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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

# &

Health Effects Divison

# ::~:E£'~:::e 

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

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: MALATHION

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMALATHION FINAL

ii TABLE OF CONTENTS

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

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

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

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

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

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................14

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................20

IV. Committee’s Assessment of Weight of Evidence..........................................22

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................22

1. Tier 1 in vivo Assays.............................................................23

2. OSRI...............................................................................24

B. Estrogen Pathway...........................................................................26

C. Androgen Pathway.........................................................................27

D. Thyroid Pathway...........................................................................27

E. Conclusions.................................................................................27

V. EDSP Tier 2 Testing Recommendations....................................................28

VI. References.......................................................................................29

LIST OF TABLES: Table 1. Tier 1 Assay for Malathion...................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Malathion ...................................10
Table 3. Androgenic/Anti-Androgenic Pathway for Malathion ..................................16
Table 4. Thyroid Pathway for Malathion ..........................................................21

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................34
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................46

APPENDIX 3. References Not Utilized in the Malathion WoE Analysis.............................64MALATHION FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

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

iv Abbreviation Terminology

F1 First filial g eneration
F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

vi Abbreviation Terminology

T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 74 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether malathion interacts with E, A or T hormone path ways, the number and type of effects induced, the ma gnitude and pattern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whet her or not endocrine-related resp onses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On April 23, 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 malathion with the E, A or T hormone signaling pathwa ys. 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).

There is no convincing evidence fo r potential interaction with th e estrogen pathway. The Tier 1 ER binding and ERTA assays were negative. The Tier 1 steroidogenesis assay was negative, however, in a steroidogenesis assa y considered as OSRI (Taxvig et al. , 2013), a ≤ 1.5-fold induction in estradiol was reported. In the Ti er 1 aromatase assay, malathion showed an equivocal response. The Tier 1 uterotrophic assa y was negative and the only effect observed in the Tier 1 female pubertal assay was a decrease in pituitary weight which was observed in the presence of systemic toxicity. The effects observe d in the FSTRA were observed in the presence of overt toxicity. There were no estrogen-related effects seen in the Part 158 mammalian toxicity
studies. In the Part 158 wildlife studies, in the bobwhite quail, reproductive effects occurred in the presence of overt toxicit y. In the mallard duck study, redu ctions in eggshell thickness ( ↓ 5%) and egg viability were observed. However, no ot her reproductive effects were noted in this study, and effects of embryo viability is limited in its ability to inform potential estrogen (or androgen) related interaction, a nd there are no other estrogen-rela ted responses observed in the
other in vivo studies.MALATHION FINAL

Page 2 of 74 There was no convincing evidence of an inte raction with the andr ogen pathway. In the in vitro Tier 1 assays, malathion was equivocal for A ndrogen Receptor (AR binding and negative in the steroidogenesis assay. In a steroidogenesis assa y considered as OSRI, malathion showed a ≤ 1.5-
fold induction in testoste rone production (Taxvig et al. , 2013). The Tier 1 in vivo Hershberger assay was negative and in the male pubertal assay, decreases in testosterone were observed at the lowest dose in the presence of systemic toxicity ; overt toxicity was obser ved at the mid and high dose. In the fish short-term reproduction assay (F STRA), effects were observed in the presence of overt toxicity. There were no androgen-related effects in the Part 158 mammalian toxicity studies. Egg viability was decreased in the Pa rt 158 mallard duck reproduction study; however, this endpoint in limited in its diagnostic capabilities.

There was no convincing evidence for an inte raction with the thyr oid pathway. The only thyroid-related effects observed in the Tier 1 pubertal assays was a non-dose-dependent increase
in T 4 concentration at the low and mid dose in the females and an isolated increase at the mid dose in the males. No thyroid- related effects were seen in the Part 158 mammalian toxicity studies. In the amphibian metamorphosis assay (AMA), there were no gross or histological effects on the thyroid or alterati ons in developmental endpoints.

Based on weight of evidence considerations, mamm alian and wildlife EDSP Tier 2 testing is not recommended since there was no evidence of potentia l interaction with the estrogen, androgen or thyroid pathways.MALATHION FINAL

Page 3 of 74 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether malathion interacts with E, A or T hormone path ways, the number and type of effects induced, the ma gnitude and pattern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whet her or not endocrine-related resp onses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On April 22, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE ) analysis of the pot ential interaction of malathion with the estrogen E, A or T hormone pathways.

Malathion’s mode of action is based on altered neurotransmission due to cholinesterase (ChE) inhibition and the oxon metabolite, malaoxon, is a mo re potent organophosphate than the parent compound. The water solubility of malathion is 145 mg/L at 20°C, and its vapor pressure is 3.97

× 10 -5 mm Hg at 30°C. It has an octa nol/water partition coefficient (log K OW ) of 2.36, and
bioconcentration has not been observed in select species [ Pseudorasbora parva or in pinfish ( Lagodon sp)]. Malathion is susceptible to photolysis a nd hydrolysis and it is not persistent in the environment, with 81 to 94% degradation occurri ng in various non-sterile soils within ten days. Volatilization is not expected to significantly co ntribute to the dissipati on of malathion in the
environment.

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

Page 4 of 74 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 for malathion are show n below in Table 1. Executive Summaries are presented in Appendix 1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on Malathion (USEPA , 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Su mmaries of the Tier 1 assays and the studies cited as OSRI are presented in Appendix 1 and 2, respectively. Additio nally, 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

Page 5 of 74 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemicMALATHION FINAL

Page 6 of 74 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

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

Page 7 of 74 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substan ce 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 malathion are shown below
in Table 1

.

Table 1. Tier 1 Screening Assays for Malathion

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied by OSRI (Chen et al ., 2002; MRID 48160705)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48617505)
AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48617503)
Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48617509)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48617504)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48617510)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No.48617507)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48685701)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48685701)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48617506)
Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48617501)MALATHION FINAL

Page 8 of 74 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

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

Malathion was negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA and steroidogenesis assays. Malathion weakly induced (113% of contro l) ER activity in an ERTA conducted in MVLN cells (Kjeldsen et al., 2013). However, malathion was negative in other
ERTAs that were considered as OSRI conducted in CHO-K1 cells (Kojima et al ., 2004) and BG1 cells (Kojima et al ., 2005). An equivocal response was noted for malathion in a published steroidogenesis assay with H295R cells (Taxvig et al. , 2013); estradiol induction of approximately 1.5-fold was observed in a non- dose-dependent response. Malathion was classified as equivocal in the aromatase assay with mean aromatase inhi bition of 75 and 65% of the control activity at concentrations of 10 -4 and 10 -3.5 M, respectively. Results from a published study (Kjeldsen et al., 2013) assessing the effects of mala thion on aromatase activity in the
human choriocarcinoma JEG-3 cell line showed that malathion did not inhibit aromatase activity at any tested concentration ≤ 5×10 -5 M (limit of cytotoxicity).

No estrogen-related effects were observed in the Tier 1 uterotrophic assay, and no estrogenrelated effects were observed in the female pubert al assay. Pituitary weights were reduced 29 to 31% at the two doses tested in the female pube rtal study. In the fema le pubertal rat assay, decreases in ChE activity (RBC a nd brain) along with increased liver and kidney weights were observed in the mid- and high-dose groups. In addition, clinical signs (excess salivation, decreased motor activity and tremors), irregular re spiration (gasping) and mo rtality were noted at the high dose (1000/750 mg/kg/day). No estrogen-related effects were observed in the Part 158
mammalian studies.

In the FSTRA, there was a significant 48% decrease in fecundity in the high-concentration group (0.69 mg/L) with no effect on fertilization. Female GSI was significantly increased at the high concentration, with an increase in gonadal oocyte atresia also noted at this dose level. No other effects ( i.e . plasma VTG concentration) were observe d. Although mortality was minimal, clinical signs of toxicity were apparent in 33% of the high-dose fish as well as observations of reduced feeding; this concentration is c onsidered to be overtly toxic.MALATHION FINAL

Page 9 of 74 In the Part 158 bobwhite quail study, a si gnificant decreases in eggs laid ( ↓ 76%), viable embryos/egg set ( ↓ 75%), and eggshell thickness ( ↓ 15.5%) were observed in the high-dose group (1200 mg/kg-diet). Subsequently, live embryos, hatchlings, and 14 d hatchlings per egg set were also significant at the high treatment, but these endpoints we re not significantly different when compared when using the preceding stage in the ratio calculation ( i.e. , live embryos/viable embryos, hatchlings/live embryos, 14 d hatchlings/nor mal hatchlings). At this same dose, clinical signs of toxicity were also observed as was a significant decrease in female and male body weight gain ( ↓ 55% & 53%, respectively), enlarged gizzards in both se xes and increased mortality. In the mallard duck, there were signi ficant decreases in eggshell thickness (5%) and decreased embryo viability (25%). In a fish life-cycl e test in the flagfish, an 18% decrease in F1 body length was observed at the mid-low dose; howeve r, no effects on fecundity or fertility were observed. In an early life stage study with the rainbow trout there were significant reductions (p<0.05) in post hatch survival, mean length, fry su rvival, and mean weight at concentrations as low as 44 μg a.i/L.

There was no treatment-related effect on the prev alence of cracked eggs; the number of eggs not cracked/eggs laid in the vehicl e-control group was generally greater compared to treatment groups. Decreases (p<0.01) in eggs laid ( ↓ 76%), viable embryos/egg set ( ↓ 75%) were observed in the high-dose group. Subsequently, live embryos , hatchlings, and 14 d hatchlings per egg set were also significant at the high treatment, but these endpoints were not significantly different when compared when using the preceding stage in the ratio calculation (i.e., live embryos/viable embryos, hatchlings/live embryos, 14 d hatchlin gs/normal hatchlings). Eggshell thickness was also decreased (p<0.05; ↓ 15.5%) at the high dose. Hatchling a nd 14-d hatchling weights were not
affectedMALATHIONFINALPage 10 of 74Table 2. Estrogenic/Anti-Estrogenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Malation1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER BindingAssay satisfied by OSRI (Chenet al., 2002; MRID 48160705, see below)ERTA(MRID 48617505)NSteroidogenesis(MRID 48617509)N4Aromatase(MRID 48617504)EUterotrophic(MRID 48617510 )N↑LW:(M,H)↓ChE(L, M, H)NFemale Pubertal Rat(MRID 48685701)NENNN↓29%(M)N N↑LW:(M,H)↓ChE(L, M, H)X(H)FSTRA(MRID 48617506)NENNNNNX5(H)OSRIER Binding(Chenet al., 2002;MRID 48160705)NERTA(Kjeldsenet al.,2013)PMALATHIONFINALPage 11 of 74Table 2. Estrogenic/Anti-Estrogenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Malation1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3ERTA(Kojimaet al., 2004)NERTA(Kojimaet al., 2005)NAromatase(Kjeldsenet al.,2013)NSteroidogenesis(Taxviget al., 2013)E21-Day Dermal(Rabbit; MRID41054201)NE N N NEX(H)N13-Week Inhalation(Rat;MRID 43266601)NNNNX(M, H)NDevelopmentaltoxicity(Rat;MRID 41160901)NNNENDevelopmentaltoxicity(Rat; Prabhakaranand Devi, 1993)NENXNDevelopmentaltoxicity(Rabbit;MRID 00152569;45626801)NENX(H)X(M, H)MALATHIONFINALPage 12 of 74Table 2. Estrogenic/Anti-Estrogenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Malation1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmentaltoxicity(Rabbit; Machin andMcBride, 1989)NENNENDevelopmentalNeurotoxicity(Rat;MRID 45646401)NNX(H)XChE Inhibition —DevelopmentalToxicity(Rat;MRIDs 45566201and 46005001)NE NE NENENEXNTwo-generationreproduction(Rat;MRID 41583401)NE NE NNENNEX(H)Chronic toxicity /carcinogenicity(Rat;MRID 43942901)NENNNEX(ML, MH,H)X(H)Carcinogenicity(Mouse;MRID 43407201)NENNNEX(ML, MH,H)X(MH, H)Chronic toxicity(Dog;MRID 40188501)NENNNEX(L, M, H)NMALATHIONFINALPage 13 of 74Table 2. Estrogenic/Anti-Estrogenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Malation1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Duck;MRID 42782101)P6N NAvian reproduction(Quail;MRID 43501501)NNX(H)Fish Life-Cycle(Flagfish;Hermanutz, 1972NN1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓r↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.LW = Liver weight; KW = Kidney weight; ChE = Cholinesterase activity2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected. Although the conditions of this assaywere not optimal to detect very weak activity, malathion responses were similar to those of the negative control corticosteroneand not comparable to theresponses of 17α-methyltestosterone, which was able to reach a maximum of 6.5-37.4% PC. Malathion was only able to reach a maximum of 3.4-3.7%PCwhen tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax< PC10in both assay runs, malathion was considerednegative in this test system.5. Although mortality was minimal, clinical signs of toxicity were apparent in 33% of the high-dose fish. The incidence of clinical signs of toxicity and reducedfeeding were suggestive of overt toxicity at the high concentration.6. In the mallard duck, there were significant decrease ineggshell thickness (5%) and decreased embryo viability (25%).E EquivocalP Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedMALATHION FINAL

Page 14 of 74 2. Effects on Androgen Pathway

The potential for malathion to interact w ith 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 androgeni c, anti-androgenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

In an evaluation of androgenic/an ti-androgenic activity from Tier 1 testing results, malathion was negative in the steroidogenesis assay, and classified as equivocal in the in vitro AR binding assay. Malathion reduced the mean specific binding of [ 3 H]-R1881 to 51.6% and 50.4% at 10 -3 M (the highest concentration) in Runs 1 and 2, but precipitation of malathion was visually observed at this concentration in Run 3. However, malathion was negative an AR transcriptional activation assays in CHO-K1 cells (Kojima et al ., 2004 and Kjeldsen et al. , 2013). An equivocal response also was determined with malathion in a published, non-guideline steroidogenesis assay with H295R cells (Taxvig et al. , 2013); testosterone induction of approximately 1.5-fold was observed in a non-dose-dependent manner.

