weights were observed for the abamectin treatment groups.

B. Estrogen Pathway

There was no evidence of potential interaction of abamectin with the estrogen pathway in the
Tier 1 or OSRI in vitro assays with the exception of the steroidogenesis assay where estradiol production was decreased (p at 1 μ M abamectin (0.72-fold). Additionally, abamectin showed no estrogen-related effects in the Tier 1 in vivo mammalian assays, in the FSTRA or the Part 158 mammalian or wildlife toxicity studies.

C. Androgen Pathway

There was no evidence of interaction of abamect in with the androgen pathway in the Tier 1 in

vitro ( i.e , AR binding and steriodogenesis a ssays were negative) or Tier 1 in vivo mammalian assays ( i.e ., Hershberger and male pubertal assays were negative) or in the FSTRA. There were no treatment-related effects on male reproductive parameters in the Part 158 mammalian or wildlife studies. While effects on the male repr oductive system (testicular damage, decreases in sperm count and motility, histopathological ch anges in testes and decreases in FSH concentrations) were observed in the OSRI literature study by Celik-Ozenki et al. (2011), no effects on gonadal histopathology were seen at simila r doses in the Tier 1 male pubertal assay or
in the Part 158 rat chroni c/carcinogenicity study. Overall, this is an isolated finding that is not corroborated by the Tier 1 or Part 158 data. Th erefore there is no convi ncing evidence for an interaction with the androgen pathway.ABAMECTIN FINAL

Page 23 of 53 D. Thyroid Pathway

There was no evidence of potential interaction of abamectin with the thyroid pathway. No
treatment-related effects were s een in thyroid, hormones, weight or histopathology in the male and female pubertal assays. In the AMA, no tr eatment-related finding were observed except for effects in growth parameters at the low concentr ation, with no effects on developmental stage or thyroid histopathology.

E. Conclusions

Abamectin demonstrates no convincing evidence of potential interact ion with the estrogen, androgen or thyroid pathways. !
V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, mamm alian or wildlife EDSP Tier 2 testing is not
recommended for abamectin since there was no convi ncing evidence of potenti al interaction with the estrogen, androgen or thyroid pathways.ABAMECTIN FINAL

Page 24 of 53 VI. References

Beavers, J. (1987) Avian Reproductive Toxicity to the Mallard Duck ( Anas platyrhynchos ). Unpublished study conducted by Wildlife Inte rnational, Ltd.; Study No. 105-135A. (MRID 40318601)

Brammer, A. (2006) Abamectin technical (MK 936) - 90 day combined oral toxicity and neurotoxicity study in rats. Unpublished study conducted by Central Toxicology Laboratory, Cheshire, U.K. Report No. PR1325-REG, August 25, 2006. (MRID 46959201)

Celik-Ozenci, C., Tasatargil, A., Tekcan, M., Sa ti, L., Gungor, E., Isbir, M., and Demir, R. (2011) Effects of abamectin exposure on male fertili ty in rats: potential role of oxidative stressrelated poly(ADP-ribose) polymerase (PARP) activation. Regul. Toxicol. Pharmacol . 61(3):310-

317.

Gledhill, A. (2005) Avermectin B1a: Comparative maternal and pup exposure following dietary and gavage dosing in the rat. Unpublished st udy conducted by Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK. La boratory Study Id.: CTL No. UR0795, April 28, 2005. (MRID 46727401)

Gordon, L. (1987) MK-0936: 53-Week Oral T oxicity Study in Dogs ; Unpublished study conducted by Merck Sharp & Dohme Research Labs.; TT#82-104-0. (MRIDs 00164022 and 40375510)

Gordon, L.R. (1982) MK-0936: Oral range-finding and teratogenic studies in rats and rabbits; TT#82-705-0; TT#82-705-1; TT#82-706-01. Unpublis hed study conducted by Merck Institute for Therapeutic Research. (MRID 00130819)

Gordon, L. (1985) Ninety-four week dietary carcinogenicity and toxicity study in mice. Unpublished study conducted by Merck Sharp & Dohme Research Laboratories; TT#83-002-0, - 1, -2, -3. (MRIDs 40069602, 40375512, and 40517802)

Gordon, L. (1985) One-Hundred-and-Five-Week Di etary Carcinogenicity and Toxicity Study in Rats with MK-0936; Final Report; Unpublis hed study conducted by Merck Sharp & Dohme Research Labs.; TT#82-099-0; A ugust 27, 1985. (MRIDs 40069601, 40375511, and 40517801)

Lee, M.R. (2012) Abamectin- Amphibian Meta morphosis Assay with African Clawed Frog ( Xenopus laevis ). Unpublished study conducted by Sm ithers Viscient, Wareham, MA. Laboratory report number 1781.6762. Study sponsor ed by Syngenta Crop Protection, LLC, Greensboro, NC. Study completed November 13, 2013. (MRID 48675001)

McAllister, W. (1986) Early Life Stage Toxicity of Avermectin B 1 (MK-0936 Technical) in Rainbow Trout ( Salmo gairdneri ) in a Flow-Through System: La boratory Project IdentificationABAMECTIN FINAL

Page 25 of 53 #33195. Unpublished study conducted by Analytical Bi ochemistry Laboratories, Inc. (MRID 40069609)

Moxon, M.E. (2005) Abamectin: Developmental ne urotoxicity study in rats. Unpublished study conducted by Central Toxicology Laboratory, Al derley Park, Macclesfield, Cheshire, UK. Laboratory Project Id.: C TL Study No. RR1048-REG, Syngenta No. T000967-05, December 16, 2005. (MRID 46727403)

Moxon, M.E. (2007) Abamectin: Assessment of developmental neurotoxicity in rats. Unpublished study conducted by Central Toxicolo gy Laboratory, Alderley Park, Macclesfield, Cheshire, UK. Laboratory Project Id.: CTL Study No. RR1086, Syngenta No. T000516-07, February 20, 2007. (MRID 47116201)

Robertson, R. (1984) Reproductive effects of MK-0936 administ ered orally by gavage to
Crl:COBS TM CD TM (SD)BR rats for two generations. Unpublished study conducted by Argus Research Laboratories, Inc. TT#82-9010. (MRID 00164151)

Sawhney Coder, P. (2012) Abamectin – Hershbe rger Assay in Orchidoepididymectomized Rats. Unpublished study conducted by WIL Research La boratories, LLC, Ashl and, Ohio. Laboratory Project ID: WIL-639124, May 10, 2012. (MRID 48614408)

Sawhney Coder, P. (2012) Abamectin – Pubertal Development and Thyroi d Function in Intact Juvenile/Peripubertal Female Rats. Unpublis hed study conducted by WIL Research, Ashland, OH. Laboratory Report No.: WIL-639122, August 9, 2012. (MRID 48926401)

Sawhney Coder, P. (2012) Abamectin - Pubertal Development and Thyroi d Function in Intact Juvenile/Peripubertal Male Rats. Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Project ID: WIL-639123, August 9, 2012. (MRID 48926402)

Sawhney Coder, P. (2012) Abamectin – Uterotrophic Assay in Ovariectomized Rats. Unpublished study conducted by WIL Research La boratories, LLC, Ashl and, Ohio. Laboratory Project ID: WIL-639125, May 10, 2012. (MRID 48614409)

USEPA (2010) Abamectin - Report of the Endocri ne Disruptor Review Team - Test Order #: EDSP-122804-252 to EDSP 122804-260. December 8, 2010. Office of Chemical Safety and
Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

USEPA. (2011) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

Wagner, H (2012) Abamectin - H295R Steroidog enesis Assay. Final Report. Unpublished study conducted by CeeTox, Inc. Kalamazoo, MI. Laboratory Report No.: 9047V-100352STER. February 29, 2012. (MRID 48614407)ABAMECTIN FINAL

Page 26 of 53 Wilga, P. (2012) Abamectin - Human Recombinant Aromatase Assay. Final Report. Unpublished study conducted by CeeTox, Inc., Ka lamazoo, MI. Laboratory Report No.: 9047V100352AROM. (MRID 48614404)

Willoughby, J.A. (2012) Abamectin - Androgen Recep tor Binding Assay (Rat Prostate Cytosol). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9047V100352ARB, January 19, 2012. (MRID 48614403)

Willoughby, J.A., Sr. (2012) Abamectin - Estroge n Receptor Binding (Rat Uterine Cytosol). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V100352ERB, January 19, 2012. (MRID 48614405)

Willoughby, J.A (2012) Abamectin-Estrogen Recepto r Transcriptional Ac tivation (Human Cell Line (HeLa-9903)) Final Report. Unpublishe d study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9047V-100352E RTA, January 19, 2012. (MRID 48614406)

York, D.O. (2012) Abamectin- Fish Short-Te rm Reproduction Assay with Fathead Minnow ( Pimephales promelas ). Unpublished study conducted by Sm ithers Viscient, Wareham, MA 02571-1037. Laboratory report number 1781.6763. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, NC 27419. St udy completed November 14, 2012. (MRID 48675002)ABAMECTIN FINAL

Page 27 of 53 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100)

The 21-day assay (MRID 48675001) of abamectin on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control a nd treatment group) were exposed to nominal
concentrations of abamectin (98.0% purity) at 0 (negative control), 0.00010, 0.0010, and 0.010 mg a.i./L. The highest nominal treatment concentration is the reported solubility of abamectin. Meanmeasured concentrations were < 0.000036 (<LOQ; control), 0.00012, 0.00094, and 0.0096 mg a.i./L. Reviewer-calculated time-weighted average (TWA) concentrations were 0.00012, 0.0010, and 0.0097 mg a.i./L. The test system was maintained at 21 to 22°C and a pH of 6.7 to 8.2.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. The survival of tadpoles exposed to abamectin was not sign ificantly affected (p>0.05) and was 100% in the control group and 99% in each of the treatment groups. Spinal
curvature was observed in the control and all trea tment groups, but did not appear to be treatmentrelated as the incidence was si milar across the control and treat ment groups except for a lower incidence in the high concentration group.

