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 Assessment Type: 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 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 61
Page 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 

Page 2 of 61
 

EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: ABAMECTIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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ABAMECTIN 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 Relevant 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-Estrogenic 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...........................52 

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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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

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iv 

Abbreviation Terminology 

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

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v 

Abbreviation Terminology 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

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vi 

Abbreviation Terminology 

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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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 potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

In addition to the available Tier 1 data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WOE) assessment. 

In determining whether abamectin interacts with E, A, or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern 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 Assay 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 (WOE) analysis of the potential interaction 

of abamectin with the E, A or T signaling pathways. 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 was decreased (p at 1 μM 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. 

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

vitro (i.e, AR binding and steriodogenesis assays 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 reproductive system (testicular damage, decreases in 

sperm count and motility, histopathological changes 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 similar doses in the Tier 1 male pubertal assay or 

in the Part 158 rat chronic/carcinogenicity study. Overall, this is an isolated finding that is not 

corroborated by the Tier 1 or Part 158 data. Therefore there is no convincing evidence for an 

interaction with the androgen pathway. 

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There was no evidence of potential interaction of abamectin with the thyroid pathway. No 

treatment-related effects were seen in thyroid, hormones, weight or histopathology in the male 

and female pubertal assays. In the AMA, no treatment-related finding were observed except for 

effects in growth parameters at the low concentration, with no effects on developmental stage or 

thyroid histopathology. 

Based on weight of evidence considerations, mammalian or wildlife EDSP Tier 2 testing is not 

recommended for abamectin since there was no convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

 

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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 potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

In addition to the available Tier 1 data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WoE) assessment. 

In determining whether abamectin interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern 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 Assay 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 pathways. 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 solubility of <0.01 mg/L at 20°C and a log KOW of 5.0-5.5. The 

available data indicate that abamectin has low mobility in soil with half-lives in soil of 2 weeks 

to 2 months. Abamectin also will be susceptible to photodegradation when exposed to sunlight 

on soil surfaces and the atmosphere. Bioconcentration factors in fish are <100. 

The available information considered in determining 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 studies 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 (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 studies recommendations are presented in Section V. 

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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 believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order 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 review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team 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 considered but not utilized 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 presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual 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 endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

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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 include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 individual study is evaluated. For the Tier 1 in vivo

assays, often multiple endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T, consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development 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 relevant, robust and consistent evidence in terms of 

complementarity and redundancy, as well as biological 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 consideration 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 effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

 

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This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA SAP that evaluated scientific issues associated with the WoE evaluation of the EDSP 

Tier 1 screening process. In October 2013, the SAP stated that , “In summary, the Panel agreed 

that little, if any, weight should be placed on signs of endocrine disruption in the presence of 

overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms of primary 

interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine 

organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally 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 included, but were not limited to: mortality; 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, especially 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/concentration 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 decreases 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 effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular 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/chemical properties and its 

known modes of action in its overall 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 determining 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 evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

 

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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 biologically 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)

ERα 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) 

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B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for abamectin to interact with the 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-relevant findings from data evaluated as OSRI. Effects that occurred in 

the presence of overt toxicity are discussed in the text but are 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 μM 

(0.92-fold) and in all three runs at 1 μM (0.72-fold). 

Abamectin was negative in the uterotrophic assay. In the female pubertal assay, the percent of 

females regularly cycling was decreased (NS and 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 in diestrus and more in estrus in treated groups 

compared to controls. Additionally, there was a slight and non-statistically significant increase 

(16%) in uterine weight at the high-dose group. No histopathological 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 studies 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 effect 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 avian 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. 

 

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Table 2. Estrogenic/Anti-Estrogenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction with the 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) 

EDSP Tier 1 Assay 

ER Binding (MRID 48614405) N ERTA (MRID 48614406) N Aromatase (MRID 48614404) N Steroidogenesis (MRID 48614407) P Uterotrophic (MRID 48614409) N Female Pubertal Rat (MRID 48926401) N N N N P4 (H) N FSTRA (MRID 48675002) NE N N N N 

OSRI 

Subchronic Oral Toxicity/ Neurotoxicity (Rat; MRID 46959201) 

N N N NE 

Developmental Toxicity (Rat; MRID 00130819) 

 N 
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Table 2. Estrogenic/Anti-Estrogenic Pathway for Abamectin

Lines of Evidence Indicating Potential Interaction with the 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) 

Developmental Toxicity (Rabbit; MRID 00130819) 

 N 

Two-Generation Reproduction (Rat; MRID 00164151) 

 NE NE N NE N 

Chronic Toxicity/ Carcinogenicity (Rat; MRID 40069601) 

N N N N 

Chronic Toxicity/ Carcinogenicity (Mouse; MRID 40069602) 

N N N N 

Chronic Toxicity (Dog; MRID 00164022, 40375510) 

 NE NE N NE 

Developmental Neurotoxicity (Rat; MRID 47116201) 

 N N 

Developmental Neurotoxicity (Rat; MRIDs 46727401-46727403) 

 N N 

Avian reproduction (Duck; MRID 40318601) 

N 
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Table 2. Estrogenic/Anti-Estrogenic Pathway for Abamectin

Lines of Evidence Indicating Potential Interaction with the 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) 

Avian reproduction (Quail; MRID 49226708) 

N 

1. Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), Medium-high treatment (MH), High 

or ↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicabl

weight 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 OS

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 

details see Section IV. A 

4. The percent of females regularly cycling was decreased (NS and p<0.05, respectively) at 0.75 and 1.5 mg/kg/day (38% and 50%, re

control). Also, a shift was noted in the cycle status at necropsy, with fewer females in diestrus and more in estrus in treated groups 

P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
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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 Tier 1 assays are delineated so 

as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. 