Malathion did not demonstrate an androgen- agonist or antagoni st effect in the in vivo Hershberger assay; no AST weights were affected. Body weights were not affected by treatment,
but RBC ChE was inhibited at all doses and brain ChE was inhibi ted at the high dose. Finally, liver weights were increased at all doses and ki dney weights were increased in the mid- and high-dose animals.

In the male pubertal rat assay, due to mortalities and clinical observations of toxicity, the initial 1000 mg/kg/day dose level was decreased to 750 mg /kg/day. Clinical signs included effects on gait ( e.g ., ataxia, lost righting refl ex and decreased motor activ ity), dyspnea, tremors, and excessive salivation; all were observed at the mid and high dose s. Increased absolute and/or relative (p<0.01 or 0.05) kidney ( ↑≤ 16%) and liver ( ↑≤ 26%) weights in the low-, mid-, and highdose groups were present with no adverse hi stological findings. ChE activity was dosedependently inhibited (p ≤ 0.01) in the brain ( ≤ 35%) and in the RBC ( ≤ 88%) in the low- and middose animals; no high-dose animals were available for analysis due to mortalities. No treatmentrelated effects were observed on age or weight at PPS. Dose-dependent decreases (p<0.01) in

testosterone concentration ( ↓ 60 to 90 %) were noted in all tr eated animals. Dose-dependent decreases (p<0.01) in one AST weight (LABC) were observed in the mid- and high-dose
animals.MALATHION FINAL

Page 15 of 74 The only androgen-related effect observed in the Part 158 mamma lian toxicity studies was a nonstatistically significant increase ( ↑ 11%) in testes weight at 359 mg/kg/day in the rat chronic/carcinogenicity study.

In the FSTRA, there were no significant effects of malathion treatment on male VTG, GSI, and body length. Male median tubercle scores were reduced (10.5 vs. 25.5 in control) and the fatpad score was reduced by 43% (p<0.05) at the high con centration (0.69 mg/L). In addition, effects on male gonadal histology was observed at the high dos e. As noted previously, clinical signs of toxicity were apparent in 33% of the high-dos e fish at his dose along with reduced feeding. Additionally, a non-signif icant reduction in male body weight (17.3%) was observed at the high
dose.

As previously indicated, avian reproductive t oxicity studies are available for the northern bobwhite quail study and a mallard duck study. Egg vi ability was significantly decreased in both species. However, in the quail, this effect was observed in conjunction w ith overt toxicity, and decreases in male body weight gain were obser ved in the mallard duck study were reproductive
effects were observed.. In a fish lif e-cycle test in the flagfish, an 18% decrease in F1 body length was observed at the mid-low dose; however, no effects on fecundity or fertility were observed.MALATHIONFINALPage 16 of 74Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / LiteratureCitation AR Binding / ARTA Steroidogenesis Sex Steroid hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48617503)ESteroidogenesis (MRID48617509)NHershberger(MRID 48617507)NEN↑LW14%,21%,32%,(L, M, H);↑KW13–18%,(M, H);↓ChE(L, M, H)NMALATHIONFINALPage 17 of 74Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / LiteratureCitation AR Binding / ARTA Steroidogenesis Sex Steroid hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Male Pubertal Rat(MRID 48685701)↓60%,(L)NNNNNNN↑LW10% (L),↑21–22%(M)↑21–26%,(H);↑KW↑12–14%,↑16%(L, M, H);↓ChE(L, M, H)X(M, H)FSTRA(MRID 48617506)NENNNNNX(H)OSRIARTA(Kojimaet al., 2004)NARTA(Kjeldsenet al., 2013)NSteroidogenesis(Taxviget al., 2013)E13-Week Inhalation(Rat; MRID 43266601)NNNENNX(H)N21-Day Dermal (Rabbit;MRID 41054201)NNNNNEX(L,M,H)NMALATHIONFINALPage 18 of 74Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / LiteratureCitation AR Binding / ARTA Steroidogenesis Sex Steroid hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmental toxicity(Rabbit; MRID00152569; 45626801NNX(H)NDevelopmentalNeurotoxicity(Rat; MRID 45646401)NNX(H)NChE Inhibition —Developmental Toxicity(Rat;MRIDs 45566201 and460050001)NENENENENENE NEX(H)NETwo-generationreproduction(Rat; MRID 41583401)NENNENNENE NNEX(H)Chronic toxicity /carcinogenicity(Rat; MRID 43942901)N N NNNE NEX(MH, H)X(ML,MH, H)Carcinogenicity(Mouse;MRID 43407201)N N NE N NE NEX(ML, MH,H)X(MH,H)Chronic toxicity(Dog; MRID 40188501)NNNENENENEX(L, M, H)NAvian reproduction(Duck;MRID 42782101)P4N NMALATHIONFINALPage 19 of 74Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / LiteratureCitation AR Binding / ARTA Steroidogenesis Sex Steroid hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail;MRID 43501501)NNX(H)Fish Life-Cycle(Flagfish;Hermanutz, 1972)NX(H)1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Levator anibulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). LW = Liver weight; KW = Kidney weight; ChE = Cholinesteraseactivity2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. In the mallard duck, there were significant decrease in embryo viability (25%).E EquivocalP Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedMALATHION FINAL

Page 20 of 74 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

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

The potential for malathion to interact with thyroid regulation are summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

In both the female and male pubertal assays, no treatment-related effects were noted for thyroid weight, histopathology, or serum TSH concentration. Concentrations of T 4 were increased at all doses in males and females, and pituitary weig ht was decreased (p<0.01) by approximately 30% in the mid- and high-dose females only. Systemic t oxicity was observed at all doses in the males and females and overt toxicity wa s seen at the high dose in females and at the mid and high dose
in males.

In the AMA, there were no gross or histological effects on the thyroid; additionally there were no effects on developmental stage or hind-limb lengt h (normalized). Day 7 snout-vent length (SVL) and body weight were increased (p<0.05) by 10.7 and 27.9%, respectively, in the lowconcentration group, but there we re no significant effects on growth parameters in the intermediate- and high-concentration treatment groups compared to control.

In the Part 158 studies, thyroid weights were in creased in the rat and dog chronic studies (and not examined in the mouse carcinogenicity study), bu t no histopathological findings were observed.MALATHION FINAL

Page 21 of 74 Table 4. Thyroid Pathway for Malathion
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Malathion 1

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

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48685701) N N ↑ 19%, ↑ 16%, (L, M) N ↓ 29 (M) N ↑ LW: 14%(M)

↓ ChE (L, M, H) X (H)

Male Pubertal Rat (MRID 48685701) N N ↑ 41%, (L) N N N ↑ LW 10% (L), ↑ 21–22%

(M), ↑ 21–26%, (H); ↑ KW ↑ 12–14%,

↑ 16% (L, M, H);

↓ ChE (L, M, H) X (M, H)

AMA (Frog; MRID 48617501) N N Day 7 SVL ( ↑ 10.7%); Day 7 BL ( ↑ 27.9 %) (M) N

OSRI

13-Week Inhalation (Rat; MRID 43266601) N N N X (L, M, H) N

Chronic toxicity / carcinogenicity (Rat; MRID 43942901) N N NE X (ML, MH, H) X (ML, MH, H)

Carcinogenicity (Mouse; MRID 43407201) NE N NE X (ML, MH, H) X (MH, H)

Chronic toxicity (Dog; MRID 40188501) ↑ (H) N NE X (L, M, H) N

1 Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not app licable in this assay. ChE = cholinesterase activity. 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. AMALATHION FINAL

Page 22 of 74 P Positive findings N Negative findings NE Not examined

IV. Committee’s Assessment of Weight of Evidence

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

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

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

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

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

Page 23 of 74 1. Tier 1 in vivo Assays

In the Hershberger assay, general systemic t oxicity was noted in bot h the mid- and high-dose groups (300 and 1000 mg/kg/day) base d on significant increases in ab solute and/or relative liver and kidney weights. At 1000 mg/ kg/day, absolute and relative liver weights were increased (p<0.01) by 21 to 32% compared to control, a nd absolute and relative kidney weights were increased by 13 to 18%. At the mid dose, relati ve liver and kidney we ights were increased (p ≤ 0.05) by 9% and 7%, respectively. Although clinical signs of t oxicity were not observed in the study, ChE activity was substant ially inhibited at all doses ( ≥ 100 mg/kg/day). Brain ChE activity was inhibited (p ≤ 0.01) by 39% at 1000 mg/kg/day, and RBC ChE activity was inhibited (p ≤ 0.01) by 22, 61, and 88% at 100, 300, a nd 1000 mg/kg/day, respectively.

In both the female and male pube rtal rat studies, the initial hi gh dose (1000 mg/kg/day) was reduced to 750 mg/kg/day by PND 24 due to mortality in the high-dose group. However, mortality was still observed in both the intermediate- (500 mg/kg/ day) and high-dose males (2/20 and 14/20, respectively) and high-do se females (2/20) during the remainder of the studies, along with clinical signs of choliner gic toxicity (excessive salivati on, tremors, irregular gait, and irregular respiration). Low-dose males and low- and mid-dose females also showed sporadic signs of clinical toxicity. Ther e were no significant effects of malathion treatment on final body weight or body weight gain for females or males. In the high-dose females, absolute and relative liver weights were increased by 14 to 16%, and absolute and relative kidney weights were increased by 11 to 12%. Absolute and relative pi tuitary weights were also decreased by 29 to 31% in both mid- and high-dose fema les. In the males, absolute a nd relative liver weights were increased by 10% at the low dose (relative weight only), 21 to 22% at the intermediate dose, and 21 to 26% at the high dose. Absolute kidney weight was increased by 12% in the mid-dose males, and relative kidney weight was increased by 14 and 16% in the mid- and high-dose males. For the cohorts used to examine ChE activity, brain and RBC ChE activit y was dose-dependently
decreased in both females and males. In the low-, mid- and high dose females (250, 500 and 750 mg/kg/day), ChE activity was inhibited in the brain by 9, 24, 42%, respectively, and in the RBC by 74, 89, 97%, respectively. In the low an d mid-dose males (250 and 500 mg/kg/day), ChE activity was inhibited in the brain by 17 and 35%, respectively, and in the RBC by 79 and 88%, respectively.

In the uterotrophic assay, no clin ical sign of toxicity were obser ved over three days of treatment at doses up to 1000 mg/kg/day, but general sy stemic toxicity was observed based on the decreases in brain and blood ChE activity. Brain ChE activity was inhibited by 16% at 1000 mg/kg/day, and RBC ChE activity was in hibited by 20, 38 and 69% at 100, 300 and 1000 mg/kg/day, respectively. In addition, absolute and relative liver weights were increased (p ≤ 0.01) by 24 and 25%, respectiv ely, at 1000 mg/kg/day.MALATHION FINAL

Page 24 of 74 In the FSTRA study, at 0.69 mg/L, 8 of 24 fish e xhibited clinical signs of toxicity, including bruising on the body, loss of color or no color in the tail, erratic swimming, lethargy, and crooked spine. In addition, feeding was redu ced on Days 5 through 16 and male body weights were reduced by 17.3% (NS) at study termination. No overt or systemic si gns of toxicity were observed in the AMA study at test concentrations up to 32 mg/L.