Abamectin caused no significant acceleration or de lay of median NF developmental stage at Day 7 or 21 at any treatment group (p>0.05) relative to the negative control. Further, no asynchronous development was observed throughout the test. By te st termination at Day 21, the median NF stage for the control and all treatment groups was NF 60. At termination, 5, 2, 0, 0% late-stage animals (NF>60) were observed among tadpoles exposed to the control and low, mid, and high treatment groups, respectively. Because the total number of organisms obser ved to be at late stage in the control were <20%, the animals > 60 NF were censored and not used in calculations of means and medians for the growth metrics presented be low (snout-vent length, hind limb length, and wet body weight).

Abamectin exposure did not signif icantly increase or decrease Da y 7 or 21 normalized (for snoutvent length) hind limb length (HLL). At Day 7, snout-vent length (SVL) and body weight were significantly increased (p=0.01) at the low treatment concentr ation relative to the negative control (10 and 24%, respectively). At Day 21, there were no significant effects indicated for normalized HLL, SVL, and body weight for any treatment concentration.

Thyroid gland histopathology changes were obser ved in the control and all treatment groups. Histopathological findings included mild follicul ar cell hypertrophy, mild follicular cell hyperplasia, and mild lumen area increases. These eff ects did not appear to be treatment-related as there was no concentration-response demonstrated in the data, and the incidences of follicular cellABAMECTIN FINAL

Page 28 of 53 hypertrophy were the same for control and hi gh treatment group. A sim ilar trend was observed with follicular cell hyperplasia diag noses (3 in negative control vers us 4 in high treatment group).

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48614403), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment,
which was conducted to measure the bi nding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M or 10

− 11 to 10

− 4 M) of abamectin (98% purity, Batch #A110628). DMSO was used as a ve hicle at a final assa y concentration of approximately 3.2%. A total of three runs were performed, and each run 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 radiolabeled reference androgen (R1881) prior to conducting AR competitive binding experiment. Saturation binding da ta were not originally provid ed in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The 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 repor ted in the EPA validation program (0.685 to 1.57 nM and 7 to 16 fmol/100 μg protein, respectively).

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 9.03 M) and the weak positive control, dexamethasone (

− 4.63 M), were within the expected ranges. The mean relative binding affinity (RBA) for the weak positive control was 0.004%, compared to R1881. Confidence in these numbers is high due to the small variation. Drift in the study assay
could not be assessed because all solvent control tubes were placed at the beginning of each run. The performance criteria for the assay were generally met.

Precipitation of abamectin was visually observed at a concentration of 10

− 3 and 10

− 4 M in Run 1, therefore the second and third runs were perf ormed at a maximum test concentration of 10

− 4 M.
Abamectin had no effect on specific binding of the [ 3 H]-ligand (>75% binding) over the concentration range of 10

− 11 to 10

− 4 M; therefore, a log IC 50 and RBA could not be calculated for
abamectin as it did not re sult in 50% reduction in [ 3 H]-ligand binding at any concentration.

Based on the results of the three runs, abamectin is classified as a N on-Binder in the Androgen Receptor Binding Assay.ABAMECTIN FINAL

Page 29 of 53 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48614404), abamectin (98.0% a.i., Lot # A110628 ) 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

− 4 M for 15 minutes to assess the pot ential of abamectin 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). Thr ee runs were conducted and each run included full activity controls, background activity controls, a positive control series in duplicate (10

− 10 to
10

− 5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and an abamectin test series in triplicate (10 –4 to 10

− 11 M). Due to precipitation observed at abamectin
concentrations of 10

− 4 M in Run 1, a half-log concentration of 10

− 4.5 M was included in Runs 2
and 3 and the 10

− 11 M concentration was dropped.

Aromatase activity in the fu ll activity controls ranged from 0.545-0.743 nmol·mg-protein

− 1 ·min

− 1for the three test runs, with a mean and standard deviation of 0.640 ± 0.067 nmol·mgprotein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.24 to 0.35% and averaged 0.30% of the full control activity. The respons e of the full activity controls and background controls was acceptable for each run.

For the positive control substance (4-OH ASDN), aromatase results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 , and %CV for replicates of each concentration within runs. The estimated log IC 50 for 4-OH ASDN averaged

− 7.3 M and the Hill slope was

− 0.95.

For abamectin, aromatase activity av eraged 0.599 ±0.054 nmol·mg-protein

− 1 ·min

− 1 at the

10

− 10 M concentration and 0.613 ± 0.021 nmol·mg-protein

− 1 ·min

− 1 at the highest successfully
tested concentration of 10

− 4.5 M. Across all three runs, aromatase activity was ≥ 90.9% of the full activity control at abamectin concentrations from 10

− 10 to 10

− 4.5 M; therefore, an IC 50 or Hill slope could not be calculated for abamectin.

Based on data from the average response curve, abamectin is classified as a non-inhibitor of aromatase activity in this assay.ABAMECTIN FINAL

Page 30 of 53 Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48614405), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A competitive binding experiment was conducted to measure the binding 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 abamectin (98% purity, Batch #A110628). DMSO was used as the solvent vehicl e at a final assay
concentration of 4%. A total of three runs were conducted, and each run included 19- norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β

estradiol as the natural ligand reference material.

The saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radiolabeled reference estrogen prior to rout inely conducting competitive binding experiment. 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. Data from two separate saturation binding assays were reported due to the use of two se parate cytosol preparations in the competitive binding assay for abamectin. For the batch of cytoso l prepared on September 27, 2010, the K d for [ 3 H]-17 β

estradiol was 0.081±0.006 nM, and the estimated B max was 52.70±1.05 fmol/100 μg protein. For the batch of cytosol prepared on July 10, 2011, the K d for [ 3 H]-17 β -estradiol was 0.303±0.090 nM, and the estimated B max was 33.59±0.70 fmol/100 μg protein. The K d for each run was within the expected range of 0.03 to 1.5 nM and there was minimal variation among runs. Although the Scatchard plots fit stra ight lines to the data, concavity was observed with the data sets from the July 10, 2011 cytosol prepar ation, which may indicate ligand depletion.

In the competitive binding experime nt, precipitation occurred at 10

− 3 M in Runs 1 and 2 and at

10

− 4 M in Run 3; therefore, data from these abam ectin concentrations were not included in the evaluation. Abamectin had no significant effect on specific radioliga nd binding (>75% binding; not interactive) at concentrations of ≤ 10

− 4 M in Runs 1 and 2 and ≤ 10

− 5 in Run 3.

The performance criteria were generally met for the assay, and the reference compounds performed as expected. The mean log IC 50 was

− 5.47 M for 19-norethindrone and

− 9.00 M for

17 β -estradiol, and the mean relative binding affinity (RBA) was 0.030% for 19-norethindrone, compared to the natural ligand.

Based on the results of the three runs, abamectin is classified as Not Interactive in the Estrogen Receptor Binding Assay.ABAMECTIN FINAL

Page 31 of 53 ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48614406), hER α -HeLa-9903 cells cultured in vitro were exposed to abamec tin (98% a.i., Lot # A 110628) at logarithmically increasing concentrations from 10

− 13 to 10

− 5 M in cell culture media containing 0.1% DMSO for 24 hours in two independent tests. The top conc entration was selected based on a preliminary cytotoxicity test, in which abamectin was tested up to the limit of solubility, 10

− 4 M, and was found to be cytotoxic at concentrations ≥ 10

− 5 M. The main assays were performed using 96-well plates, and each abamectin concentration was tested in 6 replicates per plate in each run. Cells were exposed to the test agent for 24 ± 2 hours to induce transcription of a reporter (luciferase) gene. Luciferase expression in response to ac tivation of the estrogen receptor by abamectin was measured using a proprietary luciferase assay.

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

methyltestosterone (41-47% PC) was lower th an the accepted values (at least 50% PC), indicating a decreased sens itivity to very weak agonists. In ad dition, cytotoxicity was observed at the highest tested concentrations of corticos terone in both runs. Results from cytotoxic concentrations were excluded from the analysis.

The mean RPC Max for abamectin was 1.5% for the first run and 1.3% for the second run, and the
associated PC Max values were 10

− 9 and 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, abamectin responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to
reach a maximum of 41.1-46.6% PC. Abamectin wa s only able to reach a maximum of 1.3-1.5% PC when tested up to the highest concentrati on possible based on cytotoxicity. Because the

RPC Max < PC 10 in both assay runs, abamectin was cons idered negative for estrogen receptor transcriptional activation in this test system.