This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that 

occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WOE assessment. 

Abamectin was negative (non-binder) in the Tier 1 AR binding assay 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 pubertal 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 with abamectin at 1 or 4 mg/kg/day. 

Histopathological changes in abamectin-treated rats included degenerative changes in the tubules 

and disrupted cellular organization. Sperm motility in the epididymis was decreased in a dosedependent manner in both abamectin treatment groups, and sperm motility also was decreased in 

the in vitro motility test. Finally, decreases were noted in follicle stimulating hormone (FSH) 

concentrations, but luteinizing hormone (LH) and testosterone concentrations were not affected. 

Rather than a specific anti-androgenic effect, the study authors hypothesized that the deleterious 

effects of abamectin on the male reproductive system (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. 

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Table 3. Androgenic/Anti-Androgenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Abamectin1

Study 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 

EDSP Tier 1 Assay 

AR Binding (MRID 48614403) N Steroidogenesis (MRID 48614407) N Hershberger (MRID 48614408) N Male Pubertal Rat (MRID 48926402) N N N N N N N N FSTRA (MRID 48675002) NE N N N N 

OSRI 

Subchronic Oral Toxicity/ Neurotoxicity (Rat; MRID 46959201) 

N N N N NE 

Two-Generation Reproduction (Rat; MRID 00164151) 

N N N N N 

Chronic Toxicity/ Carcinogenicity (Rat; MRID 40069601) 

 N N NE N N N 
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Table 3. Androgenic/Anti-Androgenic Pathway for AbamectinLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Abamectin1

Study 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 

Chronic Toxicity/ Carcinogenicity (Mouse; MRID 40069602) 

 N N NE N N N 

Chronic Toxicity (Dog; MRID 00164022, 40375510) 

N N NE NE NE 

Developmental Neurotoxicity (Rat; MRID 47116201) 

 N 

Developmental Neurotoxicity (Rat; MRIDs 46727401-46727403) 

 N 

Mechanistic Studies: Male Reproductive Effects (Rat; CelikOzenski et al., 2011) N N P4 P5 Avian reproduction (Duck; MRID 40318601) 

N 

Avian reproduction (Quail; MRID 49226708 

N 

1. Key to responses: Low treatment (L), Medium-Low treatment (ML), Medium treatment (M), Medium high treatment (MH), High 

or ↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicabl
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weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prosta(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 OS

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 

details see Section IV. A 

4. Dose-dependent testicular damage including degenerative tubules with disrupted cellular organization was observed at both the low 

mg/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, r

sperm motility was observed after 5 minutes of treatment with 0.42 μM abamectin in the in vitro motility test, with continued decrethe test period (p<0.01, 20 minutes). 

P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined 
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C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis 

The current EDSP Tier 1 battery 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 detect 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 thyroid 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 are not reported in the table and not considered 

further in the WOE assessment. 

There were no effects on the thyroid pathway in the Tier 1 in vivo male and female pubertal rat 

assays. In the AMA, abamectin did not delay 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 treatment-related effects on thyroid gland histopathology were 

observed. The only effects noted were an increase in Day 7 snout-to-vent length (SVL; ↑10%) 

and body weight (↑24%) at the low concentration, with no effects observed at Day 21. 

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

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Table 4. Thyroid Pathway for Abamectin

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Abamectin1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, BL, SVL) Systemic Toxicity Observd2

Overt Toxicity Obsercved3 

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 examined 

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IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight 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 recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, 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 evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those 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 are 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 evaluation of the results of the Tier 1 assays 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 endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related 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/concentration to be considered overtly toxic. 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

of toxicity (e.g., changes in organ weights or blood chemistry).

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1. Tier 1 in vivo Assays 

In the Hershberger assay androgen agonist phase, no treatment-related 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 comparable 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 material 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 survived until scheduled sacrifice, but the five 

surviving high-dose animals also exhibited 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 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 consumption was decreased by 11% in the high-dose 

group. 

There were no effects of treatment for either abamectin dose (0.75 or 1.5 mg/kg/day) on 

mortality, clinical signs, initial 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/day), 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 findings 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 was 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 weight 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 the three treatment groups, but no secondary sex characteristic or 

clinical signs were observed in males. 

Abamectin treatment had no effect on the survival of tadpoles in the 21-day AMA study. Body 

weight was increased at Day 7 in the low-concentration group only (↑24%), with no further 

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affect at Day 21. Spinal curvature was observed in the control and all treatment groups, and did 

not appear to be treatment-related (similar incidence across control and treatment groups, except 

for a lower incidence in the high-concentration group). 

2. OSRI 

In a subchronic combined oral toxicity and neurotoxicity study, there were 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 sacrificed after Week 7 due to 

adverse clinical signs and decreased body weights. Clinical signs, observed in high-dose males 

and females included: piloerection, mouth-staining, irregular breathing, sides pinched in, upward 

curvature of the spine, and tip-toe gait. Decreased body weight gain 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 decreased 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, 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 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 study in rabbits, but further effects on body weight 

from this study were not reported. 

In a two-generation reproduction study in rats, nine mortalities of F0 parental rats and five 

mortalities of F1b 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 the medium- and high-dose F0 males and females 

during the growth phase. Body weight gain increases also were reported for the medium- and 

high-dose F0 females during the F1a gestation period, with no difference noted during the F1b

gestation period. Body weight gains in F0 females during the F1b lactation period were decreased 

compared to control. High-dose F1b 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 F1b males continued to be decreased 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 F2a and F2b gestation periods. Decreased body weight gains in F1b females also were noted 

during the F2a and F2b lactation periods. 