2. OSRI

In the Part 158 studies, the most sensitive e ndpoint across studies was ChE inhibition, with
increases in liver and kidn ey weights noted in most in vivo studies as well. In the 21-day dermal toxicity study in rabbits, serum, RBC, and brain ChE activities we re decreased (p<0.01) at study termination ( ↓ 41–75%) in all high-dose males and female s. ChE activities were unaffected in low-dose animals, and only RBC ( ↓ 26%, females) and cerebellum ( ↑ 45%, males) ChE activities were affected (p<0.01) at the intermediate dose . In the 13-week subchronic inhalation study in rats, all ChE activities were decreased (p<0.01) in high-dose animals by 17–70% (plasma ChE in males, ↓ 18%, NS), and RBC ChE activity in males and females was decreased (p<0.05) at the intermediate dose by 22 and 27%, re spectively. In addition, liver ( ↑ 14 and 15%, respectively), kidney ( ↑ 9%), and lung ( ↑ 8%) weights were increased (p<0.05 or 0.01) in the high-dose males and/or females, with NS increases in liver and kidney weights occasionally observed at the
intermediate dose.

In the developmental rat study, clinical signs of toxicity consisted of red staining of the urogenital areas, excess salivation (starting at Week 4 to 5) and un-groomed oily fur (starting after Week 5). In a published study examining de velopmental toxicity in rats (Prabhakaran and Devi, 1993), ChE activity in the brain was decreased by 24 and 28%, respectively, in malathion
treated animals fed either a contro l diet or protein-deficient diet. Treatment-related clinical signs observed in the rabbit developmental study consisted of anorexia and soft stools at the high dose.

In a companion ChE inhibition study associated with the developmental neurotoxicity study, ln pups, effects on RBC ChE activity were noted at 5 mg/kg/day in both sexes on PND 21 after eleven repeated exposures. After 11 or 14 doses to young adults, effects were observed at 50 mg/kg/day in young adults and pregnant dams. In pups, brain ChE activity was inhibited after eleven repeated doses ( ↓ 16% in PND 21 pups). Based upon the results of the ChE study, it is
evident that all behavioral and neuropathological effects of treat ment observed in the dams and offspring in the developmental neurotoxicity stu dy occurred at doses at which ChE activity was, or had been, inhibited.

In a two-generation, two-litter re production study, no clinical signs related to treatment were reported. In the F 0 generation, after the first mating, body weight in females was decreased (p ≤ 0.01 or 0.05) at the high dose during gestation ( ↓ 6.6% to 7.6%) and during the entireMALATHION FINAL

Page 25 of 74 lactational period ( ↓ 6.6% to 9.2%) and at the medium-h igh dose on LD 14. After the second mating, body weight was decreased (p ≤ 0.05) at the high dose on LD 0 (8.2%) and 21 (6.2%). In
the F 1 generation, body weight was decreased (p ≤ 0.01 or 0.05) at the high dose during the entire treatment period for males ( ↓ 9.1% to 13.4%) and during premating for females ( ↓ 6.9% to 10.1%). Increased (p ≤ 0.05) weights were noted in female s at the medium-low dose during the first gestational period and at the medium-low and low doses during the second gestational period.

In the chronic rat study, the only clinical si gn observed was yellow anogenital staining among females in the high-dose group. However, increase d mortality was seen in females at the high dose, and in males at all but the low dose ( ≥ 500 ppm), with all high -dose males dead or sacrificed moribund by Week 94. Treatment-related decrements in body weight gain were also observed in males and females in the medium-hig h (3 to 13% and 4 to 5%, respectively) and high-dose (12 to 32% and 10 to 15%, respectivel y) groups. Inhibition (p<0.01 or 0.05) of ChE activity was observed in males and females in pl asma, RBC and brain at all sample periods (3, 6, and 12 months, and at study termination) in th e medium-high and high- dose groups. Inhibition was also noted for RBC ChE activity in females in the medium-low group at 3 and 12 months and at study termination, and in males and females in the low-dose group at 3 months (prior to decrease from 100 to 50 ppm). ChE activity at st udy termination in the medium-high males was decreased 64, 43 and 31% in plasma, RBC, a nd brain, respectively, and was decreased 61, 44, and 18%, respectively, in the medium-high female s. No samples were collected for high-dose males at study termination (100% mortality), butChE activity at study termination in the highdose females was decreased 89, 52, and 67% in plasma, RBC and brai n, respectively. Dosedependent inhibition (p<0.01 or 0.0 5) was also noted for plasma ChE in the medium-low dose males at study termination only ( ↓ 29%), and in RBC ChE activity in females in the medium-low group at 3 and 12 months and at study termination ( ↓ 27%). Finally, ChE effects were noted in males and females in the low-dose group at 3 mont hs (prior to decrease from 100 to 50 ppm). Organ weight effects included increased (p<0.0 1) liver weights in the medium-high and highdose males ( ↑ 22 and 36%, respectively) at the interim s acrifice, and increased (p<0.01) weights of 34% in the medium-high males at terminati on. In females, liver weights were increased (p<0.01) in the high-dose animals ( ↑ 16%) at the interim sacrifice, with increases (p<0.01) in the medium-high and high-dose females ( ↑ 30 and 31%, respectively) at termination. Kidney weights were increased (p<0.01) in the medium-high and high-dose males ( ↑ 14 and 29%, resp ectively) at the interim sacrifice, with an increase (p<0.01) of 11% in the me dium-high males at termination. In females, kidney weights were increased (p<0 .01) in the medium-high and high-dose animals ( ↑ 9 and 13%, respectively) at the in terim sacrifice, with increases (p<0.01) in the same groups of females ( ↑ 22 and 37%, respectively) at termination.MALATHION FINAL

Page 26 of 74 In the mouse carcinogenicity study, treatment-relate d clinical signs, including signs associated with ChE inhibition, were not observed. However, at the high- and medium-high dose in both males and females, treatment-related effects in cluded decreased (p<0.01) absolute body weights throughout the entire duration of the study ( ↓ 14.3 and 20.0%, respectively, in males and ↓ 9.7 and 16.1% in females at 18 months, respectively). Decreased (p<0.01) plasma ChE activity at 12 and 18 months ( ↓ 86 to 96%), decreased (p<0.01 or p<0.05) RBC ChE activity at 9, 12 and 18 months ( ↓ 61 to 92%), and decreased brain ChE activity at 18 months (37 and 43% in males and females, respectively) was present at th e high dose only. Plasma ChE activity was also decreased in the medium-low dose males (NS; ↓ 23 and 24%) and females (p<0.05; ↓ 18 and 36%), and RBC ChE activity was also decreased in the me dium-low dose males (NS or p<0.05; ↓ 37 to 44%) and females (NS, p<0.05, or p<0.01; ↓ 35 to 58%).

In the chronic dog study, all animals survived until terminal sacrif ice, with no treatment-related clinical signs and no effect s on body weight. RBC and plasma ChE activity was inhibited (p<0.05) in both sexes at all doses and sample times. Plasma ChE activity in males was decreased by 23 to 30% at all doses and samp le times; plasma ChE activity in females was decreased by 22% to 37% in the low- to high -dose groups. RBC ChE activity in both males and females was inhibited to appr oximately the same extent ( ↓ 25%) at all dose and all sample times. Brain ChE (cerebrum and cerebellum only) was not affected by treatment. Absolute kidney weights were increased in mid-and high-dose males ( ↑ 25 and 47%) and females ( ↑ 24 and 55%), and absolute liver weights were increase d in low- and high-dose females only ( ↑ 40 and 41%).

In an avian reproduction study with mallard ducks, male body weight was decreased (p<0.01, 10%) in the high-dose group at Week 20, with a time-related trend to decrease (NS, ↓ 3 to 6%) observed throughout the study. No other clinical signs were apparent. In another avian reproduction study in bobwhite quail, mortality occurred in all trea tment groups (one each in the control, low- and mid-dose groups, and nine in the high-dose group). Adult body weights were decreased (p<0.01) at Week 2 in high-dose males ( ↓ 10%) and females ( ↓ 8%) and at study termination in high-dose males ( ↓ 8%; NS) and females ( ↓ 14%).

In a published fish life-cycle study (Hermanutz, 1978) using flagfish, larval survive was decreased during the initial 30 days of testing at mala thion concentrations of ≥ 25 μ g/L, and body lengths were reduced by 11-18% at concentration ≥ 11 μ g/L.

B. Estrogen Pathw ay

There is no convincing evidence fo r potential interaction with th e estrogen pathway. The Tier 1 ER binding and ERTA assays were negative. The Tier 1 steroidogenesis assay was negative, however, in a steroidogenesis assa y considered as OSRI (Taxvig et al. , 2013), a ≤ 1.5-fold induction in estradiol was reported. In the Ti er 1 aromatase assay, malathion showed anMALATHION FINAL

Page 27 of 74 equivocal response. The Tier 1 uterotrophic assa y was negative and the only effect observed in the Tier 1 female pubertal assay was a decrease in pituitary weight which was observed in the presence of systemic toxicity. The effects observe d in the FSTRA were observed in the presence of overt toxicity. There were no estrogen-related effects seen in the Part 158 mammalian toxicity
studies. In the Part 158 wildlife studies, in the bobwhite quail, reproductive effects occurred in the presence of overt toxicit y. In the mallard duck study, redu ctions in eggshell thickness ( ↓ 5%) and egg viability were observed. However, no ot her reproductive effects were noted in this study, and effects of embryo viability is limited in its ability to inform potential estrogen (or androgen) related interaction, a nd there are no other estrogen-rela ted responses observed in the
other in vivo studies.

C. Androgen Pathway

There was no convincing evidence of an inte raction with the andr ogen pathway. In the in vitro Tier 1 assays, malathion was equivocal for AR binding assay and negative in the steroidogenesis assay. Malathion was also negative for AR activation in two OSRI ARTA assays. In a steroidogenesis assay considered as OSRI, malathion showed a ≤ 1.5-fold induction in testosterone production (Taxvig et al. , 2013). The Tier 1 in vivo Hershberger assay was negative and in the male pubertal assay, decr eases in testosterone were obser ved at the lowest dose in the presence of systemic toxicity; overt toxicity wa s observed at the mid and high dose. In the fish short-term reproduction assay (FSTRA), effects we re observed in the presence of overt toxicity. There were no androgen-related effects in the Part 158 mammalian toxicity studies. Egg viability was decreased in the Part 158 mallard duck reproduction study; however, this endpoint in limited in its diagnostic capabilities.

D. Thyroid Pathway

There was no convincing evidence for an inte raction with the thyr oid pathway. The only thyroid-related effects observed in the Tier 1 pubertal assays was a non-dose-dependent increase
in T 4 concentration at the low and mid dose in the females and an isolated increase at the mid dose in the males. No thyroid- related effects were seen in the Part 158 mammalian toxicity studies. In the AMA, there were no gross or histological effects on the thyroid or alterations in developmental endpoints.

E. Conclusions

Malathion did not demonstrate a potential to interact with the estrogen, androgen thyroid pathways in mammals or wildlife.MALATHION FINAL

Page 28 of 74 V. EDSP Tier 2 Testing Recommendations Based on weight of evidence considerations, mamm alian and wildlife EDSP Tier 2 testing is not recommended since there was no evidence of poten tial interaction with the estrogen, androgen thyroid pathways.MALATHION FINAL

Page 29 of 74 VI. References

Barnett, J. F. (2011). Oral (gavage) uterotrophic bioassay of malathion in female rats. Unpublished study conducted by Charles River Laborat ories, Preclinical Serv ices, Horsham, PA. Laboratory Project ID: 20012263, November 3, 2011. (MRID 48617510).

Barnett, J. F. (2011). Oral (gavage) Hers hberger bioassay of malathion in male rats. Unpublished study prepared by Charles River Labora tories, Preclinical Services, Horsham, PA. Laboratory Project ID 20012264, N ovember 3, 2011. (MRID 48617507).

Barnett, J.F. (2012). A Pubertal Development and Thyroid Function Study of Malathion by Oral Gavage in Peripubertal Male and Female Rats . Unpublished study conducted by Charles River Laboratories, Preclinical Serv ices, Horsham, PA. Laborat ory Project ID: 20012266, June 18, 2012. (MRID 48685701).

Barnett, J.F. (2012). Oral (Gavage) Repeat ed Dose 14-Day Dose Range-Finding Study of Malathion in Rats. Unpublished study conducted by Charles River Laboratories, Preclinical Services, Horsham, PA. Laboratory Project ID: 20014321, May 1, 2012. (MRID 48685702).

Beattie, G. (1994). A 13-week toxicity study of aerosolized malathion administered by whole body inhalation exposure to the albino rat. Labo ratory report Project Number: 90729, submitted to Cheminova Agro A/S, Denmark. Unpublished study conducted by Bio-Research Laboratories, Ltd., Canada. EPA. (MRID 43266601).

Beavers, J.B., D. Haberlein, L.R. Mitchell, and M. Jaber. (1995). Malathion: A One Generation Reproduction Study with the Northern Bobwhite ( Colinus virginianus ). Laboratory Project Number 232-112A, submitted to Cheminova Agro A/S, Denmark. Unpublished study conducted by Wildlife International, Easton, MD. (MRID 43501501).

Chen, H, Xiao, J., Hu, G., Zhaou, J., Xia o, H., and Wang, X (2002) Estrogenicity of organophosphorous and pyret hroid pesticides. J. of Tox and Environ. Health . Part A. 65, 1419-

1435.