Fish Short Term Reproduction Assay (FSTRA); (OCSPP 890.1350)

The 21-day short-term reproduction assay (MRI D 48675002) of abamectin with fathead minnow ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (16 spawning groups of 20-week-old fish; 2 males and 4 female s in each group; 4 groups per treatment) were exposed to abamectin (98.0% purity) at nominal concentrations of 0 (negative control), 0.000030, 0.00030, and 0.0030 mg a.i./L. Mean-measured concentrations were <0.000011 (<LOQ; control), 0.000030, 0.00031, and 0.0024 mg a.i./L. The reviewer-cal culated time-weighted averages (TWA)ABAMECTIN FINAL

Page 32 of 53 were 0.000025, 0.00028, and 0.0022 mg a.i./L. The test system was maintained at 24 to 26°C and a pH of 6.8 to 7.6. Unless otherwise indicated, al l effects are reported based on comparison to the negative (clean wa ter) control.

Survival at test termination was 88% for both males and females in the control and 89%, 75%, 88% for males and 93%, 75%, and 88% for female s in the low, mid, and high treatment groups, respectively. Replicate A of the mid treatment gr oup had high mortality (3 3% survival, combined sexes) but this was not considered treatment-rela ted and the replicate was excluded from analysis of all endpoints. Excluding this re plicate, mortality in the mid treatment group was 83% for males and 92% for females. Abamectin exposure was not associated with changes in body weight and/or length in either sex at any treatment level (p>0. 05). A few incidental obse rvations of lethargy, partial loss of equilibrium, and dark coloration were observed among females (1 to 2) in each of the three treatment groups. By test termination, no s econdary sex characteristic or clinical signs of toxicity were observed in males.

Mean spawning frequency and fecundity in cont rols, respectively, were every 4 days and 18 eggs/female/day/replicate (range 13-22); mean fertilization success in controls was 100% (range 99-100%). Abamectin did not signif icantly affect fecundity or fe rtility at any treatment level relative to the negative control (p>0.05). Plasma vitellogenin (VTG ) was not significantly altered in male or female fish at any treatment le vel (p>0.10 for males; p>0.05 for females). Gonadal histopathology findings were obser ved in male and female fish in the control and all groups. Histopathological effects in males included minima l to severe increased testicular degeneration, interstitial cell hyperplasia /hypertrophy, and granulomatous inflammation, and in females included minimal to severe increased oocyte atre sia, egg debris in oviduct, and granulomatous inflammation. The observations of granulomatous in flammation were consistent with infection by Mycobacteria sp . Due to this infection, hist ological effects associated with exposure to abamectin could not be determined. There were no significant changes in male or female gonado-somatic index (GSI) in any treatment group. Similarly, nuptia l tubercle score was not significantly affected in any treatment group. Plasma testosterone and 17ß-estradiol were not measured in either males
or females.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48614408) screeni ng for androgenic activity, abamectin (98.0% a.i., Batch # A110628) in aqueous 1% carboxymet hylcellulose (CMC) and 0.1% Polysorbate 80 was administered daily via oral gavage to gr oups of six 56- to 57-da y old, castrated male Sprague Dawley rats at dose levels of 0 (vehic le), 1.5 or 5 mg/kg/day. The androgenic positive control group consisted of six castrated rats exposed to the vehicle (1% CMC and 0.1%ABAMECTIN FINAL

Page 33 of 53 Polysorbate 80 by oral gavage) plus 0.2 mg/kg/ day of testosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, abamectin in 1% CMC and 0.1% Polysorbate 80 was administered daily via oral gavage to gr oups of six 56- to 57-da y old, castrated male Sprague Dawley rats at dose levels of 0 (vehic le), 0.5, 1.5 or 5 mg/kg/da y in conjunction with a daily dose of reference androgen TP at 0.2 mg/ kg/day by s.c. injection. The anti-androgenic positive control group consisted of six castrated rats exposed to 3 mg/kg/day flutamide (FT) in corn oil via oral gavage and 0.2 mg/kg/day TP (s .c. injection). The positive control animals for the androgen agonist assay served as the anti-androgenic negative controls.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were dosed for 10 consecutive days a nd terminated approximately 24 hours after the final dose administration. At necropsy, the fi ve androgen-dependent ti ssues, adrenal glands, kidneys, and liver were collected and weighed.

In the androgen agonist assay, no treatment-relate d clinical signs were not ed in either of the abamectin treated groups. Body weights and overall body weight gains for the 1.5 mg/kg/day treatment group were comparable to vehicle c ontrols throughout the duration of the assay. Animals dosed with 5 mg/kg/day had decr eased (p<0.05) overal l body weight gains ( ↓ 48%) compared to vehicle controls, and terminal body weights were decrease d (p>0.05) by 9%. For the positive TP control group, overall body weight gains were increased (p ≤ 0.05) by 24%. Food consumption in all abamectin-treated groups was statistically similar to the vehicle control; however, overall average food consumption was decreased (not signifi cantly, NS) by 17% at 5 mg/kg/day. There were no treatm ent-related effects in any of the accessory sex organs of abamectin-dosed animals. Animals in the posi tive control (vehicle + TP) group had accessory sex organ weight increases (p< 0.05) as follows: 446% in semi nal vesicles; 636% in ventral prostate; 226% in levator ani- bulbocavernosus (LABC); 438% in Cowper’s glands; and 75% in glans penis.

In the anti-androgen assay, one animal in the 5 mg/kg/day abamectin + TP group was euthanized in extremis on Day 6 following a body weight loss of 32 g and only 4.8 g of food consumed on Days 4-5. Moderate tremors and red material on the forelimbs and around the mouth were noted on Day 6. There were no macroscopic findings for this animal, but the death of this animal was
considered to be test substance related. All ot her rats in the anti-andr ogen assay survived until scheduled sacrifice, but the five surviving anim als in the 5 mg/kg/day+TP group had red material around the face, mouth, and/or forelimbs on Days 4- 11, and one of these animals also had slight tremors approximately 4 hours post-dosing on Day 6. The 5 mg/kg/day+TP animals had decreased (p<0.05) overa ll body weight gains ( ↓ 27%) compared to TP controls, and terminal body weights were decreased (p>0.05) by 6%. Body weights and overall body weight gains forABAMECTIN FINAL

Page 34 of 53 the 0.5 and 1.5 mg/kg/day groups were comparable to the TP controls th roughout the duration of the assay. Food consumption in all abamectin-tre ated groups was similar to the TP control; however, overall average food consumption was d ecreased by 11% (NS) at 5 mg/kg/day with TP. Overall mean food consumption in the positive control (FT +T P) group was decreased (p<0.05) by 12% compared to the TP controls ; no corresponding effects on mean body weight gains were noted. The seminal vesicle wei ghts at 0.5 and 1.5 mg/kg/day abamectin+TP were decreased (p<0.05) by 19 and 21%, respectively. However, there were no other treatmentrelated effects on organ weight or gross pathology in any of the accessory sex organs, including the animals dosed with 5 mg/kg/day abamectin+TP. Animals in the positive control (FT + TP) group had accessory sex organ weight decreases (p <0.05) as follows: 76% in seminal vesicles; 73% in ventral prostate; 54% in LABC; 62% in Cowper’s glands; and 35% in glans penis.

The dosing for this study (high dose of 5 mg /kg/day) was consider ed adequate based on mortality, clinical signs, a nd decreased body weights and body weight gains observed. All guideline performance criteria were met excep t that, in the anti-androgen assay, the maximum guideline-specified %CV was exceeded for the glans penis (23.1% vs. 17%) and LABC (31.2% vs. 20%) in the positive control.

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

assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48926401), 15 Sprague-Dawley [Crl:CD(SD)] rats/dose group were treated with abamectin (98.0% a. i.; Batch # A110628) in aqueous 1% (w/v) carboxymethylcellulose + 0.1% (v/v) Polysorbate 80 daily via oral gavage at doses of 0 (vehicle), 0.75, or 1.5 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on P ND 22, and age and weight on day of attainment were recorded. Following sacr ifice on PND 42, blood was collect ed for clinical chemistry analyses, and total thyroxine (T 4 ) and thyroid stimulating ho rmone (TSH) levels were determined using either an elec trochemiluminescent immunoassay (T 4 ) or a magnetic radioimmunoassay (TSH). Adrenal, liver, pitu itary, thyroid, and urogenital organs were weighed, and microscopic examinations were performed on the thyroid, ovary, kidney, and

uterus.

There were no effects of treatment at either dose level on mortality, clinical signs, initial or final body weights, overall body weight gains, food c onsumption, organ weights, clinical chemistry parameters, or gross pathology.ABAMECTIN FINAL

Page 35 of 53 The percent of females regular ly cycling was decreased at 0.75 and 1.5 mg/kg/day (38% and 50% respectively vs. 87.5% controls); however on ly the difference from control at the highest dose was statistically significant Also, a shift was noted in the cycle status at necropsy, with
fewer females in diestrus and more in estrus in treated groups compared to controls (9 diestrus per treated group vs. 13 controls; 5 estrus per trea ted group vs. 2 controls). The toxicological implications of these findings were unclear. An increase (NS) in the incidence of cysts present in the kidney were noted at 1.5 mg/kg/day (4/15 treated vs. 1/15 controls); however, there were no corroborating findings in related clinical chemistry parameters. Therefore, this increase was
considered incidental.