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In a chronic toxicity/carcinogenicity study in rats, treatment-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 considered to be related 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 control; overall body weight gains for female rats were not affected, 

but were increased for male rats in all treated groups. 

In a chronic toxicity/oncogenicity 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 control and high-dose groups were considered 

secondary and not related to treatment. There was increased mortality 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 two control groups at termination of the study. 

Similar decreases were reported to have been observed throughout the study. Decreased body 

weight gain in the high-dose females was considered to be treatment-related; no changes were 

seen at the lower doses. In male mice, body weight 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 salivation which may have 

been treatment-related as no other treated dogs exhibited this sign. 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 treatment 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 were no effects of treatment on mortality 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 animals during gestation (body weight gain and food consumption were 

comparable to control in the low-dose group). Increased body weights were still present at 

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lactation day (LDs) 1-3, with values similar to control by LD 29, but overall body weight gains 

were decreased. Pup body weights were decreased by 4-5% and 7-10% in the mid and high-dose 

groups. 

In an avian reproduction study, a single, non-treatment-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 clinical 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 effects on hatching or growth. 

In a published, non-Guideline study (Celik-Ozenki et al., 2011), no mortality or signs of toxicity 

were observed in male rats treated with abamectin at 1 or 4 mg/kg/day for up to 6 weeks. In 

addition, no changes in body weights 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 μM 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 abamectin with the androgen pathway in the Tier 1 in 

vitro (i.e, AR binding and steriodogenesis assays 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 reproductive system (testicular damage, decreases in 

sperm count and motility, histopathological changes 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 similar doses in the Tier 1 male pubertal assay or 

in the Part 158 rat chronic/carcinogenicity study. Overall, this is an isolated finding that is not 

corroborated by the Tier 1 or Part 158 data. Therefore there is no convincing evidence for an 

interaction with the androgen pathway. 

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D. Thyroid Pathway 

There was no evidence of potential interaction of abamectin with the thyroid pathway. No 

treatment-related effects were seen in thyroid, hormones, weight or histopathology in the male 

and female pubertal assays. In the AMA, no treatment-related finding were observed except for 

effects in growth parameters at the low concentration, with no effects on developmental stage or 

thyroid histopathology. 

E. Conclusions 

Abamectin demonstrates no convincing evidence of potential interaction with the estrogen, 

androgen or thyroid pathways. 

!

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, mammalian or wildlife EDSP Tier 2 testing is not 

recommended for abamectin since there was no convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

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VI. References 

Beavers, J. (1987) Avian Reproductive Toxicity to the Mallard Duck (Anas platyrhynchos). 

Unpublished study conducted by Wildlife International, Ltd.; Study No. 105-135A. (MRID 

40318601) 

Brammer, A. (2006) Abamectin technical (MK936) - 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., Sati, L., Gungor, E., Isbir, M., and Demir, R. 

(2011) Effects of abamectin exposure on male fertility 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 study conducted by Central Toxicology Laboratory, 

Alderley Park, Macclesfield, Cheshire, UK. Laboratory Study Id.: CTL No. UR0795, April 28, 

2005. (MRID 46727401) 

Gordon, L. (1987) MK-0936: 53-Week Oral Toxicity 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. Unpublished 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 Dietary Carcinogenicity and Toxicity Study in 

Rats with MK-0936; Final Report; Unpublished study conducted by Merck Sharp & Dohme 

Research Labs.; TT#82-099-0; August 27, 1985. (MRIDs 40069601, 40375511, and 40517801) 

Lee, M.R. (2012) Abamectin- Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis). Unpublished study conducted by Smithers Viscient, Wareham, MA. 

Laboratory report number 1781.6762. Study sponsored by Syngenta Crop Protection, LLC, 

Greensboro, NC. Study completed November 13, 2013. (MRID 48675001) 

McAllister, W. (1986) Early Life Stage Toxicity of Avermectin B1 (MK-0936 Technical) in 

Rainbow Trout (Salmo gairdneri) in a Flow-Through System: Laboratory Project Identification 

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Page 25 of 53 

#33195. Unpublished study conducted by Analytical Biochemistry Laboratories, Inc. (MRID 

40069609) 

Moxon, M.E. (2005) Abamectin: Developmental neurotoxicity study in rats. Unpublished study 

conducted by Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK. 

Laboratory Project Id.: CTL 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 Toxicology 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 administered orally by gavage to 

Crl:COBSTMCDTM (SD)BR rats for two generations. Unpublished study conducted by Argus 

Research Laboratories, Inc. TT#82-9010. (MRID 00164151) 

Sawhney Coder, P. (2012) Abamectin – Hershberger Assay in Orchidoepididymectomized Rats. 

Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, Ohio. Laboratory 

Project ID: WIL-639124, May 10, 2012. (MRID 48614408) 

Sawhney Coder, P. (2012) Abamectin – Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Female Rats. Unpublished 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 Thyroid 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 Laboratories, LLC, Ashland, Ohio. Laboratory 

Project ID: WIL-639125, May 10, 2012. (MRID 48614409) 

USEPA (2010) Abamectin - Report of the Endocrine 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 Screening Program – Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

Wagner, H (2012) Abamectin - H295R Steroidogenesis Assay. Final Report. Unpublished study 

conducted by CeeTox, Inc. Kalamazoo, MI. Laboratory Report No.: 9047V-100352STER. 