Cohle, P. (1989). Early Life Stage T oxicity of Cythion to Rainbow Trout ( Oncorhynchus mykiss ) in a Flowthrough System: Lab Report Number: 37400. Unpublished study prepared by Analytical Bio-Chemistry Laboratories, Inc. (MRID 41422401).

Daly, I.W. (1996). A 24-month oral toxicity/o ncogenicity study of mala thion in the rat via dietary administration. Laborat ory report Project Number: 90- 3641, submitted to Cheminova Agro A/S. (MRID 43942901).MALATHION FINAL

Page 30 of 74 Fulcher, S.M . (2001). Malathion: Effects on cholinesterase in th e CD rat (adult and juvenile) by oral gavage administration. Unpublished study prepared by Huntingdon Life Sciences, Ltd., Huntingdon, Cambridgeshire, England. Do c. No. CHV067/012452. November 30, 2001. (MRID 45566201).

Fulcher, S.M. (2002). Malathion developmental ne urotoxicity study in the CD rat by oral gavage administration. Laboratory report Project Number: CHV066/013331, submitted to Cheminova A/S. (MRID 45646401).

Fulcher, S.M. (2003). Amendment 1 to the final report; Malathion: effects on cholinesterase in the CD rat (adult and juvenile ) by oral gavage administrati on. Unpublished study prepared by Huntingdon Life Sciences, Ltd., Cambridgeshir e, England. Doc. No. CHV067/012452. June 19, 2003. (MRID 46005001).

Hermanutz, R.O. (1978). Endrin and malathion toxicity to flagfish ( Jordanella floridae ). Environ. Contam. Toxicol. 7:159–168.

Ishihara, A., N. Nishiyama, S. Sugiyama, and K. Yamauchi. (2003). The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor. General and Comparative Endocrinology 134:36–43.

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112 (5): 524-531.

Kojima, M., K Fukunaga, M. Sasaki, M. Nakamura, M. Tsuji, T. Nishiyama. (2005) Evaluation of estrogenic activities of pesticides us ing an in vitro reporter gene assay. Int. J. Environ. Health

Res

. 15: 271-280. Krause, W., K

. Hamm, and J. Weissmuller

. (1976). Damage to sperma togenesis in juvenile rat
treated with DDVP and malathion. Bulletin of Environmental Contamination and Toxicology 15(4):458–462. (MRID 48160711).

Krause, W. (1977). Influence of DDT, DDVP and malathion on FSH, LH and testosterone
serum levels and testosterone concentration in testis. Bulletin of Environmental Contamination and Toxicology 18(2):231–242. (MRID 48160710).

Kjeldsen, L. S., Ghisari, M., a nd Bonefeld-Jørgensen, E. C. (2013). Currently used pesticides
and their mixtures affect the function of sex hormone receptors and aromatase enzyme activity. Toxicol Appl Pharmacol . 272(2): 453–464.

Lillie, R.J. (1973). Studies on the reproductive performance and progeny performance of caged white leghorns fed malathion and carbaryl. Poult. Sci. 52:266–272.MALATHION FINAL

Page 31 of 74 Lochry, E.A. (1989). A development toxicity study with AC6,601 in rats. Laboratory report Project Number: 101-005, submitted to Americ an Cyanamid Company and A/S Cheminova. Unpublished study conducted by Argus Research Laboratorie s, Inc., Pennsylvania. (MRID 41160901).

Machin, M.G.A., and W.G. McBride

. (1989). Teratological study of malathion in the rabbit. Journal of Toxicology and Environmental Health 26:249–253. (MRID 48160712).

Moreno, O.M. (1989) 21-day dermal toxicity st udy with AC 6,601 in rabbits. Laboratory report Project Number: MB 88-9191, submitted to Malathion Reregistration Task Force. Unpublished study conducted by MB Research Laboratories, Inc., Pennsylvania. MRID 41054201.

Neal, B., Williams, A., Lamb, J.C., Freeman, E ., Staveley, J. (2013). Weight of the Evidence Discussion of Potential Endocrine Pathway Interactions for Malathion. Cheminova A/S, Arlington, VA 22209. MRID No. 49079701

Palmer, S., Schneider, S., Kendall, T. and Kr ueger, H. (2011). Malathion: Amphibian Metamorphosis Assay Final Report. Unpublishe d study conducted by Wildlife International, Easton, MD. Laboratory Project Number 232A -124. Study sponsored by Cheminova A/S. October 31, 2011. (MRID 48617501).

Palmer, S., Schneider, S., Kendall, T. and Kruege r, H. (2011). Malathion: Fish Short-Term Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Unpublished study conducted by Wildlife International, Ltd., East on, MD. Lab Project No.: 232A-121. Submitted by Cheminova A/S. October 27, 2011. (MRID 48617506).

Pedersen, C.A. and Fletcher, D.W. (1993). AC6,601 Technical: Toxicity and Reproduction Study in Mallard Ducks. Laboratory Project Nu mber 90DR39; Submitted to Cheminova Agro A/S, Lemvig, Denmark. Unpublished study conducted by Biolife Associates Ltd., Neillsville, WI. (MRID 42782101).

Piramanayagam S., Manohar, B.M., and Sundararaj, A. (1996). Pathology of malathion toxicity
in rats. Indian Vet. J. 73 (7): 734–737.

Robinson, K. (2002). Evaluation of the Embryo-Le thal Potential of Mala thion in the Rabbit. Unpublished study prepared by ClinTrials BioResearch, Ltd. 12 p. (MRID 45626801).

Schroeder, R.E. (1990). A two- generation (two-litters) reproducti on study with AC 6,601 to rats. Laboratory report Project Number: 87-3243, submitted to American Cyanamid Company and
A/S Cheminova. Unpublished study conducted by Bio/dynamics, Inc., New Jersey. (MRID 41583401).MALATHION FINAL

Page 32 of 74 Siglin, J.C., K.A. Voss, and P.J. Becci. (1985b) . A teratology study with AC 6,601 in rabbits. Laboratory report Project Number: 8171, subm itted to American Cyanamid Company, New Jersey. Unpublished study conducted by Food and Drug Research Labora tories, Inc., New York. ( MRID 00152569).

Stauter, R.W. (1994). 18-month oral (dietary ) oncogenicity study in mice. Laboratory report Project Number: 668-001, submitted to Cheminova Agro A/S, Denmark. Unpublished study conducted by International Research and Deve lopment Corporation, Michigan. (MRID 43407201).

Taxvig, C., Hadrup, N., Boberg, J., Axelstad, M., Bossi, R., Bonefeld-Jorgensen, E.C., Vinggaard, A.M. (2013). In vitro – in vivo correlations for endocrine activity of a mixture of currently used pesticides. Toxicol Appl Pharmacol . 272: 757–766.

Tegeris Laboratories, Inc. (1987). One-year oral toxic ity study in purebred beagle dogs with AC 6,60l. Laboratory report Project Number: 85010, s ubmitted to American Cyanamid Company. Unpublished study conducted by Tegeris Laboratories, In c., Maryland. (MRID 40188501).

USEPA (2006). Reregistration Eligibility Deci sion (RED) for Malathion. Office of Chemical Safety and Pollution Prevention, Office of Pestic ide Programs, U.S. Environmental Protection Agency, Washington, D.C. EPA 738-R-06-030, July, 2006.

USEPA (2010). Malathion — Report of the Endo crine Disruptor Review Team; Test Order# EDSP-057701-44. September 23, 2010. Office of Chem ical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

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

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

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

Uzun F., Kalender, S., Durak, D., Demir, F. and Kalender, Y. (2009). Malathion-induced testicular toxicity in male rats and th e protective role of vitamins C and E. Food Chem. Toxicol

. 47: 1903–1908.

Wagner, H. (2011). Malathion: H295R ster oidogenesis assay. U npublished study conducted by CeeTox, Inc, Kalamazoo, MI. Laborator y Report No.: 9148V-100372STER, Nov 3, 2011. (MRID 48617509).MALATHION FINAL

Page 33 of 74 Wilga, P.C. (2011). Malathion: human recombinant aromatase assay. Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9148V-100372AROM, November 3, 2011. (MRID 48617504).

Willoughby, J.A. (2011). Malathion: Androgen Receptor Binding (Rat Prostate Cytosol). Unpublished study conducted by CeeTox, Inc., Ka lamazoo, MI. Laboratory Study Number: 9148V-100372ARB, November 4, 2011. (MRID 48617503).

Willoughby, J.A. (2011). Malathion: Estrogen re ceptor transcriptional activation (human cell line (HeLa-9903)). Unpublished st udy conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9148V-100372ERTA, Nove mber 4, 2011. (MRID 48617505).MALATHION FINAL

Page 34 of 74 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100)

The 21-day assay (MRID 48617501) of malathi on on amphibian metamorphosis of South African clawed frogs ( Xenopus laevis ) was conducted under fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) St age 51 (80 per control or treatment group, 20/replicate) were exposed to malathion (96.0% pur ity) at nominal concentr ations of 0 (negative control), 0.040, 0.13, and 0.40 mg a.i./L; mean-meas ured concentrations were <0.0250 (<LOQ; control), 0.028, 0.11, and 0.32 mg a.i. /L. The test system was maintained at 20.2 to 23.5°C and a pH of 8.0 to 8.2.

There were no treatment-related effects on surv ival of tadpoles during the 21-day test. From Days 0 through 7, survival was 98.8, 98.8, 100, and 100% in the negative control and 0.028, 0.11, and 0.32 mg a.i./L treatment groups, respectiv ely. From Day 8 through test termination, survival was 100, 100, 100, and 98.3% in the negative control and 0.028, 0.11, and 0.32 mg a.i./L treatment groups, respectivel y. At test termination, tail cu rvature was observed in 92% of tadpoles in the negative contro l and 86, 93, and 95% of tadpoles in the low, intermediate, and high concentration groups, respec tively. The incidence of tail curvature did not appear to be
treatment-related.

There were no significant differenc es in developmental endpoints ( i.e. , NF developmental stage, hind limb length, normalized hind leg length) in any treatment group when compared to the negative control. No asynchronous developm ent was observed. There were no diagnostic abnormalities observed by histopathological examination of the thyroids of tadpoles in any treatment group when compared to the negative c ontrol at the terminati on of the 21-day test.

Day 7 snout-vent length (SVL) and body weight were significantly increased (p<0.05) by 10.7 and 27.9%, respectively, in the low-concentra tion group relative to the negative control. However, there were no significant effects on grow th parameters in the intermediate and high concentration groups compared to the negative control. Furthermore, SVL and body weight in the treated groups were comparable to negativ e controls on Day 21 (effects on body weight were observed at 0.2 and 0.6 mg a.i./L in the rangefi nder suggesting the high concentration in this study was sufficient).

Late-stage (NF>60) tadpoles we re excluded from analyses with respect to body weight, SVL, and HLL (including normalized HLL) by the revi ewer; these included 4 (6.8%), 1 (1.7%), 4 (6.7%) and 1 (1.7%) tadpoles in the negative control and low, in termediate, and high treatment groups, respectivelyMALATHION FINAL

Page 35 of 74 Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48617503), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct saturation and competitive binding experiments for malathion (96.0%, Batch# 9010501) . The saturation binding experiment was
conducted to demonstrate that the AR in the ra t prostate cytosol was present in reasonable numbers and was functioning with appropriate affi nity for the radiolabeled reference androgen (R1881). The competitive binding assay was co nducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the pr esence of increasing concen trations of malathion (logarithmic increase from 10

− 10 to 10

− 3 M). Dimethyl sulfoxide (DMSO) was used as a vehicle at a final assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The mean dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM, and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was used in the assay. Only one of the three K d values was within the range reported in the EPA validati on program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for individual runs was acceptable (R 2 = 0.880-0.986), there was a large variation in K d estimates

among runs.

In the competitive binding experiment, malathion was classified as equivocal in the first two runs
and a non-binder in the third run. Malathion re duced the mean specific binding of the ligand, [ 3 H]-R1881 to 51.6% and 50.4%, at 10 -3 M in the first two runs, re spectively. The results from these two runs were considered equivocal as the average specific binding at the lowest portion of the curve was between 50-75%. Precipitation was visually observed at the highest concentration tested (10

− 3 M) in Run 3; therefore, the highest con centration used for data evaluation in Run 3

was 10

− 4 M. The specific binding in the presence of malathion was consistently >98% at all
concentrations ≤ 10 -4 M in all three runs.