No effects of treatment were observed on serum T 4 or TSH levels, and th ere were no effects of treatment on thyroid follicular cell height or colloid area.

The doses tested were determined to be adequa te based on data from two previously performed studies showing tremors and toni c/clonic convulsions after 1-2 dos es at 15 mg/kg/day and above, body weight losses and/or decreased body weight gains and food consumption at 7.5 mg/kg/day, and slight to moderate tremors, and lowe r mean body weights and food consumption at 2.5 mg/kg/day.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48926402), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral ga vage (5.0 mL/kg) with abamectin technical (98.0%; Lot # A110628) in aqueous 1% carboxymethylcellulose with Polysorbate (0.1%) at doses of 0, 0.75 or 1.50 mg/kg/day from post-natal day (PND) 23 to PND 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and thei r age and weight at day of attainment was recorded. Following sacrifice on PND 53, blood was collected for clinical chemistry analyses, including total thyroxine (T 4 ), testosterone, and thyroid stimulating hormone (TSH).
Testosterone and T 4 concentrations were analyzed using an electrochemiluminescent assays and TSH concentrations were analyzed using a ra dioimmunoassay. Liver, ad renals, pituitary, and urogenital organ weights were recorded, and mi croscopic examinations were performed on the thyroid, kidney, testis and epididymis.

No treatment-related effects were observed on mortality, body weights, b ody weight gain, organ weights, or gross pathology. Ch anges observed in thyroid, testis and seminal vesicle weights were either not statistically significant or did not occur in a dose-dependent manner and were not supported by microscopic changes in the same or gans. Age and body weight at PPS in the treated groups were similar to controls. Thyroid hormone levels, colloid area an d follicular cell heightABAMECTIN FINAL

Page 36 of 53 scores for the thyroid were similar in the treated groups relative to control. Serum testosterone
levels were similar in the trea ted groups to the control group.

Although no toxicity was observed in the curr ent study at doses of abamectin up to 1.50 mg/kg/day, this dose was considered adequate based on previous studies showing decreased body weight and body weight gain at 7.5 mg/kg/da y in young adult rats, and tremors, decreased body weight, and food consumption at 2.5 mg/kg/day in juvenile rats.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48614407), H295R cells cultured in vitro in 24-well plates were incubated with abamectin (98.0% a.i., Lo t # A110628) at log concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulf oxide (DMSO) was used as the vehicle, at a
final concentration of 0.05%.

Testosterone and estradiol levels were measured with HPLC/MS-MS analysis. A total of eight assay runs were reportedly conducted; however, four of the runs we re reported to be unsuccessful and the hormone concentrations were not analyzed, and another run did not meet the assay’s performance criteria and was not stat istically analyzed. Ther efore, the study report includes data from only three independent r uns. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels; positive controls included the known inhibitor (proch loraz) and inducer (forskolin) of estradiol and testosterone production.

No evidence of cytotoxicity was present from the MTT cell viability test conducted on the QC test plates (97.2 to 111.8% viability). Cell viability was also acceptable at abamectin
concentrations of 0.001 to 1.0 μ M (91.2-104.0%), but excessive cytotoxicity was observed at
abamectin concentrations of ≥ 10μM (13.4-50% viability). Viability for the methanol positive
control was 4.9 to 6.0%.

Guideline acceptability recommendations and requirements were met, including adequate production of testosterone and estradiol, acceptabl e reproducibility (low % CV), and appropriate
induction and inhibition w ith positive controls.

Significant (p ≤ 0.05) changes in testosterone production af ter incubation with abamectin were not observed, although the mean fold-change decreased at the highest testable concentration. Significant (p ≤ 0.05) decreases in estradiol production were observed in Run 1 at 0.1 μM abamectin, and in all three runs at 1 μM abamectin. The mean fold-differences showed a concentration-dependent response with decreases of 0.9-fold at 0.1 μM and 0.7-fold at 1 μM.ABAMECTIN FINAL

Page 37 of 53 Based on the hormone responses in each of the three independent runs, abamectin treatment resulted in statistically signifi cant and reproducible decreases in estradiol production, but did not result in statistically signif icant and reproducible alterations in testosterone production.

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48614409) conducted to screen for potential estrogenic activity, abamectin (98.0% a.i., Batch# A110628) in aque ous 1% carboxymethyl cellulose (CMC) and 0.1% Polysorbate 80 was administered daily via oral gava ge to groups of six ovariectomized Sprague Dawley rats at dose levels of 0 (vehic le), 2.5 or 7.5 mg/kg/day on post-natal days (PND) 57-59. A positive control group was tr eated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.3 mg/kg/day by or al gavage. Body weights and f ood consumption were determined daily. All animals were terminated and necrops ied on PND 60 approximately 24 hours after the
final dose administration to determine wet and blotted uterine weights.

All animals survived until sche duled termination. No treatment -related effect was observed at 2.5 mg/kg/day, and wet and blotted uterine weights were comparab le to the vehicle controls.

At 7.5 mg/kg/day, all six females had slight to m oderate tremors on Days 1 or 2, and the tremors persisted for three females until sacrifice on Day 4. Additional findings included pale body and red material around the nose, mouth and forelimbs. Body weights were decreased (p ≤ 0.05) by 14% and 21% on Days 3 and 4, respectively, and th ere was an overall body weight loss of 37.3 g for Days 1-4, compared to a body weight gain of 16.1 g for the vehicle control. Overall mean food consumption in was also decreased (p ≤ 0.05) by 60% compared to the vehicle controls. However, wet and blotted uterine weights we re comparable to the vehicle controls.

In the positive control (EE) group, the mean body weights were decreased (p ≤ 0.05) by 10% and 13% on Days 3 and 4, respectively. The overall body weight loss was 13.5 g for Days 1-4, and the overall mean food consumption was decreased (p ≤ 0.05) by 48%. Absolute wet and blotted uterus weights were increased (p ≤ 0.05) by 432% and 167%, respectively, as expected.

The mean blotted uterine weight for the vehicle control group (0.037% of body weight) was within the performance criteria (<0.04%) of the test guideline.

At the high-dose of 7.5 mg/kg/day, the rats lost 15% of their initial body weight during the 3-day treatment period and clinical signs of neurotoxicity (tremors) pers isted in at least half of the animals through the study. In a range-fi nding study (MRID No. 48614402; Appendix 2), no
treatment-related effects were seen when treate d at 5.0 mg/kg/day for 3 consecutive days. BasedABAMECTIN FINAL

Page 38 of 53 on the results of the range findi ng and the main study, the dose tested were deemed to be adequate to assess the potential estrogenic activity of abamectin.

No statistically significant changes were seen in uterine weight in this assay. Abamectin is negative in the uterotrophic assay.ABAMECTIN FINAL

Page 39 of 53 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

The Part 158 general toxicity studies considered relevant for determining the potential interaction of abamectin with the endocrine system are summ arized below. Given the breadth of toxicity data available on abamectin, the only Part 158 stud ies discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

Combined 90–Day Oral Toxicity and Neurot oxicity in Rodents (Rat; OCSPP 870.3100 and 870.6200)

In a combined subchronic oral toxicity and neurotoxicity study ( MRID 46959201 ), abamectin (96.2% purity) was administered in sesame s eed oil (1 mL/100 g) by gavage to Alpk:APfSD (Wistar-derived) rats (16/sex/group) at doses of 0, 0.4, 1.6, and 4.0 mg/kg/day for 90 days. All rats in the 4.0 mg/kg/day group we re sacrificed after Week 7 due to adverse clinical signs and decreased body weights. Neurobehavioral assessment [functional observational battery (FOB) and motor activity testing] was conducted on ten rats/sex/group in Weeks -1, 2, 5, and 9, and on all surviving animals at Week 14. At study termination, five anim als/sex in the remaining groups were euthanized and perfused in situ for neuropathological examination, and the brain and peripheral nervous system tissues were examined from the control and medium-dose groups. All other rats were subjected to gross pathological examinations after sacrifice, including those sacrificed prematurely. Organs that were weighed included the liver, kidneys, testes, epididymides, ovaries, uterus, and adrenals (c ontrol, low-, and medium-dose animals only). Gross and microscopic pathological examinations were conducted on these tissues as well as the prostate, seminal vesicles, mammary gland, pituitar y, and thyroid.

There were no treatment-related mortalities in the low- and medium-dose groups during the study. Clinical signs, observed in the high-dose females starting at Week 2, included: irregular breathing, sides pinched in, upward curvature of the spine, and tip -toe gait; pilo erection, upward curvature of the spine, and m outh-staining were prevalent in high-dose males. Decreased (NS) body weight gain was observed in both the high-do se males and females in the first four weeks of treatment, although the effects were minimal in the males ( ↓ 11%) and more severe in females ( ↓ 40%). The high-dose males had mini mally decreased body weight (NS, ↓ 5%) at Week 4, with weight similar to control by Week 6. Body wei ghts in the high-dose females were decreased (NS) by 4-7% through Week 7, and the overall (Week 1-7) body weight gain was decreased (NS) by 26%. No clinical signs, or decreases in body weight or body weight gain, were observed in the low- or medium-dose groups. Food consumption was not affected in treated males, but was decreased (p<0.05, ↓ 14%) in high-dose females at Week 2, and increased (p<0.01, ↑ 12%) at
Week 4.ABAMECTIN FINAL

Page 40 of 53 Some treatment-related behavior al effects were observed for high-dose males and females during
the FOB evaluations conducted at Weeks 2 and/or 5; no effects were observed in the lower-dose groups. There were no treatment-related effects in any animals on the measured clinical chemistry, hematology or urinalysis parameters; motor activity; brain weight; organ weights; gross and microscopic pat hology; or neuropathology.