February 29, 2012. (MRID 48614407) 

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Wilga, P. (2012) Abamectin - Human Recombinant Aromatase Assay. Final Report. 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9047V100352AROM. (MRID 48614404) 

Willoughby, J.A. (2012) Abamectin - Androgen Receptor 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 - Estrogen 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 Receptor Transcriptional Activation (Human Cell 

Line (HeLa-9903)) Final Report. Unpublished study conducted by CeeTox, Inc., Kalamazoo, 

MI. Laboratory Report No.: 9047V-100352ERTA, January 19, 2012. (MRID 48614406) 

York, D.O. (2012) Abamectin- Fish Short-Term Reproduction Assay with Fathead Minnow 

(Pimephales promelas). Unpublished study conducted by Smithers Viscient, Wareham, MA 

02571-1037. Laboratory report number 1781.6763. Study sponsored by Syngenta Crop 

Protection, LLC, Greensboro, NC 27419. Study completed November 14, 2012. (MRID 

48675002) 

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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 and 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 effects are reported based on comparison to the negative (clean 

water) control. The survival of tadpoles exposed to abamectin was not significantly 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 treatment groups, but did not appear to be treatmentrelated as the incidence was similar across the control and treatment groups except for a lower 

incidence in the high concentration group. 

Abamectin caused no significant acceleration or delay 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 test 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 observed 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 below (snout-vent length, hind limb length, and wet 

body weight). 

Abamectin exposure did not significantly increase or decrease Day 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 concentration 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 observed in the control and all treatment groups. 

Histopathological findings included mild follicular cell hypertrophy, mild follicular cell 

hyperplasia, and mild lumen area increases. These effects did not appear to be treatment-related as 

there was no concentration-response demonstrated in the data, and the incidences of follicular cell 

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hypertrophy were the same for control and high treatment group. A similar trend was observed 

with follicular cell hyperplasia diagnoses (3 in negative control versus 4 in high treatment group). 

Androgen Receptor (AR) Binding 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 binding 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 vehicle at a final assay 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 was functioning with appropriate affinity for the 

radiolabeled reference androgen (R1881) prior to conducting AR competitive binding 

experiment. Saturation binding data were not originally provided 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 (Kd) for [3

H]-R1881 

was 0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for the single 

batch of prostate cytosol that was prepared. The mean and individual Kd and Bmax values were 

below the expected ranges reported 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 IC50s 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 performed 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 IC50 and RBA could not be calculated for 

abamectin as it did not result in 50% reduction in [3

H]-ligand binding at any concentration. 

Based on the results of the three runs, abamectin is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

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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 tritiated androstenedione ([1β- 3

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

concentrations from 10−11 to 10−4 M for 15 minutes to assess the potential 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. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three 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 full activity controls ranged from 0.545-0.743 nmol·mg-protein−1·min−1

for 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 response 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 IC50, and %CV for 

replicates of each concentration within runs. The estimated log IC50 for 4-OH ASDN averaged 

−7.3 M and the Hill slope was −0.95. 

For abamectin, aromatase activity averaged 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 IC50 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. 

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Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (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 [3H]-17βestradiol (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 vehicle 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 17βestradiol 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 was functioning with appropriate affinity for the 

radiolabeled reference estrogen prior to routinely conducting competitive binding experiment. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the 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 separate cytosol preparations in the competitive binding assay for 

abamectin. For the batch of cytosol prepared on September 27, 2010, the Kd for [3H]-17βestradiol was 0.081±0.006 nM, and the estimated Bmax was 52.70±1.05 fmol/100 μg protein. For 

the batch of cytosol prepared on July 10, 2011, the Kd for [3H]-17β-estradiol was 0.303±0.090 

nM, and the estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Kd 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 straight lines to the data, concavity was observed with the data 

sets from the July 10, 2011 cytosol preparation, which may indicate ligand depletion. 

In the competitive binding experiment, precipitation occurred at 10−3 M in Runs 1 and 2 and at 

10−4

 M in Run 3; therefore, data from these abamectin concentrations were not included in the 

evaluation. Abamectin had no significant effect on specific radioligand 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 IC50 was −5.47 M for 19-norethindrone and −9.00 M for 

17β-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. 

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ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48614406), hERα-HeLa-9903 

cells cultured in vitro were exposed to abamectin (98% a.i., Lot # A110628) 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 concentration 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 activation of the estrogen receptor by abamectin was 

measured using a proprietary luciferase assay. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone (41-47% PC) was lower than the accepted values (at least 50% PC), 

indicating a decreased sensitivity to very weak agonists. In addition, cytotoxicity was observed at 

the highest tested concentrations of corticosterone in both runs. Results from cytotoxic 

concentrations were excluded from the analysis. 

The mean RPCMax for abamectin was 1.5% for the first run and 1.3% for the second run, and the 

associated PCMax 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 the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions 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 17α-methyltestosterone, which was able to 

reach a maximum of 41.1-46.6% PC. Abamectin was only able to reach a maximum of 1.3-1.5% 

PC when tested up to the highest concentration possible based on cytotoxicity. Because the 

RPCMax < PC10 in both assay runs, abamectin was considered 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 (MRID 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 females 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-calculated time-weighted averages (TWA) 

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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, all effects are reported based on comparison to the 

negative (clean water) 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 females in the low, mid, and high treatment groups, 

respectively. Replicate A of the mid treatment group had high mortality (33% survival, combined 

sexes) but this was not considered treatment-related and the replicate was excluded from analysis 

of all endpoints. Excluding this replicate, 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 observations 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 secondary sex characteristic or clinical signs of 

toxicity were observed in males. 