The estimated log IC 50 s for R1881 and the weak positive control (dexamethasone) were

− 9.3 and

− 4.4 M, respectively. The mean RBA for the w eak positive control was 0.0022%. Confidence in these numbers is high due to the small variation. However, it is noted that the variations between Run 1 compared to Runs 2 and 3 are likel y due to the concentration differences used for
R1881 and dexamethasone. The curve for dexameth asone was not fully defined in the first run
and therefore the ca lculation for the IC 50 may be affected. The range of concentrations for R1881 in the first run fully defined the curve, bu t the concentration range was off by more than an order of magnitude.MALATHION FINAL

Page 36 of 74 The performance criteria were generally met; however, the bottom percent binding for
dexamethasone was not achieved in the first r un. The concentrations prepared for R1881 and dexamethasone were lower than the targeted (guide line) levels because of an error in the stock preparation. Therefore, non- specific binding was not evaluated with 100X of cold (nonradiolabeled) R1881 but with 30X cold R1881, a nd the resulting curve for dexamethasone fit only two of the three criteria (H ill slope and Top plateau). All performance criteria were met for
the other two runs.

Based on the results from the three runs, malathi on is classified as Equivocal in the Androgen Receptor Binding Assay.

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48617504) , malathion (96% a.i., Batch #: 9010501) in dimethyl sulfoxide (DMSO; ≤ 1%) was incubated with human recombinant
aromatase and tritiate d androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) for 15 minutes at 37°C to assess the po tential of malathion to inhib it aromatase activity. Malathion was tested at logarithmic concentrations from 10 –10 M to 10

− 3 M (Run 1) and from 10 –9 M to
10

− 3.5 M (Runs 2 and 3), due to precipitation observed at 10

− 3 M in Run 1.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each con centration of chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted,
and each run included a full activ ity control, a background activity control, a positive control series (10

− 10 M to 10

− 5 M) using a known inhibitor (4- hydroxyandrostenedione; 4-OH ASDN), and the test chemical series (10

− 10 M to 10

− 3 M) with 3 repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.170 to 0.237 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a mean and standard deviation of 0.198 ± 0.024 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.55% to 1.8% and averaged 0.75% of the full activ ity controls. The response of the full activity controls and background controls was acceptable for each run.

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, hill slope, log IC 50 , and coefficient of varia tion for replicates of each concentration within runs. Also, the coefficients of variations (%CVs) fo r replicates of each
concentration of malath ion within a run were within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 averaged –7.16 M, and the slope was –1.01.MALATHION FINAL

Page 37 of 74 For malathion, aromatase activity av eraged 0.170 ± 0.066 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration of 10 –9 M (the lowest concentra tions tested from Run 1, 10 –9 and 10 –10 , wereexcluded from analysis because of low % control values) and 0.133 ± 0.010 nmol·mgprotein -1 ·min

− 1 at the highest successfully tested concentration of 10

− 3.5 M (highest nonprecipitating dose). Precipitati on was observed in Run 1 at 10

− 3 M. The data for malathion were not suitable for modeling. The mean aromatase activity of 10 –3.5 M malathion was 64.6% of control activity, and the be st fit mean inhibition response curve was a straight line (essentially no inhibition was observed at other doses). The re sponse for malathion was considered equivocal since the average lowest porti on of the response curve across runs was between 50 and 75% activity.

Based on the data from the average response curv e, malathion is classified as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

Requirement satisfied by OSRI Chen et al ., 2002; MRID 48160705); see Appendix 2.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48617505), hER α -HeLa-9903
cells cultured in vitro were exposed to malathion (96.0% a.i., Batch # 9010501) at logarithmically increasing concentrations from 10 -11 to 10 -4 M in 0.1% DMSO for 24±2 hours. The experiments were performed using 96-well plates and each malathion concentration was tested in 6 wells/plate. Cells were exposed to the test agent for 24± 2 hours to induce reporter (luciferase) gene products. Lu ciferase expression in response to activation of the estrogen receptor was measured upon addition of a lucifera se substrate and detection with a luminometer.

Malathion was tested up to the limit of solubility, which was 10 -5 M. The mean RPC Max for
malathion was 3.4% for the first run and 3.7% for the second run, and the associated PC Max was 10 -7 M for both runs.

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

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, malathion responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to
reach a maximum of 6.5-37.4% PC. Malathion wa s only able to reach a maximum of 3.4-3.7% PC when tested up to the highest concentrati on possible based on cytotoxicity. Because theMALATHION FINAL

Page 38 of 74 RPC Max < PC 10 in both assay runs, malathion was cons idered negative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (MRID 48617506) of malathion technical with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 females in each group; ca. 5 months old) were exposed to malathion (96.0% purity) at nominal concentrat ions of 0 (control), 0.10, 0.32 and 1.0 mg a.i./L. The mean-measured concentrations, calculate d by the study authors and excluding Day 21 recoveries, were <LOQ (0.0400), 0.08, 0.25 and 0.82 mg a.i/L, with %CVs of 4.4, 8.7, and 6.1%, respectively. The study authors excluded Day 21 sa mples due to suspected handling or analysis error, however, no supporting data were provid ed. Therefore, the revi ewer calculated meanmeasured concentrations, including Day 21 samples, of <LOQ (0.0400), 0.08, 0.22, and 0.69 mg a.i/L, with %CV’s of 7.6, 26, and 29%, respectiv ely. EPA requires a CV for measured test concentrations of <20% over 21 days. The test system was maintained at 24.7 to 25.3oC and a pH of 7.9 to 8.2. Unless otherwise indicated, all eff ects are reported based on comparison to the negative (clean wa ter) control.

Male and female survival was 100% in the ne gative control and in the lowest treatment concentration (0.08 mg a.i/L). There was a singl e male and female mortality each at the mid
treatment concentration and a si ngle female mortality at th e high treatment concentration. Observed clinical signs of toxicity included one fish at the mid concentration exhibiting a crooked spine, which was not attributed to trea tment. However, in the high concentration group, 8 of 24 fish (33%) exhibited clin ical signs of toxicity that in cluded bruising on the body, loss of color or no color in the tail, erratic swimming, lethargy, and crooked spine. These effects were observed as early as the third da y of exposure and increased in in cidence as the test continued.
Excess feed was noted in the test chamber on Da ys 7 through 9 for the mid concentration and on Days 5 through 16 for the high concentration. Male body weight was decreased (not significant) by 19.8 and 17.3% in the mid and high concentrati on groups, respectively; there was no effects on female body weight and length or male length.

Spawning and fecundity in contro ls, respectively, were every 3 days and 26.3 eggs/female/day;
fertilization success in contro ls was 99.3%. There was a statis tically significant (p<0.05) reduction in fecundity ( ⇘ 48%) at the high concentration compared to the negative control. There were no significant effects of malathion treatment on, female and male vitellogenin (VTG), fertilization success, or male gonado-somatic index (GSI). Female GSI exhibited a concentration-dependent monotonic increasing tr end, with an increase of 21% at the high concentration compared to the negative cont rol (p<0.05; Jonckheere-Te rpstra). At the high concentration, male tubercle scor es were significantly reduced co mpared to the negative controlMALATHION FINAL

Page 39 of 74 (10.5 vs. 25.5 in control) (p<0.01; Jonckheere-Terp stra). The study author’s also reported a statistically significant decrease in the fatpad score was reduced (2.3 versus 4.0 for control; p<0.05) compared to the control.

Malathion significantly impacted the multiple end points involved with the development of the testes at the highest treatment le vel but did not appear to affect ovarian development to the same degree, with the exceptio n of increased oocyte atresia. In the high-concentration group males, there were treatment-related effects (p<0.05; Jonc kheere-Terpstra trend te st) including increased spermatogonia, altered proportion of spermatocyte s and spermatids, as well as decreases in gonad size, Sertoli cell spermatocyst number/tubule and spermatocyst size, germinal epithelium thickness, tubule diameter, efferent duct diameter , spermatozoa, and milt. Although these effects were not statistically analyzed by the reviewer, the re viewer’s observations were validated by the study authors’ sta tistical analysis.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48617507) screeni ng for androgenic activity, malathion (96.0% a.i., batch/lot 9010501) in corn oil was administered daily via oral gavage to groups of eight 60- day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day (limit dose). An androgenic positive c ontrol group consisted of eight castrated rats exposed to 0.4 mg/kg/day of test osterone propionate (TP) by s ubcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, malathion (96.0% a.i., batch/lot 9010501) in corn oil was administered daily via oral gavage to groups of eight 60-d ay old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive c ontrol group consisted of eight castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT) . TP alone was used as the an ti-androgenic negative control.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were dosed for 10 consecutive days a nd terminated approximately 24 hours after the final dose. At necropsy, the fi ve androgen-dependent tissues, liv er, and kidneys were collected and weighed. Brain and red bl ood cell (RBC) samples were also collected from rats in the androgen agonist assay for determinati on of cholinesterase (ChE) activity.
There were no treatment-related clin ical signs of toxicity or gross pathological findings in either

assay.

In the androgen agonist assay, there were no tr eatment-related effects of malathion on accessory sex organ weights. At 1000 mg/kg/day, absolu te and relative (to body weight) liver weights were increased (p ≤ 0.01) by 29% and 32%, respectively, comp ared to controls; absolute andMALATHION FINAL

Page 40 of 74 relative kidney weights were also increased (p ≤ 0.05) in this group ( ↑ 18% and ↑ 16%). Relative liver and kidney weights were increased (p ≤ 0.05) at 300 mg/kg/day ( ↑ 9% and ↑ 7%, respectively). At 1000 mg/kg/day, br ain ChE activity was inhibited (p ≤ 0.01) by 39%, while RBC ChE activity was inhibited (p ≤ 0.01) by 22-88% at 100 mg/kg/day and above.

Percent CVs for the vehicle control and 1000 mg /kg/day group of the androgen agonist assay were compared to the performan ce criteria in the Guidelines. The %CV values were less than the maximum permissible values for all accesso ry sex organs, with the exception of ventral prostate (CV = 50.5% for control; maximum %CV = 45) and seminal vesicles (CV = 41.6% for control and 42.8% for 1000 mg/kg/day group; co mpared to maximum %CV = 40). The study author noted that the same prosec tor dissected these organs for ev ery rat in the androgen agonist

assay.

In the anti-androgen assay, there were no treatment -related effects for ma lathion on the weights of accessory sex organs. At 1000 mg/kg/day, ab solute and relative (to body weight) liver weights were increased (p ≤ 0.01) by 21% and 27%, respectively ; relative kidney weights were also increased by 13% (p ≤ 0.01) at this dose. Absolute and re lative liver weight s were increased (p ≤ 0.05) by 14% and 10%, respective ly, at 100 mg/kg/day. The pe rformance criteria indicated that this assay was performing as expected.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Malathion was negative for androgenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48685701), 20 Crl:CD(SD) Sprague Dawley rats/dose group were treated daily via oral gavage with malathion (95.8-96.0% purity, Lot #s 9010501 or D2014- OSJ-MLT-01-S) in corn oil at doses of 0, 250, 500 or 1000/750 mg/kg/day from post-natal day (PND) 22 to 42. The 1000 mg/kg/day dose leve l was decreased to 750 mg/kg/day on PND 24 due to mortality and clinical observations at th e high-dose. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age a nd weight on the day of attainment of VO was recorded. After sacrifice on PND 42, blood wa s collected for clinical chemistry analyses, including total thyroxine (T 4 ) and thyroid stimulating hormone (TSH), which were determined using radioimmunoassays. Kidney, liver, adrenal gland, thyroid, pituitar y, uterus, and ovary weights were recorded, and microscopic examina tions were performed on the thyroid, ovaries, uterus, and kidneys. In addition to the main study groups, additional groups of eight female
rats/dose were used in a cohort that examined blood and brain cholinesterase (ChE) activity at
2 hours after the last dose on PND 42.MALATHION FINAL

Page 41 of 74 Two animals in the high-dose group from the main study were found dead on PND 25 and 41, and four animals in the high-dose group from the cholinesterase cohort we re found dead between
PNDs 23-25. All other rats survived until schedule d sacrifice. Treatment-related clinical signs observed in the high-dose group included e ffects on gait (decreased motor activity and prostration), irregular respiration (dypsnea), ne rvous system (whole body tremors, tremors in the head, slight excess salivation) , and general toxicity (urine stained abdominal fur, chromodacryorrhea, laceration on the neck and s cab on the neck). In the low- and mid-dose groups, sporadic clinical signs included slight excess salivation, urine stained abdominal fur, and/or chromodacryorrhea.

There were no statistically signifi cant effects on age or weight at VO, mean age at first vaginal estrous, mean cycle length, number of females cy cling, number of females regularly cycling, or cycle status at necropsy. However, there were apparent dose-related trends toward decreased percentages in the number of anim als cycling and the number of an imals regularly cycling in the
malathion-treated rats.

Serum T 4 levels in the low-, mid- and high-dose groups were increased (p ≤ 0.01) by 19, 16 (NS), and 25%, respectively, compared to the control. However, serum TSH levels were not affected by treatment, and there were no ch anges in thyroid weight or hist ology. Therefore, the biological significance of the changes in T 4 levels is unclear.