The LOAEL for abamectin was 4.0 mg/kg/day based on adverse clinical signs in male and female rats, and decreases in body weight gain and fore- and hind-limb grip strength in the females; the NOAEL was 1.6 mg/kg/day.

Prenatal Development in Rodents (Rat; OSCPP 870.3700)

In a developmental toxicity study in rats ( MRID 00130819 ), abamectin (94% purity) in sesame oil was administered by gavage to SD rats (2 5/dose group) at concentrations of 0, 0.4, 0.8, or 1.6 mg/kg/day on GDs 6 through 19. All dams were sacrificed on GD 20 and the number of corpora lutea, implantations, and resorptions were recorded. Fetuses were examined for external abnormalities, sexed, and weighed. Visceral exam ination was performed on one-third of the
fetuses in each litter. Free-hand sections of the h ead of one-third of the fe tuses in each litter also were conducted. Finally, all fetuses were pro cessed, stained, and examined for skeletal
abnormalities.

There were no mortalities during the study, and no clinical signs were noted. No decreases in body weight or body weight gain were observed; body weight gain was increased among all treatment groups, but the extent was not repor ted. There were no effects of treatment on implantations or percent pre-implantation loss. Resorptions were increased in the medium-dose group only, and are not considered to be related to treatment. The total number of fetuses, sexratio, number dead (n=0 for all groups), and fe tal body weight were unaffected by treatment. Although some external abnormalities were observed, the malformations were not considered to
be related to treatment. No treatment-related malformations or variat ions were noted during
visceral examinations. An increased incidence of alterations of the lumbar ribs was observed in the high-dose group; however, when compared with historical control data, it was concluded that
increased lumbar ribs was not a fetotoxic eff ect. The maternal and fetotoxic NOAEL is the high dose ( ≥ 1.6 mg/kg/day). An LOAEL is not established. The teratogeni c potential was negative.

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 00130819 ), abamectin (94% purity) was administered to impregnated New Zealand White rabbits (18/dose group) by gavage at doses of 0, 0.5, 1.0, and 2.0 mg/kg/day from GDs 6 through 27. In an initial dose-range finding study, abamectin (94% purity) was administered to impregnated New Zealand White rabbits (10/dose group) by gavage at doses of 0, 0.5, 1.0, 2.0, a nd 3.0 mg/kg/day from GDs 6 through 18. On GD 28, all surviving does were sacrificed in both studies; maternal toxi city was reported from the dose-range finding study, and fetotoxicity from the developmental study. Maternal bodyABAMECTIN FINAL

Page 41 of 53 weights were recorded during both studies; the numbers of corpora lutea, implantations, and resorptions were recorded in the developmen tal study. In addition, all fetuses were weighed, sexed, and external examinations were conducted. Each fetus was examined for gross external observations, sacrificed, and examined fo r visceral and skeletal malformations.

Deaths, sacrifices due to mori bundity, and abortions occurred acr oss the control and treatment groups, with no respect to treatment. Live pregna nt rabbits available for evaluation in the study ranged from 11 (medium dose) to 15 (contro l and high-dose groups). Treatment-related decreases in body weight, and feed and water cons umption (extent and sign ificance not reported) were noted in the high-dose dams. There were no effects of treatment on implantations, percentage of implantation losses, or resorptions . The total number of fetuses were increased (significance not reported) in the high-dose grou p, and the number of dead fetuses was increased (significance not reported) in the medium- dose group. Sex ratio and fetal body weight were unaffected by treatment.

Developmental toxicity was repor ted in fetuses of the high-dose group (the maternal toxicity level); findings included cleft palate, clubbed foot, and delayed ossifi cation of sternebrae, metacarpals, and forefoot phalanges. No effect s were seen at doses of 1.0 and 0.5 mg/kg/day. The maternal and developmental toxicity L OAELs were 2.0 mg/kg/day, and the NOAELs were 1.0 mg/kg/day.

Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation reproduction study in rats ( MRID 00164151 ), abamectin (purity not reported) was administered daily by gava ge to SD rats (30/sex/dose for the F 0 generation;
32/sex/dose for the F 1 b parental generation) at concentrations of 0 (vehicle control, sesame oil), 0.05, 0.12, and 0.40 mg/kg/day. F 0 rats were mated twice, in order to produce the F 1a and the F 1b litters, and pups selected for the F 1b parental generation were also mated twice to produce the F 2 a and F 2b litters. F 0 rats were given the test material beginning approximately 68 days prior to cohabitation and continuing through weaning of F 1b pups, with at least a 16-day maternal rest period prior to the second mating. At weaning of the F 1b rats, five males and five female pups per dosage group were sacrificed for subsequent complete gross necropsy and histopathology evaluations; body weights and weight s of liver, kidney, testes, hear t, brain, and spleen were recorded. Also at weaning, one or two pups per sex, from each F 1b generation litter with surviving pups, were randomly selected for continued study.

The dosing period of the F 1b generation began at weaning, at l east 70 days prior to cohabitation,
and continued to at least 30 days after the last F 2b pups were weaned, with at least a 14-day maternal rest period prior to the seco nd mating. Ten male and 25 female adult F 1b generation animals per dosage group were randomly sele cted for complete ne cropsy and histopathology procedures; body weights and weights of brain, spl een, heart, kidneys, liver, and testes were recorded. The testes and epididymides from remaining F 1b male rats were removed, weighed, andABAMECTIN FINAL

Page 42 of 53 fixed; eyes were retained from these F 1b male rats and from the remaining F 1b female rats. At weaning of the F 2 b rats, ten male and ten female wean ling rats per dosage group were randomly selected for skeletal examination, and five male and five female F 2 b weanling rats per dosage group were randomly selected for complete gr oss necropsy and histopathology evaluations. The eyes from the F 2 a and F 2 b weanling rats were retained.

Clinical observations, body weights, and reprodu ctive parameters were recorded for the F 0 and

F 1b parental rats. Mating confir mations, delivery and lactation observations, as well as pup viability, sex, and body weights were recorded for the F 1a and F 1b pups until weaning; the same procedures were conducted for the F 2a and F 2b pups. Gross external and visceral examinations were conducted on pups that died during the l actation period, and they were examined for skeletal anomalies. No signs of clinical toxici ty were noted for the parental generations during the study.

Nine mortalities of F 0 parental rats and five mortalities of F 1b parental rats during dosing occurred, but the mortalities were not dose-related and were not at tributed to test material. Body weight gain increases (NS) were re ported for the medi um- and high-dose F 0 males; body weight gain increases (NS) also were reported for the medium- and high-dose F 0 females during the growth phase. Body weight gain increases (NS) also were reported fo r the medium- and highdose F 0 females during the F 1a gestation period, with no di fference noted during the F 1b gestation period. Body weight gain in F 0 females during the F 1b lactation period were decreased (NS) compared to control. High-dose F 1b pups (both sexes) and medium-dose F 1b females selected for the parental generation had lower mean body wei ghts compared to control at dose initiation. Body weight in the high-dose F 1b males continued to be decreas ed (p<0.01) up to Day 28, but were then similar to control through Day 168, and were then increased (significance not reported) compared to the control between Days 175-238 and Days 245-272 (NS). Body weight gain in high-dose females was decreased (NS) dur ing the growth phase, with no differences in overall body weight gains during the F 2a and F 2b gestation periods. Decreased (NS) body weight gains in F 1b females also were noted during the F 2a and F 2b lactation periods.

In the first mating of the F 0 generation, the male rats showed no effects of treatment on mating or days of cohabitation. However, in the second mating of the F 0 generation, males exhibited a decrease (p<0.01) in mating at the high dose ( ↓ 29%) compared to control, and an increase (p<0.05 or 0.01) in the number of da ys in cohabitation in the low- ( ↑ 1.5 days), medium- ( ↑ 1.5 days), and high-dose ( ↑ 1.5 days) groups compared to control.

For F 0 females, there were no effects of treatment on fertility, days in c ohabitation, or gestation length with the first mating. In the F 1a litters, increases in live pups dying (p<0.01, ↑ 567%), pup mortality, and decreases in viability indices and the lactation index were observed at the high dose. In addition, pup body weight in the high -dose group was decreased (p<0.01) on LDs 7, 14, and 21. In the second mating of the F 0 generation, females showed a decrease (p<0.01) in mating at the high dose ( ↓ 26.7%), and an increase (significance not reported) in the number of days inABAMECTIN FINAL

Page 43 of 53 cohabitation ( ↑ 5.7 days) compared to control. There was no effect of treatment on fertility index or duration of gestation, but th ere were increases in live pups dying, pup mort ality, and decreases in the viability and lactat ion indices were observed at the high dose in the F 1b litters. Pup body weights in the high-dose group also were decreased (p<0.01) on LDs 7, 14, and 21.