Mean spawning frequency and fecundity in controls, 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 significantly affect fecundity or fertility 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 level (p>0.10 for males; p>0.05 for females). Gonadal 

histopathology findings were observed in male and female fish in the control and all groups. 

Histopathological effects in males included minimal to severe increased testicular degeneration, 

interstitial cell hyperplasia/hypertrophy, and granulomatous inflammation, and in females 

included minimal to severe increased oocyte atresia, egg debris in oviduct, and granulomatous 

inflammation. The observations of granulomatous inflammation were consistent with infection by 

Mycobacteria sp. Due to this infection, histological 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, nuptial 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) screening for androgenic activity, abamectin (98.0% 

a.i., Batch # A110628) in aqueous 1% carboxymethylcellulose (CMC) and 0.1% Polysorbate 80

was administered daily via oral gavage to groups of six 56- to 57-day old, castrated male 

Sprague Dawley rats at dose levels of 0 (vehicle), 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% 

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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-androgenic activity, abamectin in 1% CMC and 0.1% Polysorbate 80

was administered daily via oral gavage to groups of six 56- to 57-day old, castrated male 

Sprague Dawley rats at dose levels of 0 (vehicle), 0.5, 1.5 or 5 mg/kg/day 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 assay, body weights and food consumption were determined daily. 

The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the 

final dose administration. At necropsy, the five androgen-dependent tissues, adrenal glands, 

kidneys, and liver were collected and weighed. 

In the androgen agonist assay, no treatment-related clinical signs were noted 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 controls throughout the duration of the assay. 

Animals dosed with 5 mg/kg/day had decreased (p<0.05) overall body weight gains (↓48%) 

compared to vehicle controls, and terminal body weights were decreased (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 significantly, NS) by 17% at 5 

mg/kg/day. There were no treatment-related effects in any of the accessory sex organs of 

abamectin-dosed animals. Animals in the positive control (vehicle + TP) group had accessory 

sex organ weight increases (p<0.05) as follows: 446% in seminal 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 other rats in the anti-androgen assay survived until 

scheduled sacrifice, but the five surviving animals 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) overall 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 for 

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the 0.5 and 1.5 mg/kg/day groups were comparable to the TP controls throughout the duration of 

the assay. Food consumption in all abamectin-treated groups was similar to the TP control; 

however, overall average food consumption was decreased by 11% (NS) at 5 mg/kg/day with 

TP. Overall mean food consumption in the positive control (FT +TP) 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 weights 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 considered adequate based on 

mortality, clinical signs, and decreased body weights and body weight gains observed. All 

guideline performance criteria were met except 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 androgenicity 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 PND 22, and age and weight on day of attainment 

were recorded. Following sacrifice on PND 42, blood was collected for clinical chemistry 

analyses, and total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were 

determined using either an electrochemiluminescent immunoassay (T4) or a magnetic 

radioimmunoassay (TSH). Adrenal, liver, pituitary, 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 consumption, organ weights, clinical chemistry 

parameters, or gross pathology. 

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The percent of females regularly cycling was decreased at 0.75 and 1.5 mg/kg/day (38% and 

50% respectively vs. 87.5% controls); however only 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 treated 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 T4 or TSH levels, and there were no effects of 

treatment on thyroid follicular cell height or colloid area. 

The doses tested were determined to be adequate based on data from two previously performed 

studies showing tremors and tonic/clonic convulsions after 1-2 doses 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 lower mean body weights and food consumption at 2.5 

mg/kg/day. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48926402), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage (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 their age and weight at day of attainment was 

recorded. Following sacrifice on PND 53, blood was collected for clinical chemistry analyses, 

including total thyroxine (T4), testosterone, and thyroid stimulating hormone (TSH). 

Testosterone and T4 concentrations were analyzed using an electrochemiluminescent assays and 

TSH concentrations were analyzed using a radioimmunoassay. Liver, adrenals, pituitary, and 

urogenital organ weights were recorded, and microscopic examinations were performed on the 

thyroid, kidney, testis and epididymis. 

No treatment-related effects were observed on mortality, body weights, body weight gain, organ 

weights, or gross pathology. Changes 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 organs. Age and body weight at PPS in the treated 

groups were similar to controls. Thyroid hormone levels, colloid area and follicular cell height 

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scores for the thyroid were similar in the treated groups relative to control. Serum testosterone 

levels were similar in the treated groups to the control group. 

Although no toxicity was observed in the current 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/day 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 (MRID 48614407), H295R cells cultured in vitro in 24-well plates 

were incubated with abamectin (98.0% a.i., Lot # A110628) at log concentrations from 0.0001 to 

100 μM in triplicate for 48 hours. Dimethyl sulfoxide (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 were 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 statistically analyzed. Therefore, the study report 

includes data from only three independent runs. 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 (prochloraz) 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 μM (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, acceptable reproducibility (low % CV), and appropriate 

induction and inhibition with positive controls. 

Significant (p≤0.05) changes in testosterone production after 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. 

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Based on the hormone responses in each of the three independent runs, abamectin treatment 

resulted in statistically significant and reproducible decreases in estradiol production, but did not 

result in statistically significant 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 aqueous 1% carboxymethylcellulose (CMC) and 

0.1% Polysorbate 80 was administered daily via oral gavage to groups of six ovariectomized 

Sprague Dawley rats at dose levels of 0 (vehicle), 2.5 or 7.5 mg/kg/day on post-natal days (PND) 

57-59. A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) in 

corn oil at 0.3 mg/kg/day by oral gavage. Body weights and food consumption were determined 

daily. All animals were terminated and necropsied on PND 60 approximately 24 hours after the 

final dose administration to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No treatment-related effect was observed at 

2.5 mg/kg/day, and wet and blotted uterine weights were comparable to the vehicle controls. 