Compared to control, mean liver weight in the high-dose group was increased (p ≤ 0.01) by 16%, and the relative liver weight (to body) was increased (p ≤ 0.01) by 6% and 14% in the mid- and high-dose groups, respectively. In the mid- and high-dose groups, mean kidney weights were increased (p ≤ 0.01) by 7% and 12%, respectively, and re lative kidney weights were increased (p ≤ 0.01) by 7% and 11%, respectively. Given that the weight change s in liver and kidneys were minor in magnitude and there were no adverse hi stopathological findings in the liver or kidneys, these weight increases are considered to be adaptive in nature.

In addition, both absolute and relative pituitary weights were decreased (p ≤ 0.01, 29 to 31%) in the mid- and high-dose groups, but these changes were not associated with any histological changes in the pituitary glands. There were no other significant eff ects on organ weights, including the uterus and ovaries, and there were no microscopic ch anges in any of the examined

organs.

There were a number of treatment -related changes in clinical chemistry parameters that were concomitant with the changes in liver weight; these included increases in GGT (NS, 94 to 128%), albumin (p ≤ 0.01 or 0.05; 4 to 8%), and globulin (p ≤ 0.01 or 0.05; 5 and 11%), and dosedependent decreases in the A/G ratio (p ≤ 0.01; 7 to 18%). In addition, there were decreases (p ≤ 0.01) in total bilirubin (17 to 31%) and triglyceride (40 and 53 %). Finally, other treatmentMALATHION FINAL

Page 42 of 74 related changes were also noted in cholesterol, creatinine, phosphorus, calcium, potassium, and urea nitrogen; however, the releva nce of these changes is unclear as similar changes were not observed in the male pubertal rat assay.

In the ChE cohort (250, 500 and 750 mg/kg/day), ChE activity was dose-dependently inhibited (p ≤ 0.01 or 0.05) in the brain by 9, 24, and 42%, respectively, and in the RBC by 74, 89, and 97%, respectively. The high dose (750 mg/kg/da y) was considered adequate based on the mortality, clinical signs a nd cholinesterase inhibition observed at 1000 mg/kg/day.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48685701), 20 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral gavage with ma lathion technical (95.8- 96.0% purity, Lot #s 9010501 or D2014-OSJ-MLT-01-S) in corn oil at doses of 0, 250, 500 or 1000/750 mg/kg/day from postnatal day (PND) 23 to 53. Due to mortalities and clinical observations of toxicity, the initial high-dose level (1000 mg/kg/day) was decreased to 750 mg/kg/day by PND 24. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and weight on the day of attainment was recorded. Following sacr ifice on PND 53, blood was collected for clinical chemistry analyses, including total thyroxine (T 4 ), testosterone, and thyroid stimulating hormone (TSH). Testosterone, T 4 , and TSH were analyzed using radioimmunoassay kits. Kidney, liver, adrenals, pituitary, thyroid, and urogenital or gan weights were reco rded, and microscopic examinations were performed on the thyroid, kidne y, testis and epididymis. In addition to the main study groups, additional groups of 8 male rats/dose were used in a cohort th at examined
blood and brain cholinesterase (ChE) activity at 2 hours af ter the last dose on PND 53.

In the main study, 14 rats from the high-dose gr oup were found dead during the study, from PND 25 (3 doses) to PND 53 (31 doses), and one rat was sacrificed in extremis due to adverse clinical
observations on PND 32. Two animals in the mid-dose group (500 mg/kg/day) were also found
dead on PNDs 45 and 50. In addition, 6 rats from the high-dose group in the ChE cohort were found dead between PNDs 24 and 49 (2 to 27 doses) . All other rats survived until scheduled sacrifice. In the mid- and high-dose groups, th e observed clinical signs included effects on gait ( e.g. , ataxia, lost righting reflex, and decreased motor activity), ir regular respiration (dyspnea), nervous system ( e.g. , whole body tremors, tremors in the head, extreme excess salivation, and lacrimation), and general toxicity ( e.g. , urine-stained abdominal fur, ungroomed coat, chromodacryorrhea, dehydration, and chromorhi norrhea). In the low-dose group, sporadic clinical signs included slight excess salivation, ungroomed coat , and urine-stained abdominal fur.

There were no treatment-related effects on fina l body weight, overall body weight gain, and body weight at PPS. Although the first day of PPS was delayed (p ≤ 0.01) by 1.4 to 1.6 days in the lowand mid-dose groups, there was no effect on fi rst day of PPS in the high-dose group.MALATHION FINAL

Page 43 of 74 Compared to the control group, absolu te liver weights were increased (p ≤ 0.05) by 21% in the mid- and high-dose groups, and relative (to body) liver weights were increased (p ≤ 0.01) by 10, 22, and 26% in the low-, mid- and high-dose groups , respectively. Absolute kidney weight in the mid-dose group was increased (p ≤ 0.01) by 12%, and relative kidney weights were increased (p ≤ 0.01) by 6, 14, and 16% in the low-, mid-, and high-dose groups, respectiv ely. The liver and kidney weight effects are treatment related and ar e likely an adaptive response to malathion. The mean LABC weights for the mid- and high-dose groups were also decreased (p ≤ 0.01) by 14% and 17%, respectively, but there were no other ch anges in weights of accessory sex tissues. There were no other significan t effects on organ weights.

Serum T 4 concentrations in the low-, mid-, and high-dose groups were increased (p ≤ 0.01) by 41, 35, and 32%, respectively, but these cha nges were not dose-dependent, the T 4 levels were within the range of historical control values, and ther e were no related changes in TSH levels. In addition, there were no changes in thyroid weight or any microscopi c findings in the thyroids of malathion-treated rats. Therefore, the bi ological relevance of the changes in T 4 levels is unclear. However, there were clear, dose-dependent decreases (p ≤ 0.01) in testosterone levels of 60, 78, and 90% in the low-, mid-, and high-dose groups, respectively, which may correlate with the observed delay in PPS and the reductions in weight of the LABC.

In all malathion-treated animals, albumin was decreased (p ≤ 0.01) by 5-6%, globulin was increased (p ≤ 0.01) by 8-17%, and the A/G ratio was decreased (p ≤ 0.01) by 13-19%. There were also dose-dependent decreases (p ≤ 0.01) in total bilirubi n levels of 16-36% and in triglycerides of 33-68%, and cholesterol levels were increased (p ≤ 0.05) by 11 and 19% in the mid- and highdose groups. These changes were concurrent with the increases in liver weights and likely reflect adaptive changes in liver metabolism. Although significant changes were observed in other clinical chemistry parameters, the values we re generally within the historical control ranges
and are not considered to be biologically relevant.

In the ChE cohort, the ChE levels on PND 53 in the low- and mid-dose groups were decreased (p ≤ 0.01) dose-dependently by 79 and 88% in red blood cells and by 17 and 35% in brain tissue.
ChE levels were not eval uated in the high-dose group.

Based on the ChE data, and the mortality and c linical signs, both the mi d- and high-doses (500 and 750 mg/kg/day) of malathion were clearly ex cessive for immature ma le rats; however, the low-dose level was adequate.

Effects on androgen-related endpoints included dose -dependent decreases in testosterone in all treated animals and dose-dependent decreases in LABC weights in mid- and high-dose animals.
Concentrations of T 4 were increased to a similar (non-dos e dependent) extent at all doses.MALATHION FINAL

Page 44 of 74 Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48617509), H295R cells cultured in vitro in 24-well plates were incubated with malathion (96% purit y, Lot# 9010501) at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1%.

Testosterone and estradiol le vels were measured using HP LC/MS-MS. Four independent experiments were performed, although only Runs 2, 3 and 4 were reported. The media from Run 1 was not analyzed for testoster one and estradiol con centrations because one of the forskolin concentrations on the Quality Control (QC) plate was different from the concentration specified in the Test Guideline. A QC plate was run conc urrently with each indepe ndent run of the test chemical plate to demonstrate that the assay resp onded properly to positive control agents at two concentrations; positive contro ls included the known inhibito r (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate induction an d inhibition with positive contro ls. It was stated that all assays were performed at CeeTox by personnel th at have demonstrated proficiency performing this assay, however, proficienc y records were not provided.

The only significant changes noted in hormone production of malathion treated cells were a decrease in testosterone produc tion at 100 μM of malathion (p<0.05; 0.81-fold change) in Run 4
and an increase in estradiol production at 10 μM of malath ion in Run 2 (p<0.05; 1.12-fold change). The validity of the obser ved decrease in testosterone is questionable as precipitation of
malathion was observed at 100 μ M in Runs 2 and 3, and there was no other concentration dependent effects noted in test osterone production. Similarly for the observed increase in estradiol, the effect was observed at 10 μ M in only one of three runs, and no concentration dependent changes in estradio l production were observed.

Based on hormone response in at least three inde pendent runs, malathion treatment did not result in statistically significant and re producible alterations in testosterone or estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

In a uterotrophic assay (MRID 48617510) conducted to screen for potenti al estrogenic activity, malathion (96.0% a.i., Batch/lot 9010501) in corn o il was administered daily via oral gavage to groups of eight ovariectomized female Sprague Da wley rats at dose levels of 0 (vehicle), 100,MALATHION FINAL

Page 45 of 74 300, or 1000 (limit dose) mg/kg/day on post-nata l days (PND) 68-70. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 5 μ g/kg/day by subcutaneous (s.c.) injections. Body weights and food consumption were determined daily. All animals were terminated and necropsied on PND 71 approximate ly 24 hours after the final dose to determine wet and blotted uterine weights and liver weights. Brain and red blood cell (RBC) samples were
also collected from rats in the vehicle contro l and malathion treatment groups for determination of cholinesterase (ChE) activity.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any dose group.

Body weights in all malathion treatment groups were comparable to controls over the duration of the study. At 1000 mg/kg/day, the ra ts displayed a body weight loss (p ≤ 0.05) during Days 2-3 (

− 0.8 g treated vs. +2.9 g controls), resulting in a decrease in overall (Days 1-4) body weight gains (NS) of 35%. Additionally at this dose, daily mean food consumption was decreased (p ≤ 0.01) during Days 2-3 ( ↓ 10%) and Days 3-4 ( ↓ 8%), leading to decreased (p ≤ 0.01) average food consumption over the duration of the study ( ↓ 6%). Uterine weights in the malathion treatment groups were comparable to controls . Absolute and relativ e liver weights were increased (p ≤ 0.01) at 1000 mg/kg/day ( ↑ 24 and 25%); relative (to body) liver weights were also increased (p ≤ 0.05) at 300 mg/kg/day ( ↑ 10%). These increases were attributed to malathion treatment. Brain ChE activity was significantly inhibited (p ≤ 0.01) at 1000 mg/kg/day (16% inhibition), and RBC ChE activity was significantly inhibited (p ≤ 0.01) in rats at 100 mg/kg/day and above (20-69% inhibition).

In the positive control group, overall body weight gain was decreased (p ≤ 0.01; ↓ 85%); however, body weights in this group were comparable to the vehicle control over the duration of the study. Mean food consumption was increased (p ≤ 0.01) on Days 1-2 ( ↑ 10%) and decreased (p ≤ 0.01) on Days 2-3 ( ↓ 10%); average food consumption over the dur ation of the study was comparable to the control. Absolute wet and blotted uterine weights were increased (p ≤ 0.01) by 509% and 196%, respectively. The increased uterine we ights were in the expected range.

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

Page 46 of 74 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of malathi on with the endocrine system are summarized below. Given the breadth of toxicity data available on malathi on, 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.).

ER Binding

In a published non-Guideline study, Chen et al., (2002) tested malathion in an ER binding assay with receptor derived from rat uterine cytosol. Test concentrations ranged from 10 -12 to 10 -3 M. A log plot of competitor concentration versus response showed no inhibition of estradiol (E 2 ) binding to the receptor. The general procedures us ed were clearly described and were similar to the procedures specified in th e test Guideline, including th e concentration range tested.

Receptor Transactivation and Aromatase Assays

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of 200 chemicals (including malathion; purity 95-100%) to interact with two human ER subtypes (hER α and
hER β ) and one human androgen receptor (hAR) in tr ansactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was inve stigated. For detection of hER α or hER β activity, cells were transfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZ eoSV2AR, pIND-ARE, an d pRL-SV40. Flutolanil was tested over a concentration range from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 ; purity >97%) or 5 α -dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For meas urement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures along with the test compound, respectively. A gonistic activities were eval uated by the relative activity expressed as 20% relative effective concentration (REC 20 ), the concentration of the compound demonstrating 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20% relative i nhibitory concentration (RIC 20 ), the concentrat ion of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. Malathion did not show agonist or antagonist activity for ER α , ER β , or AR in this study.