In the first mating of the F 1b generation, treated males did not show any adverse effects on fertility, days in cohabitation, or other reproductive parameters. In the second mating of the F 1b generation, males did not show any treatment-related effects in fertility, bu t did show differences in days in cohabitation with increased (p<0.05) mating during Da ys 1-4 at the high dose (90.9% vs. 63.6% control) and decreased (p<0.05) mati ng during Days 5-8 at the high dose (9.1% vs 13.6% control).

For the first and second matings of the F 1b females, no adverse effects were seen in mating, days in cohabitation, fertility index, a nd duration of gestation. In the F 2a and F 2b litters, there were increases (NS and significance not reported, respectively) in total pup mortality at the high dose, as well as decreases (significance not reported) in the viability and lacta tion indices at the high dose. Pup body weights in the hi gh-dose groups were decreased (significance not reported) on LDs 7, 14, and 21 for both litters.

There were no treatment-related effects on organ weights of F 0 adults, F 1b weanlings and adults,

or F 2b weanlings. This included no treat ment-related lesions in the testes and epididymides of the

F 0 /F 1 adults and F 1b /F 2b weanlings; prostate glands of the F 1b /F 2b weanlings and F 1b adults;
ovaries and uterus of the F 1b /F 2b weanlings and F 1 b adults; cervix of the F 1b adults and F 2b weanlings; and the thyroid, adrenal and pituitary glands of the F 1b /F 2b weanlings and F 1b adults.
Treatment-related retinal anomalie s (unilateral intra-retinal rosett es) were observed in males and females in the high-dose F 2b weanlings.

The overall NOAEL for the study is 0.12 mg/kg/day. The LOAEL is 0.40 mg/kg/day (high-dose) based on increased incidence of retinal rosettes (F 2b weanlings) and increased pup mortality, decreased viability and lacta tion indices, and decreased pup body weight in all litters.

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic toxicity/carci nogenicity study in rats ( MRIDs 40069601, 40375511, and
40517801 ), abamectin (91.1% purity) was administered to albino Crl:CD (SD) rats (65/sex/dose)
in the diet at nominal concentrations of 0 (acetone-treated feed; two groups), 0.75, 1.5, and 2.0 mg/kg/day for two years. Fifteen rats/sex/group we re selected prior to study initiation for the 53- week interim sacrifice. At Week 11, the dos e for the high-dose group was increased to 2.5 mg/kg/day, and then decreased back to 2.0 mg/k g/day at Week 13 due to the appearance ofsevere signs of CNS toxicity; to prevent excessive mortality the males and females in the highdose group only were placed on control diet for one day during Weeks 12 and 13, respectively. Organs that were weighed at inte rim and final sacrifice included th e liver, adrenals, kidneys, and testes; at final sacrifice, the thyroid, pituitary, pr ostate, seminal vesicles, ovaries, and uterus wereABAMECTIN FINAL

Page 44 of 53 also weighed. Gross and microsc opic pathological examinations were conducted on these tissues, as well as the mammary gland and epididymides (with testes), for the control and high-dose group at interim sacrifice and all do se groups at final sacrifice.

Compound-related whole body tremors and overall unthr ifty appearance were observed in some rats in the high-dose group. In some cases, the tremors correlated with the increase in dosage from 2.0 to 2.5 mg/kg/day; however, the tremor s persisted intermittently until the time of sacrifice, despite the reduction in the high-dos e to 2.0 mg/kg/day. The tremors were considered
to be related to treatment because there were no pathological lesions to correlate with the

tremors.

There were no treatment-related effects on morta lity or survival. Body weights were increased (significance not reported) for both male and fe male rats in all treated groups compared to control; overall body weight gains for female rats were not affected, but we re increased for male rats in all treated groups. Although increased body weight is considered to be a treatment-related effect, it is not adverse. There were no effects of treatment on food consumption, ophthalmological lesions, hematological parameters , serum biochemical parameters, urinalysis, organ weights, gross necropsy fi ndings, or histopathology in any treated male or female rats.
There also were no treatmen t-related neoplastic findings.

The oncogenic potential was negative for abamec tin. The systemic NOAEL is 1.5 mg/kg/day, and the LOAEL is 2.0 mg/kg/day based on tr eatment-induced tremors. The high dose (2.0 mg/kg/day) is also cons idered the MTD due to th e observation of tremors.

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Mouse; OCSPP 870.4300)

In a chronic toxicity/car cinogenicity study in mice ( MRIDs 40069602, 40375512 and

40517802 ), abamectin (91.1% purity) was administered to CrL:CD-1 (ICR) BR mice (74/sex/dose) in the diet at nomin al concentrations equivalent to 0 (vehicle control; two groups), 2.0, 4.0, and 8.0 mg/kg/day for 93-94 weeks. Mean compound intake was determined to be 2.0, 4.1, and 8.1 mg/kg/day in male mice and 2.1, 4.2, a nd 8.3 mg/kg/day in female mice. At 25/26 weeks and 52 weeks, twelve mice/sex/group were sacrificed for blood examinations. Due to increased mortality in male rats in the high- dose group, dosing of this group (males only) was
discontinued at Week 90 when survival had reach ed 40%. Organs that were weighed at final sacrifice included the liver, adre nals, kidneys, thyroid, pituitary, testes, prostate, ovaries, and uterus. Gross and microscopic pa thological examinations were c onducted on these tissues as well as the mammary gland, epididymides, and seminal vesicles.

Tremors observed in two females in the high- dose group late in the study (Weeks 89 and 91)
were considered to be related to treatment b ecause there were no gross or microscopic findings
associated with the tremors. Tremors also were observed in a medium-dose female the day prior to death; tremors in this animal were consider ed secondary to a metast atic lymphoma of theABAMECTIN FINAL

Page 45 of 53 pituitary. Tremors observed in moribund male rats in the control and high-dose groups were considered secondary and not related to treatment.

There was increased (p<0.05) mortality in ma le mice in the high-dose group compared to control, which was considered to be possibly related to treatment. There were no treatmentrelated effects on mortality in female mice. Overall body weight gain was decreased (p<0.05, ↓ 23-25%) in high-dose females compared to cont rols at termination of the study. Similar decreases were reported to have been obs erved throughout the study. Decreased body weight gain in the high-dose females was considered to be treatment-related; no changes were note at the lower doses. In male mice, body weight ga ins in the mid- and high-dose groups were decreased (NS; ↓ 4-5%) compared to control group I and increased (NS; ↑ 3-4%) compared to control group II. There were no effects of treatment on food cons umption in male mice, but food consumption was increased (NS; ↑ 2-8%) in treated female mice.

There were no treatment-related effects on opht halmological lesions, he matological and serum biochemical parameters, organ weights (absolute or relative), or gross necropsy findings in male or female mice. In male mice in the high-dos e group, there was an increased extramedullary hematopoiesis of the spleen (3-fold greater than control), an increased incidence of dermatitis (22% vs. 4-6% in controls), and an increase d incidence of myeloid hyperplasia of the bone marrow. There were no treatment-related neoplastic lesions.

The systemic NOAEL is 4.0 mg/kg/day. The LOA EL is 8.0 mg/kg/day based on an increased incidence of dermatitis in male mice, an increased incidence of extramedullary hematopoiesis in the spleen of males, increased mortality in male s, and tremors and decreased body weight gain in
females.

Chronic Oral Toxicity in No n-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study in dogs ( MRIDs 00164022 and 40375510 ), abamectin (>89% purity) was administered to Beagle dogs (6 /sex/dose) in the diet at nomina l concentrations of 0 (acetone, vehicle control), 0.25, 0.50, and 1.0 mg/kg/day for 53 weeks. Organs that were weighed included the liver, adrenals, kidneys, and testes. Gross an d microscopic pathological examinations were conducted on these tissues as well as the thyr oid, pituitary, ovaries, prostate, uterus, and mammary gland.

There was a high incidence of mydriasis in male and female dogs administered the medium and high doses during the study (occurrences of 3 and 15%, respectively). In addition, three highdose dogs exhibited salivation which may have b een treatment-related as no other treated dogs exhibited this sign. Three male dogs in the high-do se group died or were sacrificed prior the end of the study; the deaths were attributed to abam ectin treatment. Overall body weight gain in dogs in the high-dose group were decreased ( ↓ 23%) compared to control. Food consumption also was decreased (significance not repor ted) in the high-dose group; decreased food consumption may have contributed to observed decreases (NS) in BUN and total protein in these dogs. There wereABAMECTIN FINAL

Page 46 of 53 no effects of treatment on hematological parameters . The results of urinalyses did not show any
effects of abamectin treatment. There also we re no effects of treatment on organ weights (absolute and relative), or gross necrops y and histological examination results.

The NOAEL is 0.25 mg/kg/day, and the LOAEL is 0.5 mg/kg/day based on increased mydriasis.