At 7.5 mg/kg/day, all six females had slight to moderate 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 there 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 were 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) persisted in at least half of the 

animals through the study. In a range-finding study (MRID No. 48614402; Appendix 2), no 

treatment-related effects were seen when treated at 5.0 mg/kg/day for 3 consecutive days. Based 

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on the results of the range finding 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. 

 

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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 summarized below. Given the breadth of toxicity 

data available on abamectin, the only Part 158 studies discussed in this appendix are those that 

fulfill current guideline requirements and are directly relevant to assessing potential endocrine 

effects (i.e., multiple-generation rodent reproductive studies, development studies, chronic 

toxicity studies, etc.). 

Combined 90–Day Oral Toxicity and Neurotoxicity 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 seed 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 were 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 animals/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 (control, 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, pituitary, 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; piloerection, upward 

curvature of the spine, and mouth-staining were prevalent in high-dose males. Decreased (NS) 

body weight gain 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 more severe in females 

(↓40%). The high-dose males had minimally decreased body weight (NS, ↓5%) at Week 4, with 

weight similar to control by Week 6. Body weights 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. 

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Some treatment-related behavioral 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 pathology; 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 (25/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 examination was performed on one-third of the 

fetuses in each litter. Free-hand sections of the head of one-third of the fetuses in each litter also 

were conducted. Finally, all fetuses were processed, 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 reported. 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 fetal 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 variations 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 effect. The maternal and fetotoxic NOAEL is the high 

dose (≥1.6 mg/kg/day). An LOAEL is not established. The teratogenic potential was negative. 

Prenatal Development in Non-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, and 3.0 mg/kg/day from GDs 6 through 18. On 

GD 28, all surviving does were sacrificed in both studies; maternal toxicity was reported from 

the dose-range finding study, and fetotoxicity from the developmental study. Maternal body 

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weights were recorded during both studies; the numbers of corpora lutea, implantations, and 

resorptions were recorded in the developmental study. In addition, all fetuses were weighed, 

sexed, and external examinations were conducted. Each fetus was examined for gross external 

observations, sacrificed, and examined for visceral and skeletal malformations. 

Deaths, sacrifices due to moribundity, and abortions occurred across the control and treatment 

groups, with no respect to treatment. Live pregnant rabbits available for evaluation in the study 

ranged from 11 (medium dose) to 15 (control and high-dose groups). Treatment-related 

decreases in body weight, and feed and water consumption (extent and significance 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 group, 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 reported in fetuses of the high-dose group (the maternal toxicity 

level); findings included cleft palate, clubbed foot, and delayed ossification of sternebrae, 

metacarpals, and forefoot phalanges. No effects were seen at doses of 1.0 and 0.5 mg/kg/day. 

The maternal and developmental toxicity LOAELs 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 gavage to SD rats (30/sex/dose for the F0 generation; 

32/sex/dose for the F1b parental generation) at concentrations of 0 (vehicle control, sesame oil), 

0.05, 0.12, and 0.40 mg/kg/day. F0 rats were mated twice, in order to produce the F1a and the F1b

litters, and pups selected for the F1b parental generation were also mated twice to produce the F2a 

and F2b litters. F0 rats were given the test material beginning approximately 68 days prior to 

cohabitation and continuing through weaning of F1b pups, with at least a 16-day maternal rest 

period prior to the second mating. At weaning of the F1b rats, five males and five female pups per 

dosage group were sacrificed for subsequent complete gross necropsy and histopathology 

evaluations; body weights and weights of liver, kidney, testes, heart, brain, and spleen were 

recorded. Also at weaning, one or two pups per sex, from each F1b generation litter with 

surviving pups, were randomly selected for continued study. 

The dosing period of the F1b generation began at weaning, at least 70 days prior to cohabitation, 

and continued to at least 30 days after the last F2b pups were weaned, with at least a 14-day 

maternal rest period prior to the second mating. Ten male and 25 female adult F1b generation 

animals per dosage group were randomly selected for complete necropsy and histopathology 

procedures; body weights and weights of brain, spleen, heart, kidneys, liver, and testes were 

recorded. The testes and epididymides from remaining F1b male rats were removed, weighed, and 

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fixed; eyes were retained from these F1b male rats and from the remaining F1b female rats. At 

weaning of the F2b rats, ten male and ten female weanling rats per dosage group were randomly 

selected for skeletal examination, and five male and five female F2b weanling rats per dosage 

group were randomly selected for complete gross necropsy and histopathology evaluations. The 

eyes from the F2a and F2b weanling rats were retained. 

Clinical observations, body weights, and reproductive parameters were recorded for the F0 and 

F1b parental rats. Mating confirmations, delivery and lactation observations, as well as pup 

viability, sex, and body weights were recorded for the F1a and F1b pups until weaning; the same 

procedures were conducted for the F2a and F2b pups. Gross external and visceral examinations 

were conducted on pups that died during the lactation period, and they were examined for 

skeletal anomalies. No signs of clinical toxicity were noted for the parental generations during 

the study. 