In a published study by Kojima et al . (2005) , the response of an in vitro ER–related reporter gene assay was investigated af ter exposure to thirty two pest icides either alone or in combination. The source of the test compounds was not reported; however, malathion had a reported purity of >96%. The test compounds were prepared in DMSO at 10

− 2 M.MALATHION FINAL

Page 47 of 74 The E-CALUX Assay (Xenobiotic Detection Systems, Inc., NC, USA) uses a human ovarian carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a mouse mammary tumor virus (MMTV) promoter . Estrogen-responsive concatemers (repetitive DNA sequences), inserted directly above the pr omoter, confer estrogen responsiveness to the viral promoter and reporter gene. The transfected cells are made st able by an antibiotic resistance gene resulting in the cell lin e used in the assay (BG1Luc4E 2 ). Compounds that interact with estrogen-responsive DNA are identif ied by the luciferase gene re sponse and quantified in terms of Relative Light Units (RLU).

The BG1Luc4E 2 cells were grown in medium for f our days, then the assay medium was replaced, and cells were transferred (2 × 10 5 cells/mL) into 96-well pl ates and incubated at 37  ̊ C
with 5% CO 2 for 24 hours. The medium was removed, and the test compound or estrogen standard (E2) was added. After incubation at 37  ̊ C with 5% CO 2 for another 24-hour period, the medium was removed and the cells were lyse d. Luciferase activity was measured with a luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes,
the EC 10 (Effective Concentration at which 10% of the maximum response was observed) was
used. The data were not sufficien t to allow calculation of an EC 50 . Malathion was negative for ER activity in this test system.

In a study by Kjeldsen et al. (2013), the effects of malathion on estrogen receptor (ER) and androgen receptor (AR) function, as well as ar omatase activity were investigated. The androgenic, anti-androgenic, estrogenic, and an ti-estrogenic properties were determined from in

vitro tests including an ER transa ctivation assay usi ng MCF-7 derived MVLN cells and an AR transactivation assay using the Chinese hamste r ovary cells (CHO-K1). Effects on aromatase enzyme activity were assessed using the human choriocarcinoma JEG-3 cell line. Malathion was
assessed alone in various concentr ations within the range of 1×10 -10 to 1×10 -4 M. In the ER transactivation assay with MVLN cells, malathion weakly (113% of contro l) induced luciferase activity. Co-exposure with 25 pM E 2 (E 2 -EC 65; 65% of maximum response) did not alter the E2- induced response of malathion; malathion did not exert an ER-antagonistic effect. In the AR transactivation assay with CHO-K1 cells, ma lathion was not cytotoxic at the highest concentration tested (1×10 -5 M) and did not elicit an AR agoni st or antagonist response in this assay. In the aromatase activ ity assay with JEG-3 cells, malathion was cytotoxic at ≥ 1×10 -5 M. Malathion did not alter aromatase activity in this test system.

Thyroid hormone binding assay (Avian)

In a published study, ( Ishihara et al., 2003 ) the effects of malathion (98% purity) on binding of

125 I-labeled T 3 to Japanese quail ( Coturnix japonica ) thyroid transthyreti n (TTR) and the ligandbinding domain of the quail thyroid r eceptor (TR) were examined in an in vitro cell-free assay

. Blood was collected from quail, serum was sepa rated, and the quail TTR was isolated with a column chromatographic method. A recombinant plasmid contai ning the ligand binding domainMALATHION FINAL

Page 48 of 74 (LBD) of the quail TR was cloned into Escherichia coli to generate a GST-fusion protein containing the LBD of TR. The binding of T 3 to TTR and TR was measured by using
radiolabeled T 3 in the presence and absence of increasing concentrations (range not reported) of malathion. Malathion inhibited binding of T 3 to quail TTR with an IC 50 of 1,400±370 nM. The

IC 50 for T 3 in this system was 9.7±0.9 nM. Malathion had no effect on T 3 binding to the quail

TR.

21-Day (Subchronic) Dermal Toxici ty in Non-Rodents (Rabbit)

In a subchronic dermal toxicity study ( MRID 41054201, 1988 ), New Zealand white rabbits (6/sex/dose; except for control females, n=5) were administered malathion (94% purity) by dermal application (once a day for 6 hours, 5 da ys/week) for 21 days at concentrations of 0, 50, 300, or 1000 mg/kg/day. Hair was clipped from 10 to 15% of the body to allow application. ChE activity was determined prior to treatmen t (serum and RBC) and at study termination [serum, RBC, and brain regions ( i.e. , cerebrum and cerebellum)]. Liver, kidneys, ovaries, testes/epididymides, and adrena ls were weighed, and examined microscopically for the control and high-dose groups.

One low-dose male was sacrificed on Day 15 due to poor health (not treatment related) and one high-dose male died on Day 17 (treatment effect could not be ruled out) . Clinical signs, body weight, food consumption, hematology, clinical chem istry, organ weights (except for adrenals in males), macro- and microscopic lesions were co mparable between the tr eated groups and control (both sexes). Slight dermal irritation was observed, but it was not dose dependent and not considered treatment related. Adrenal weights were decreased to a similar extent across all doses in male rabbits [NS ( ↓ 24 to 33%), absolute and p<0.05 ( ↓ 33 to 35%), relative to body weight] with no dose response; no effect was noted for fe males. Statistical re-analysis demonstrated significance (with or without this value included), but the pertin ent treatment comparisons were not reported. The only consistent effects observed were on inhibition of ChE activity. Serum, RBC, and brain ChE activities were decr eased (p<0.01) at study termination ( ↓ 40.8 to 74.9%) in all high-dose males and females. ChE activities were unaffected in low-dose animals, and only RBC ( ↓ 25.6%, females) and cerebellum ( ↑ 45.4%, males) ChE activities were affected (p<0.01)
at the intermediate dose.

The NOAEL for systemic effects is 300 mg/kg/da y, and the LOAEL is 1000 mg/kg/day based on the death in the high-dose male. The NOAEL for ChE inhibition is 50 mg/kg/day, and the LOAEL is 300 mg/kg/day based on decreased cerebellum ChE activity in males and decreased RBC ChE activity in females.MALATHION FINAL

Page 49 of 74 13-Week (Subchronic) Inhalation Toxicity in Rodents (Rat)

In a 13-week subchronic inha lation study in SD rats ( MRID 43266601 ), groups of rats (15/sex/dose) were exposed by inhalation in whole body exposure chambers to malathion (96.4% purity) aerosols at nominal concentratio ns of 0 (room air), 0.24, 1.10 or 4.94 mg/L for 6 hours/day, 5 days/week for 13 weeks. The mean -measured concentrations were 0, 0.1, 0.45 or 2.01 mg/L. The mass median aerodynamic diameters of the malathion particles were 1.6 μM at 0.1 mg/L and 1.7 μM at 0.45 and 2.01 mg/L.

No mortalities occurred in the study. Clinical si gns of toxicity, consiste nt with decreased ChE activity, were observed in most an imals at the high dose and sporadically in a few animals at the intermediate and low doses. These signs, which occu rred in both males and females, consisted of red staining of the urogenital area s, excess salivation (starting at Week 4 to 5) and un-groomed oily fur (starting afte r Week 5). Body weight and food consumption were not affected by treatment. At 13 weeks, dose-related and treatment-related decreases in ChE activity were seen in both sexes, although large vari ability impacted stat istical analyses. In general, all ChE activities were decreased (p< 0.01) in high-dose animals by 17 to 70% (plasma ChE in males, ↓ 18%, was NS), and RBC ChE activity in males and females was decreased (p<0.05) at the intermediate dose by 22 and 27%, respectiv ely. For males, the NOAEL and LOAEL, respectively, are 0.45 mg/L and 2.01 mg/L for pl asma and brain ChE inhibition and 0.1 mg/L and 0.45 mg/L for RBC ChE inhibition. For fema les, the LOAEL for plasma and RBC ChE inhibition is 0.1 mg/L and a NOAEL is not determined. Th e NOAEL and LOAEL for brain inhibition are 0.45 mg/L and 2.01 mg/L.

Absolute and/or relative liver ( ↑ 14 to 20%), kidney ( ↑ 9 to 12%), and lung ( ↑ 8 to 9%) weights were increased (p<0.05 or 0.01) in the high-dose ma les and females. Non-significant increases in liver and kidney weights were occasionally observed at the intermediate dose. No treatmentrelated effects on organ weight s or histopathology were obser ved in any endocrine-related tissues. The only treatment-related histopathological lesions noted in the study were lesions in the nasal cavity and larynx observe d in nearly all animals (10 to 15/15 at the low dose and 14 to
15/15 at the intermediate and hi gh doses). There was no evidence of neoplastic lesions in the nasal cavity or larynx in any of the treated or control rats. For overall inhibition of ChE activity in this study, a NOAEL is not determined for i nhibition of either plasma or RBC ChE activity ( i.e. , <0.1 mg/L, LDT); the LOAEL is 0.1 mg/L . For brain ChE activity, the NOAEL is 0.45 mg/L and the LOAEL is 2.01 mg/L. For system ic effects, a NOAEL is not determined in either males or females (<0.1 mg/L). The LOAE L is 0.1 mg/L based on microscopic lesions of the nasal cavity and larynx in nearly all animals. Since similar microscopic lesions of the nasal cavity were also observed in long-term oral stud ies in rats with both malathion and malaoxon, it
is not clear whether these lesions are local or systemic in nature.MALATHION FINAL

Page 50 of 74 Prenatal Development in Rodents (Rat)

In a developmental toxicity study ( MRID 41160901 ), malathion (94% purity) was administered to presumed-pregnant female SD rats (25/dose) by oral gavage at concentr ations of 0 (corn oil), 200, 400, and 800 mg/kg/day from gestation days (GD) 6 to 15. On GD 20, the dams were euthanized and subjected to gross necropsy. All fetuses were weighed, sexed and examined for gross external alterations. One-half of the fetuse s in each litter were examined for visceral soft tissue alterations using a varia tion of Wilson’s sectioning technique. The remaining one-half of
the fetuses in each litter were ex amined for skeletal alterations.

There were no mortalities; clin ical signs of toxicity observe d in the high-dose only included
urine-stained abdominal fur in 5/25 dams and chromodacryorrhea/chromorhinorrhea in one dam. At the high dose, mean maternal body weight gains were decreased (p<0.05 or 0.01) by 26, 14, and 19% for GD 6 to 9, 9 to 12, and 6 to 12, resp ectively, and increased by 18% (p<0.01) for GD 16 to 20. Food consumption in the high-dose group was decreased (p<0.05 or 0.01) by 8 and 13% for GD 6 to 12 and 9 to 12, and increased by 5% (p<0.05 or 0.01) for GD 16 to 20. There
were no treatment-related effects of treatment on corpora lutea, impl antations, litter size, live/dead fetuses, resorptions, or live fetal body weights. There were also no treatment-related external, visceral, or skeletal malformations or variations.

The maternal NOAEL is the intermediate dose (4 00 mg/kg/day), and the maternal LOAEL is the high dose (800 mg/kg/day) based on decreased body weight gain and food consumption. The developmental NOAEL is 800 mg/kg/day (HDT ), and a developmental LOAEL is not
determined.

In a published non-Guideline study examini ng developmental toxicity in rats ( Prabhakaran and Devi, 1993 ), confirmed pregnant SD rat dams (12/dose) were administered malathion (98% purity ) by gavage at 0 or 500 mg/kg/day in p eanut oil on GD 6, 10, and 14. Two treatment groups were fed diets with either 16% (control) or 5% (protein deficient) casein. Dams were sacrificed on GD 20, and reproductive and de velopmental parameters were examined.

The number of implantations/dam and the number of live fetuses/litter were decreased (p<0.05)
similar to each other in the cont rol and low-protein diet groups (8 and 18%, respectively) with
malathion treatment. In rats fed the control diet s plus malathion, significa nt (p<0.0001) decreases were seen in fetal weights, crown-to-rump lengths , tail lengths, umbilical cord lengths, placental weights, and amniotic fluid wei ghts. In rats fed the protein- deficient diets plus malathion, significant (p<0.0001) decreases were seen only the tail lengths and placen tal and amniotic fluid weights. No skeletal anomalies were seen excep t for poor ossification in the protein-deficient dams, which were exacerbated by malathion expo sure. ChE activity in the brain was decreased by 24 and 28%, respectively, in the control and protein-deficient an imals treated with malathion. The fetal effects showed that protein defici ency intensified malathion-induced toxicity.MALATHION FINAL

Page 51 of 74 Prenatal Development in Non-Rodents (Rabbit)

In a developmental toxicity study ( MRID 00152569 and 45626801 ), malathion (92.4% purity) was administered by oral gavage at a dos e volume of 1.5 mL/kg to New Zealand White (DLI:NZW) rabbits (20/treatment) at doses of 0 (corn oil), 25, 50, or 100 mg/kg/day from GD 6 to 18. On GD 29 of gestation, the does were eu thanized and subjected to complete gross necropsy. All fetuses were weighed, sexed and examined for external abnormalities, visceral soft tissue abnormalities and skeletal abnormalities by standard procedures.