Developmental Neurotoxicity in Rodents (Rat; OCSPP 870.6300)

In a developmental neurotoxicity study ( MRID 47116201 ), abamectin (96.2% purity) in sesame oil was administered by gavage to pregnant Al pk:APfSD rats (30/dose) from GD 7 through LD 22 at doses of 0 (vehicle c ontrol), 0.12, 0.20, or 0.40 mg/kg/day. The pups were not directly dosed. Dams were allowed to deliver naturally and were killed on LD 29. On PND 5, litters were standardized to 8 pups/litter; th e remaining offspring were sacrificed and not examined further. Subsequently, 1 pup/sex/litter/group (at least 10 pups/sex/dose when av ailable) were allocated to the following subsets: Subset 1: FOB on P ND 5, 12, 22, 36, 46, and 61; motor activity on PND 14, 18, 22, and 60; and brain weight and neuropa thology on PND 63; Subset 2: FOB on PND 5, 12, 22, 36, 46, and 61; water maze on PND 24 and 27; and brain weight and neuropathology on PND 63; Subset 3: FOB on PND 5 and 12; audito ry startle on PND 23 and 61 (for animals not sacrificed on PND 12); and brain weight and neuropathology on PND 12; and Subset 4: FOB on PND 5, 12, 22, 36, 46, and 61; water maze on PND 59 and 62.

There were no effects of treatment on mortalit y, clinical signs, FOB parameters, reproductive performance, or gestation length in maternal rats. Body weights ( ↑ 1-5%), body weight gains ( ↑ 10-18%), and food consumption ( ↑ 8-12%) were increased (p<0.01 or 0.05) in all treated animals during gestation, with body weights and body weight gains comparable to control during lactation (except for a continued increase (NS) in body weight gain at the high dose). Food consumption was decreased (p<0.01 or 0.05) in the high-dose dams during lactation. The maternal LOAEL is not observed; th e maternal NOAEL is 0.40 mg/kg/day.

No treatment-related effects were observed on li tter parameters [number born live, number born dead, sex ratio (% male), mean litter size, live birt h index, and viability index] or clinical signs (including FOB) in the offspring from PND 1 through 5. After culling on PND 5, mortality and associated clinical signs of t oxicity were observed in the high-dose pups. A total of 32 males and 27 females were missing and presumed dead, found dead, or killed due to clinical signs of toxicity. The pups were generally small in si ze and presented with dehydration and tremors. Additionally at this dose, pup body weights were decreased (p ≤ 0.01) by 10-36% from PND 8 to 36. There were no surviving pups in this dose group after PND 38. As th ese deaths left an insufficient number of pups to complete all of the study objectives , all dams and pups at this dose were removed from the study during PNDs 15-38.

No treatment-related effects were observed on litt er parameters, clinical signs, FOB parameters, motor activity, auditory startle reflex, lear ning and memory, brain weight, or gross or microscopic pathology (neuropath ological tissues) in the low- and medium-dose pups. The ageABAMECTIN FINAL

Page 47 of 53 when VO occurred was delayed (p ≤ 0.05) in the low- and medium-dose pups ( ↑ 1.3 days). Although the body weights at VO for these groups we re similar to control, examination of the body weight data revealed a 7-8% decrease (p ≤ 0.01) in body weights at approximately the time of VO (PND 36). Therefore, the delay in VO may be related to a delay in development associated with decreased growth. No effect of treatment was observed on the time to PPS.

Decreases (p ≤ 0.01 or 0.05; ↓ 4-8%) in post-weaning body weights we re observed in the low- and medium-dose pups throughout the post-weaning in terval (PNDs 36-63). Re-evaluation of the body weight data indicated that there was a stat istically significant di fference for low- and medium-dose males compared to control, but no statistically significant difference for females. However, the decrease in low-dose males was appr oximately 3% relative to control, and it was determined not to be toxicologically relevant. Th e offspring LOAEL for this effect is established at 0.2 mg/kg/day based on body weight reductions ( ↓ 6%). The NOAEL for this effect is 0.12 mg/kg/day.

In an earlier developmental neurotoxicity study ( MRIDs 46727401-46727403 ), abamectin (96.2% purity) in sesame oil was administered by gavage to pregnant Alpk:APfSD rats (30/dose) from GD 7 through LD 22 at doses of 0 (veh icle control), 0.12, 0.20, or 0.40 mg/kg/day. The pups were not directly dosed. Dams were allowed to deliver naturally and were killed at weaning on LD 29. On PND 5, litters were standardized to 8 pups/litter; the remaining offspring and dams
were sacrificed and not examined further. S ubsequently, one pup/sex/li tter/group (at least 10 pups/sex/dose when available) were allocated to the following subsets: Subset 1: FOB on PND 5, 12, 22, 36, 46, and 61; motor activity on PND 14, 18, 22, and 60; and brain weight and neuropathology on PND 63; Subset 2: FOB on PND 5, 12, 22, 36, 46, and 61; water maze on PND 24 and 27; and brain weight and neuropath ology on PND 63; Subset 3: FOB on PND 5 and 12; auditory startle on PND 23 and 61 (for animal s not sacrificed on PND 12); and brain weight and neuropathology on PND 12; and Subset 4: FOB on PND 5, 12, 22, 36, 46, and 61; water
maze on PND 59 and 62.

There were no effects of treatment on mortalit y, clinical signs, FOB parameters, reproductive performance, or gestation length in maternal rats. Body weights ( ↑ 3-7%), body weight gains ( ↑ 10-16%), and food consumption ( ↑ 8-14%) were increased (p<0.01 or 0.05) in all treated animals during gestation (body weight gain and fo od consumption were comp arable to control in the low-dose group). Increased body we ights were still present at LD s 1-3, with values similar to control by LD 29. Food consumption also was in creased (p<0.01 or 0.05) in the medium- and high-dose dams during lactation, but overall body weight gains were decreased. The maternal LOAEL is not observed; the maternal NOAEL is 0.40 mg/kg/day.

No treatment-related effects were observed on li tter parameters (number born live, number born dead, sex ratio (% male), mean litter size, live bi rth index, and viability index), clinical signs, FOB parameters, motor activity, auditory startl e reflex, learning and memory, sexual maturation, brain weight, or gross or microscopic pathology.ABAMECTIN FINAL

Page 48 of 53 Non-dose-dependent body weight increases during PNDs 1-22 ranged from 4-11% in all treatment groups. On the day of weaning (PND 29), minor decreases (p ≤ 0.05 or 0.01) in pup body weights of 5 and 7% were observed at 0.40 mg/kg/day in the male and female pups, respectively. Pup body weight s at the high dose conti nued to be decreased (p ≤ 0.01) by 5-6% in the males and by 8-10% in the females thr oughout the post-weaning interval (PND 36-63). Minor decreases (p ≤ 0.05) were also observed in the medium-dose females on PND 36 ( ↓ 5%) and PND 63 ( ↓ 4%).

The age when VO occurred was delayed (p ≤ 0.01) in the medium- and high-dose pups ( ↑ 1.9 and 2.8 days, respectively). Although the body weights at VO for these groups were similar to control, examination of the body weight data revealed a 5-10% decrease (p ≤ 0.01) in body weights at approximately the time of VO (PND 36 ). Therefore, the delay in VO may be related to a delay in development associated with decreased growth. No effect of treatment was observed on the time to PPS. The offspring LOAEL is 0.4 mg/kg/day due to decreased body weights in both sexes, and the NOAEL is 0.2 mg/kg/day.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 40318601 ), Mallard ducks ( Anas platyrhynchos ; 19 weeks old) were administered abamectin (94.7% purity) in the feed at nominal concentrations of 0 (acetone), 3, 6, and 12 ppm for 19 weeks (mean-measured concentrations were 3.2, 6.21, and 12.71 ppm, respectively). Each treatment group was comprised of sixteen mating pairs, with one pair per pen. A progressive photo period was employed in this study: 8 hours/day through Week 8, 16 hours/day through Week 10, and then 17 hours/day until the end of the study. Mean temperature was 19.7±2.5°C throughout the study a nd mean humidity was 65%. Individual adult body weights were recorded at study initiati on, biweekly through Week 8, and at study termination. Food consumption in each replic ate was measured weekly throughout the study.
Adult birds were observed routin ely for mortality and clinical signs. The following reproductive parameters were observed and reco rded: eggs laid, eggs cracked, eggs set, viable embryos, live 3-week embryos, hatchlings, 14-day-old survivors, body weight of 14-day-old survivors, and egg
shell thickness.

A single mortality occurred with a hen in the medium-concentration group. There were no
differences between the control and treatment groups for any reproductive parameter. The reproductive NOEL is ≥ 12 ppm; the LOAEL is >12 ppm. In a pilot reproduction study, there was a marked reduction in number of eggs laid at an abamectin concentration of 64 ppm.

The one-generation reproductive toxicity ( MRID 49226708 ) of MK936 (abamectin) to 16 pairs per level of 35-week old Northern bobwhite quail ( Colinus virginianus ) was assessed over ca. 20
weeks. Abamectin was administered to the birds in the diet at nominal concentrations of 0 (control), 5, 10 and 20 mg ai/kg. Mean-measured c oncentrations were <1.00 (<LOD, control), 5.10, 9.93 and 20.0 mg ai/kg diet, resp ectively. There were no treatm ent-related effects observedABAMECTIN FINAL

Page 49 of 53 upon any reproductive or offspring parameter at any treatment level. Food consumption, however, exhibited a dose-dependent reduction with significant inhibition noted at all treated levels (William’s test; p<0.05). Because no other adul t or offspring endpoint was affected in this study, including no effects on adult body weight as a result of reduced food consumption, the reviewer assumed that this effect was likely due to taste aversion and concluded the NOAEC and LOAEC to be 20 and >20 mg ai/kg diet, respectively.