Nine mortalities of F0 parental rats and five mortalities of F1b parental rats during dosing 

occurred, but the mortalities were not dose-related and were not attributed to test material. Body 

weight gain increases (NS) were reported for the medium- and high-dose F0 males; body weight 

gain increases (NS) also were reported for the medium- and high-dose F0 females during the 

growth phase. Body weight gain increases (NS) also were reported for the medium- and highdose F0 females during the F1a gestation period, with no difference noted during the F1b gestation 

period. Body weight gain in F0 females during the F1b lactation period were decreased (NS) 

compared to control. High-dose F1b pups (both sexes) and medium-dose F1b females selected for 

the parental generation had lower mean body weights compared to control at dose initiation. 

Body weight in the high-dose F1b males continued to be decreased (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) during the growth phase, with no differences in 

overall body weight gains during the F2a and F2b gestation periods. Decreased (NS) body weight 

gains in F1b females also were noted during the F2a and F2b lactation periods. 

In the first mating of the F0 generation, the male rats showed no effects of treatment on mating or 

days of cohabitation. However, in the second mating of the F0 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 days in cohabitation in the low- (↑1.5 days), medium- (↑1.5 

days), and high-dose (↑1.5 days) groups compared to control. 

For F0 females, there were no effects of treatment on fertility, days in cohabitation, or gestation 

length with the first mating. In the F1a 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 F0 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 in 

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cohabitation (↑5.7 days) compared to control. There was no effect of treatment on fertility index 

or duration of gestation, but there were increases in live pups dying, pup mortality, and decreases 

in the viability and lactation indices were observed at the high dose in the F1b 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 F1b generation, treated males did not show any adverse effects on 

fertility, days in cohabitation, or other reproductive parameters. In the second mating of the F1b

generation, males did not show any treatment-related effects in fertility, but did show differences 

in days in cohabitation with increased (p<0.05) mating during Days 1-4 at the high dose (90.9% 

vs. 63.6% control) and decreased (p<0.05) mating during Days 5-8 at the high dose (9.1% vs 

13.6% control). 

For the first and second matings of the F1b females, no adverse effects were seen in mating, days 

in cohabitation, fertility index, and duration of gestation. In the F2a and F2b 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 lactation indices at the high 

dose. Pup body weights in the high-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 F0 adults, F1b weanlings and adults, 

or F2b weanlings. This included no treatment-related lesions in the testes and epididymides of the 

F0/F1 adults and F1b/F2b weanlings; prostate glands of the F1b/F2b weanlings and F1b adults; 

ovaries and uterus of the F1b/F2b weanlings and F1b adults; cervix of the F1b adults and F2b

weanlings; and the thyroid, adrenal and pituitary glands of the F1b/F2b weanlings and F1b adults. 

Treatment-related retinal anomalies (unilateral intra-retinal rosettes) were observed in males and 

females in the high-dose F2b 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 (F2b weanlings) and increased pup mortality, 

decreased viability and lactation indices, and decreased pup body weight in all litters. 

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

In a chronic toxicity/carcinogenicity 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 were selected prior to study initiation for the 53-

week interim sacrifice. At Week 11, the dose for the high-dose group was increased to 2.5 

mg/kg/day, and then decreased back to 2.0 mg/kg/day at Week 13 due to the appearance of 

severe 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 interim and final sacrifice included the liver, adrenals, kidneys, and 

testes; at final sacrifice, the thyroid, pituitary, prostate, seminal vesicles, ovaries, and uterus were 

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also weighed. Gross and microscopic 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 dose groups at final sacrifice. 

Compound-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; however, the tremors persisted intermittently until the time of 

sacrifice, despite the reduction in the high-dose 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 mortality or survival. Body weights were increased 

(significance not reported) for both male and female rats in all treated groups compared to 

control; overall body weight gains for female rats were not affected, but were 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 findings, or histopathology in any treated male or female rats. 

There also were no treatment-related neoplastic findings. 

The oncogenic potential was negative for abamectin. The systemic NOAEL is 1.5 mg/kg/day, 

and the LOAEL is 2.0 mg/kg/day based on treatment-induced tremors. The high dose (2.0 

mg/kg/day) is also considered the MTD due to the observation of tremors. 

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

In a chronic toxicity/carcinogenicity 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 nominal 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, and 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 reached 40%. Organs that were weighed at final 

sacrifice included the liver, adrenals, kidneys, thyroid, pituitary, testes, prostate, ovaries, and 

uterus. Gross and microscopic pathological examinations were conducted 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 because 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 considered secondary to a metastatic lymphoma of the 

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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 male 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 controls at termination of the study. Similar 

decreases were reported to have been observed 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 gains 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 consumption in male mice, but food 

consumption was increased (NS; ↑2-8%) in treated female mice. 

There were no treatment-related effects on ophthalmological lesions, hematological and serum 

biochemical parameters, organ weights (absolute or relative), or gross necropsy findings in male 

or female mice. In male mice in the high-dose 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 increased 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 LOAEL 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 males, and tremors and decreased body weight gain in 

females. 

Chronic Oral Toxicity in Non-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 nominal 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 and microscopic pathological examinations were 

conducted on these tissues as well as the thyroid, 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 been treatment-related as no other treated dogs 

exhibited this sign. Three male dogs in the high-dose 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. Food consumption also was 

decreased (significance not reported) in the high-dose group; decreased food consumption may 

have contributed to observed decreases (NS) in BUN and total protein in these dogs. There were 

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no effects of treatment on hematological parameters. The results of urinalyses did not show any 

effects of abamectin treatment. There also were no effects of treatment on organ weights 

(absolute and relative), or gross necropsy 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 Alpk:APfSD rats (30/dose) from GD 7 through LD 

22 at doses of 0 (vehicle 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 on LD 29. On PND 5, litters were 

standardized to 8 pups/litter; the remaining offspring were sacrificed and not examined further. 