Fish Early Life-Cycle Toxicity (OCSPP 850.1400)

In a fish early life-cy cle toxicity study ( MRID 40069609 ), Rainbow trout ( Oncorhynchus mykiss ) were exposed in a flow-through system to fi ve concentrations of abamectin (91% purity) in a carrier solvent of acetone; mean-measur ed concentrations were 0.15 (±0.051), 0.28 (±0.071), 0.52 (±0.21), 0.96 (±0.39), and 2.2 (±0.78) μ g/L. No significant reduction was seen in hatchability at 12 days. Fry survival at 60 days and growth (standard length) at 36 days were decreased (p<0.05) at the high c oncentration. Wet weight at 60 days was decreased (p<0.05) at the medium-high concentration. The NOAEC is 0.52 μ g/L, and the LOAEC was 0.96 μ g/L based on decreases in wet weight.

The 34-day chronic toxicity ( MRID 49226707 ) of abamectin to the early life-stage of sheepshead minnow ( Cyprinodon variegatus ) was studied under flow-through conditions. Fertilized eggs/embryos (120/level, <24 hours old) were exposed at nominal concentrations of 0 (negative control), 0 (20 μ L/L DMF solvent control), 0.081, 0.16, 0.33, 0.65 and 1.3 μ g/L. Meanmeasured concentrations were <0.058 (<LOQ, controls), 0.076, 0.15, 0.30, 0.67 and 1.3 μ g ai/L, respectively. The test system was maintained at 24 to 26°C and a pH of 7.4 to 8.0. Using meanmeasured concentrations, the overall NOAEC and LOAEC were 0.30 and 0.67 μ g ai/L, respectively, based upon a treatmen t-related reduction in post-hatc h larval survival at the 0.67
and 1.3 μ g ai/L levels.

No treatment-related effect was observed on hatching success, which averaged 75 to 83% for all control and treatment levels. In addition, no trea tment-related adverse effects were observed on time to hatch (Day 6 for all viable embryos); pe rcentage of live, normal larvae at hatch (99-100% for all levels); or terminal growth (21 .4-23.3 mm and 0.0341-0.0510 g for all levels). The only adversely affected endpoint wa s post-hatch larval survival, which averaged 97 and 92% for the negative and solvent control levels, respectively, and 92, 95, 95, 41 and 45% for the meanmeasured 0.076, 0.15, 0.30, 0.67 and 1.3 μ g ai/L levels, respectiv ely. Differences were statistically-significant compared to the negative control at the 0.67 and 1.3 μ g ai/L treatment
levels.ABAMECTIN FINAL

Page 50 of 53 Mechanistic Studies: Male Repr oductive Effects (Rodent)

In a published, non-guideline study, Celik-Ozenki et al. (2011) investigated the relationship between male reproductive function and exposure to abamectin in male ra ts and the potential mechanisms for gonadal effects caused by abamectin exposure. Male SD rats (14 weeks old; 8 rats/dose) were dosed with abamectin (97.1% purity ) in sesame oil by oral gavage at 0 (vehicle control), 1, and 4 mg/kg/day for 1 week (subacute effects) or 6 weeks (subchronic effects). At test termination, rats were anes thetized and the liver, testes and epididymis, and blood samples were collected. Abamectin concentrations in pr epared plasma and tissue samples were assessed with by HPLC analysis with fluorescence detection. Blood samples taken from the abdominal
aorta were evaluated for concentrations of se rum testosterone, LH, and FSH with an ELISA
method.

For histopathological evaluations, the left testicular tissues we re fixed, embedded, and stained. The tubule degeneration was evaluated, and sperma togenesis was categorized by determining the Johnsen score (1=no germ or Sertoli cells pres ent to 10=complete spermatogenesis and perfect tubules). The epididymal sperm count and motili ty were determined from left epididymis samples. In vitro sperm motility was evaluated with sper m suspensions prepared with either
saline imidazole or with human tubal fluid and incubated with 0.42 μM abamectin; sperm motility was assessed at 5, 10, 15, and 20 minutes.

In order to assess direct effect s of abamectin on testicular function, the study also examined the expression of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, which is involved in cell death program either by its over-activatio n or by participation to an extracellular signal regulated cascade. In addition, testes were examined for the presence of 4-hydroxy-2-nonenal (4- HNE), which is an indicator of oxidative stress. The presence and expression of PARP and
4-HNE in the testes of treated rats was eval uated using western blot and immunohistochemistry analyses. For the immunohistochemical analyses, sections of testes were deparaffinized and blocked for endogenous peroxidase activity ; mouse monoclonal poly(ADP-ribose) (PAR) primary antibody was applied in a dilution of IgG3 and incubated overnight at 4°C, and then incubated with biotinylated horse anti-mouse Ig G for 45 min at room temperature. The antigenantibody complex was detected using an avidin -biotin horseradish peroxidase complex and diaminobenzidine as the chromogen. Wester n blotting procedure was conducted using homogenized right testis supern atants. Samples were evaluated for PARP activity and oxidative stress by incubating with mouse monoclonal PA R and mouse anti-4HNE Mab (HNEJ-2) primary antibody, respectively; after overn ight incubation, the membranes were washed and incubated with anti-mouse IgG horseradish peroxidase co njugate. Immunolabelling was visualized using
chemiluminescence.

No mortality or clinical signs of toxicity were observed for the abamectin treatment groups. In addition, no changes were observed in body weights or the weights of the liver and testes for the abamectin treatment groups. For the subacu te, low-dose group, the mean abamectinABAMECTIN FINAL

Page 51 of 53 concentrations were 5.12 ng/mL for plasma, 22.51 ng/g for testis, and 59.64 ng/g for epididymis.
The mean abamectin concentrations for the subacute high-dose group were 9.88 ng/mL for plasma and 105.23 ng/g for testis. The mean abamectin concentrations for the subchronic dose group were 77.14 ng/mL for plasma and 148.81 ng/g for testis, compared to high-dose group mean abamectin concentrations of 77.10 ng/ mL for plasma and 105.23 ng/g for testis.

Based on histological evaluation of the testes, testicular damage including degenerative changes in the tubules and disrupted cellular organizatio ns was observed in the low- and high-treatment groups. The Johnsen scores, used to evaluate tu bule degeneration, were decreased (p<0.001) in a dose-dependent manner for both abamectin treatmen t groups compared to control. For both the subacute and subchronic tests, sperm motility in the epididymis was decreased (p<0.01) in a dose-dependent manner in both abamectin treatment groups ( ↓ 13 and 23% at the low dose and ↓ 21 and 31% at the high dose, respectively). For the subacute test, the sperm count was decreased (NS) in both treatment groups by 13 and 11%, respectively. For the subchronic test, the sperm counts were decreased (p<0.01) in both abamectin treatment groups by 17 and 21%, respectively. Decreased sperm motility was observe d after 5 minutes of treatment with 0.42 μM
abamectin in the in vitro motility test, with cont inued decreases through the end of the test period (p<0.01, 20 minutes). Decreases (p <0.01) were noted in FSH concentrations for both abamectin treatment groups in th e subchronic test ( ↓ 29 and 16%, respectively), but were comparable to
control in the subacute test. No differences in serum LH or testosterone concentrations were observed in the abamectin treatment groups in eith er test. For the Wester n blot analysis, 4-HNE and PAR expressions showed specific bands co rresponding to molecular weights of 43 and 58 kDA, respectively. Significant (p<0.001) increases in these proteins were observed in both subchronic abamectin treatment groups compared to the control. Immunohistochemistry analyses confirmed that PAR expression increased af ter abamectin exposure, particularly in spermatogenic cells of the testes. The authors concluded that these results demonstrate that abamectin exposure induces testicular damage and affects sperm dynamics. Oxidative stressrelated poly(ADP-ribose) polymerase activation might be one of the possible mechanisms underlying testicular damage induced by abamectin.ABAMECTIN FINAL

Page 52 of 53 APPENDIX 3: References Not Utilized in the Abamectin WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for abamectin but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Argus Research Laboratories, In c. (1984) Oral range-finding study (multigeneration) in rats MK0936 (TT#82-707-0). Study Director : Gordon, L.R. MRID 00164154

Celik-Ozenci, C., Tasatargil, A., Tekcan, M., Sati, L., Gungor, E., Isbir, M., Usta, M. F., Akar, M. E., and Erler, F. (2012) Effect of abam ectin exposure on semen parameters indicative of reduced sperm maturity: a study on fa rmworkers in Antalya (Turkey). Andrologia . 44(6):388-

395

Sawhney Coder, P. (2012) Abamectin – A Dose Range-Finding Oral (Gavage) Toxicity Study in Young Adult Rats. Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Project ID: WIL-639121, May 9, 2012. (MRID 48614401)ABAMECTIN FINAL

Page 53 of 53 Sawhney Coder, P. (2012) Abamectin – A Dose Range-Finding Oral (Gavage) Toxicity Study in Young Adult Rats. Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Project ID: WIL-639159, May 10, 2012. (MRID 48614402)