Subsequently, 1 pup/sex/litter/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 neuropathology on 

PND 63; Subset 3: FOB on PND 5 and 12; auditory 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 mortality, 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; the maternal NOAEL is 0.40 mg/kg/day. 

No treatment-related effects were observed on litter parameters [number born live, number born 

dead, sex ratio (% male), mean litter size, live birth 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 toxicity 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 size 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 these 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 litter parameters, clinical signs, FOB parameters, 

motor activity, auditory startle reflex, learning and memory, brain weight, or gross or 

microscopic pathology (neuropathological tissues) in the low- and medium-dose pups. The age 

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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 were 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 were observed in the low- and 

medium-dose pups throughout the post-weaning interval (PNDs 36-63). Re-evaluation of the 

body weight data indicated that there was a statistically significant difference for low- and 

medium-dose males compared to control, but no statistically significant difference for females. 

However, the decrease in low-dose males was approximately 3% relative to control, and it was 

determined not to be toxicologically relevant. The 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 (vehicle 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. Subsequently, one pup/sex/litter/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 neuropathology on PND 63; Subset 3: FOB on PND 5 and 

12; auditory 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 mortality, 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 food consumption were comparable to control in 

the low-dose group). Increased body weights were still present at LDs 1-3, with values similar to 

control by LD 29. Food consumption also was increased (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 litter parameters (number born live, number born 

dead, sex ratio (% male), mean litter size, live birth index, and viability index), clinical signs, 

FOB parameters, motor activity, auditory startle reflex, learning and memory, sexual maturation, 

brain weight, or gross or microscopic pathology. 

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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 weights at the high dose continued to be decreased (p≤0.01) by 5-6% in 

the males and by 8-10% in the females throughout 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 and mean humidity was 65%. Individual adult 

body weights were recorded at study initiation, biweekly through Week 8, and at study 

termination. Food consumption in each replicate was measured weekly throughout the study. 

Adult birds were observed routinely for mortality and clinical signs. The following reproductive 

parameters were observed and recorded: 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 concentrations were <1.00 (<LOD, control), 

5.10, 9.93 and 20.0 mg ai/kg diet, respectively. There were no treatment-related effects observed 

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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 adult 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-cycle toxicity study (MRID 40069609), Rainbow trout (Oncorhynchus 

mykiss) were exposed in a flow-through system to five concentrations of abamectin (91% purity) 

in a carrier solvent of acetone; mean-measured concentrations were 0.15 (±0.051), 0.28 (±0.071), 

0.52 (±0.21), 0.96 (±0.39), and 2.2 (±0.78) μg/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 concentration. Wet weight at 60 days was decreased (p<0.05) at 

the medium-high concentration. The NOAEC is 0.52 μg/L, and the LOAEC was 0.96 μg/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 μL/L DMF solvent control), 0.081, 0.16, 0.33, 0.65 and 1.3 μg/L. Meanmeasured concentrations were <0.058 (<LOQ, controls), 0.076, 0.15, 0.30, 0.67 and 1.3 μg 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 μg ai/L, 

respectively, based upon a treatment-related reduction in post-hatch larval survival at the 0.67 

and 1.3 μg 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 treatment-related adverse effects were observed on 

time to hatch (Day 6 for all viable embryos); percentage 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 was 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 μg ai/L levels, respectively. Differences were 

statistically-significant compared to the negative control at the 0.67 and 1.3 μg ai/L treatment 

levels. 

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Mechanistic Studies: Male Reproductive 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 rats 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 anesthetized and the liver, testes and epididymis, and blood samples 

were collected. Abamectin concentrations in prepared 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 serum testosterone, LH, and FSH with an ELISA 

method. 

For histopathological evaluations, the left testicular tissues were fixed, embedded, and stained. 

The tubule degeneration was evaluated, and spermatogenesis was categorized by determining the 

Johnsen score (1=no germ or Sertoli cells present to 10=complete spermatogenesis and perfect 

tubules). The epididymal sperm count and motility were determined from left epididymis 

samples. In vitro sperm motility was evaluated with sperm 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 effects 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-activation 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 evaluated 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 IgG for 45 min at room temperature. The antigenantibody complex was detected using an avidin-biotin horseradish peroxidase complex and 

diaminobenzidine as the chromogen. Western blotting procedure was conducted using 

homogenized right testis supernatants. Samples were evaluated for PARP activity and oxidative 

stress by incubating with mouse monoclonal PAR and mouse anti-4HNE Mab (HNEJ-2) primary 

antibody, respectively; after overnight incubation, the membranes were washed and incubated 

with anti-mouse IgG horseradish peroxidase conjugate. 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 subacute, low-dose group, the mean abamectin 

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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 organizations was observed in the low- and high-treatment 

groups. The Johnsen scores, used to evaluate tubule degeneration, were decreased (p<0.001) in a 

dose-dependent manner for both abamectin treatment 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 observed after 5 minutes of treatment with 0.42 μM 

abamectin in the in vitro motility test, with continued 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 the 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 either test. For the Western blot analysis, 4-HNE 

and PAR expressions showed specific bands corresponding 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 after 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. 

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APPENDIX 3: References Not Utilized in the Abamectin WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support 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 compound of interest was not used 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 adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was 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) the study contained only data on invertebrates. 

Argus Research Laboratories, Inc. (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 abamectin exposure on semen parameters indicative of 

reduced sperm maturity: a study on farmworkers 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) 

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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) 

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