No mortalities in this study were attributed to malathion treatment. Four deaths occurred in the low-dose group and three deaths at the intermedia te dose; causes of death were not reported. Two deaths in high-dose animals were attrib uted to pulmonary intubation during dosing. Treatment-related clinical signs observed in rabbits administ ered the high dose consisted of anorexia and soft stools. Although no treatmen t-related effects on body weight were noted, decreased (p<0.05) body weight gains of 116% in the mid- and high-dose animals were noted during GD 6 to 18.

The only treatment-related effect noted for repr oductive and gestation in dices was an increase (significance not reported) in th e number of resorptions per doe (mean percent resorptions) in the mid- and high-dose does ( ↑ 87 and 82%, respectively). No tr eatment-related abortions were observed. Live fetal weights were not affected by malathion treatment. No treatment-related skeletal retardations were noted during the fe tal examinations, and there were no treatmentrelated external, visceral, or skel etal variations or malformations.

The maternal NOAEL is 25 mg/kg/day (low dose) , and the maternal LOAEL is 50 mg/kg/day (intermediate dose) based on decreased doe body weight gain during GD 6 to 18, the treatment period during gestation. The developmental NO AEL is 25 mg/kg/day (low dose), and the LOAEL is 50 mg/kg/day based on an increase in the mean number of resorptions at the intermediate and high doses.

In a published non-Guideline study examining developmental toxicity in rabbits ( Machin and McBride, 1989 ), 12 New Zealand white rabbit does were confirmed pregnant and administered malathion (70% purity with 30% calcium carbonate ) by gavage at doses of 0 (n=5) or 143 mg/kg/day (n=7; 100 mg/kg/day dose of malath ion) in corn oil from GD 7 through 12. Does were sacrificed on GD 27, and re productive and developmental parameters were examined. No
treatment-related effects were seen on the live fetuses/litter, litters with resorptions, fetal sex ratio or fetal weight, fetal length (crown-rump) or fetal eye bulge.

Developmental Neurotoxicity in Rodents (Rat)

In a developmental neurotoxicity study ( MRID 45646401 ), malathion (96% purity) was administered to 24 parental female Crl:CD ® BR SD rats per dose by gava ge at doses of 0, 5.0, 50,MALATHION FINAL

Page 52 of 74 or 150 mg/kg/day in corn oil from GD 6 thr ough PND 10, and to the offspring from PND 11 to 21 inclusive. A Functional Observational Battery (FOB) was performed on 10 dams/dose on GD 12 and 18 and LD 4 and 10. Offspring were ev aluated as follows: ag e-appropriate FOBs were conducted on PND 4, 11, 21, 35, 45, and 60, automated motor activity on PND 13, 17, 22, and 60; assessment of auditory startle respons e on PND 23/24 and 60/61, assessment of learning and memory (Morris Water Maze) at PND 23/24 and 61/62 (separate groups), brain weights on PND 11, 21, and 65, and brain histopathology and morphometries on PND 21 and 65. Pup physical development was assessed by body weight . Sexual maturation of females was assessed by age at VO, and sexual maturation of male s was assessed by age at completion of PPS.

There were no treatment-related maternal deaths before scheduled termination. Clinical signs were limited to transient post-dosing salivation (5/24 control, 4/24 and 3/24 at the low and intermediate doses, and 20/24 at the high dose). Th ere were no other treatm ent-related effects on cholinergic signs, and there were no effects on maternal body weight, food consumption, or reproductive indices. The maternal LOAEL for ma lathion in rats is 150 mg/kg/day based on an increased incidence of post-dosing saliva tion, and the maternal NOAEL is 50 mg/kg/day.

There were no apparent treatment-related clinic al signs noted in offspring prior to direct treatment starting on PND 11. In addition, no ef fects on pup body weights were observed pre- (through PND 21) or post-weaning (through PND 63), and no effects on age at VO or PPS. In the mid- and high-dose pups, there was an increased incidence of slightly flattened gait in PND 60 males, and motor activity counts [rearing (NS) and ambulator y (p<0.05 or 0.01)] were decreased in female pups at PND 17 and 22. Additional treatment-related findings included post-dosing
clinical observations on PND 17 and 18 (whole body tremors, hypo-activity, prostrate posture, partially closed eyelids, and/or abnormal gait), delayed surfac e righting reflex in PND 11 female pups, and increased th ickness of the corpus callosum in PND 63–67 male s and females at the high dose. No treatment-related findings were noted in brain weights, or macroscopic or microscopic findings.

The offspring NOAEL is not determined (<5 mg/kg/day, LDT). The offspring LOAEL is 5 mg/kg/day, based upon increased auditory startle reflex peak amplitude in PND 23/24 male and female offspring, and decreased habituation in PND 60/61 females.

In a companion ChE inhibition study ( MRIDs 45566201 and 46005001 ), malathion (96% purity) was administered to Crl:CD ® BR SD rats per dose by gavage at doses of 0, 5.0, 50, or 150 mg/kg/day. Treatment groups consisted of 9 pregnant dams treated from GD 6 through 20 and terminated; 10 pregnant dams treated from GD 6 through PND 10, followed by treatment of one male and one female offspring/litter on PND 11 through PND 21; and groups of eight untreated dams whose offspring were treate d on PND 11. Repeated exposure to malathion resulted in decreases in ChE activity in the bl ood and/or brain in dams, fetuses, weanling pups, and adult male and female SD rats. In pups , effects on RBC ChE activ ity were noted atMALATHION FINAL

Page 53 of 74 5 mg/kg/day in both sexes on PND 21 after 11 re peated exposures. After 11 or 14 doses to young adults, effects were observed at 50 mg/kg/day in young adults and pregnant dams. In pups, brain ChE activity was inhibited after 11 repeated doses ( ↓ 16% in PND 21 pups). Based upon the results of the ChE study, it is evident that al l behavioral and neuropa thological effects of treatment observed in the dams and offspring in the developmental neurotoxicity study occurred at doses at which ChE activity was, or had been, inhibited.

For repeated exposures, the overall adult LOA EL for ChE inhibition is 50 mg/kg/day for RBC; the adult NOAEL is 5 mg/kg/day. For repeated exposures, the overall offspring LOAEL for ChE inhibition is 5 mg/kg/day for RBC; the offspring NOAEL is not determined (<5 mg/kg/day).

Multi-Generation Reproduc tion in Rodents (Rat)

In a two-generation, two-lit ter reproduction study ( MRID 41583401 ), male and female SD rats (25/sex/dose) were administered malathi on (94.0% purity) in the diet to the F 0 and F 1 parental generations at dosages of 0, 550, 1700, 5000, or 7500 ppm. Mean-measured intake during the premating period in the F 0 parents was 42.7, 131.8, 393.5, and 595 mg/kg/day in males and 49.9, 151.9, 438.1, and 655 mg/kg/day in females. Mean-meas ured intake during the premating period
in the F 1 parents was 42.6, 130, 394.5, and 628 mg/kg/day in males and 51.2, 154.3, 464.7, and 751.8 mg/kg/day in females. Average intake for both parental groups wa s equivalent to 0, 43, 131, 394 or 612 mg/kg/day in males and 0, 51, 153, 451 or 703 mg/kg/day in females. The F 0 generation was treated for 63 days prior to th e initial mating and the subsequent mating to produce the second litter was init iated after a 2-week resting period following weaning of the
first litter. The F 1 generation was treated for 79 days (100 da ys of age) prior to mating, with the same schedule described previous ly to produce the second litter.

After weaning of the second lit ters and selection of the F 1 parental animals, the F 0 parents were sacrificed and subjected to a gross examination. Tissues collected for possible histopathological examination included: vagina, uterus, ovaries, te stes, epididymides, seminal vesicles, prostate, and pituitary. The F 1 parental animals were sacrificed according to the same regimen after weaning and sacrifice of the F 2 litters. The tissues listed above were examined microscopically
from all F 0 and F 1 control and high-dose animals.

There was no evidence of a treatment-related eff ect on survival; five animals in each parental generation died or were sacrif iced moribund in the control and treatment groups. Clinical signs reported for all animals (unrelated to treatmen t) included chromodacryorrhea, excess lacrimation, and alopecia (females only). In the F 0 generation, after the first mating, body weight in females was decreased (p ≤ 0.01 or 0.05) at the high dose on GD 7, 14, and 20 ( ↓ 6.6% to 7.6%) and during the entire lactational period ( ↓ 6.6% to 9.2%), and at the medium-high dose on LD 14. After the second mating, body weight was decreased (p ≤ 0.05) at the high dose on LD 0 (8.2%) and 21 (6.2%). In the F 1 generation, body weight was decreased (p ≤ 0.01 or 0.05) at the high dose duringMALATHION FINAL

Page 54 of 74 the entire treatment period for males ( ↓ 9.1% to 13.4%) and during premating Weeks 1, 8, and 11 for females ( ↓ 6.9% to 10.1%). Increased (p ≤ 0.05) weights were noted in females at the mediumlow dose on GD 14 and 20 during the first gestati onal period, and at th e medium-low and low doses on GD 14 and 20, respectively, during th e second gestational period. Mean food consumption was occasionally decreased (p<0.05 or 0.01) during the study in both sexes. No consistent evidence of treatment-related effects on fertility, gestation or lactation indices were
observed. In the F 1 generation, the lactati on index was decreased (p ≤ 0.05) in the high-dose group during the first mating. Pup body weight was decreased (p ≤ 0.01 or 0.05) in all first litters (except medium-low) on LD 21 and in the second litters at the high dose on LD 0 ( ↓ 8.3%) and 21 ( ↓ 10.7%). In the F 2 generation, fertility i ndices in the control a nd treated animals for F 2b litters were slightly reduced (range: 70 to 83%) compar ed to the other litter s (range: 88 to 100%). Viability indices were decreased (p ≤ 0.05) at the medium-high dos e during the second mating. Pup body weight was decreased (p ≤ 0.01 or 0.05) in the first litter at the high dose on LD 21 and in the second litter at the medium-high and high doses on LD 7 to 21 and 21, respectively.

Determination of ChE activity and organ we ights were not conducted in this study. No treatment-related histopathol ogical lesions were seen in any examined tissues.

The parental toxicity NOAEL is 5000 ppm (394 mg/kg/day in males and 451 mg/kg/day in females) and the parental toxicity LOA EL is 7500 ppm (612 mg/ kg/day in males and 703 mg/kg/day in females) based on decreased body weights in F 0 females during gestation and lactation and on decreased body weights in F 1 males and females during the pre-mating period. The developmental toxicity NOAEL is 1700 ppm (131 mg/kg/day in males and 153 mg/kg/day in females) and the developmental toxicity LOAEL is 5000 ppm (394 mg /kg/day in males and 451 mg/kg/day in females) based on decreased pup body weights during th e lactation period in

F 1A and F 2B pups. The reproductive toxicity NOAEL is >7500 ppm (612 mg/kg/day in males and 703 mg/kg/day in females). The reproductive toxi city LOAEL is >7500 pp m (612 mg/kg/day in males and 703 mg/kg/day in females). No repr oductive toxicity was observed in this study.

In a published study examining male reproductive toxicity ( Choudhary et al. , 2008 ), male Wistar rats (6/dose) were administered mala thion (technical grade, purity NR) by gavage at
doses of 0, 50, 150, or 250 mg/kg/day in olive oil for 60 days. Treated males were mated with virgin females; females were sacrificed on GD 16 to determine the number of implantation sites and the males were sacrificed at the end of treatment. Parameters evaluated included: fertility; sperm motility; sperm density; testosterone, protein, sialic acid, glycogen, and cholesterol levels; and the weights of testes, epididymides , seminal vesicle and prostate glands.

Fertility and sperm motility were decreased (p<0.001) in a dose-dependent manner by malathion treatment; the treatment effect on fertility is uncl ear as it is reported as a negative effect, instead of a decrease from control (100%). Dose-rela ted decreases (p<0.001) in testicular and epididymidal sperm density were also observe d. Serum testosterone concentrations wereMALATHION FINAL

Page 55 of 74 decreased at all doses (NS, low and intermediate doses; p<0.001, high dose). Testes and epididymal weights were decreased (p<0.001) in the mid- and high-dose groups, seminal vesicle weights were decreased (p<0.001) at the intermed iate dose only, while the ventral prostate weights were decreased (p<0.001) at all doses.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a combined chronic toxicity/oncogenicity study ( MRID 43942901 ), malathion (97.1% purity)
was administered to 90 Fischer 344 rats (55/sex/dose) in the diet for up to 24 months at nominal doses of 0, 100/50, 500, 6000 or 12,000 ppm [equivalen t to respective mean values of 0, 4, 29, 359 and 739 mg/kg/day (males) and 0, 5, 35, 415 and 868 mg/kg/day (females)]. Interim
sacrifices were conducted at 3 and 6 months (10/sex/dose at e ach period) and at 12 months (15/sex/dose). The initial low dose (100 ppm) wa s decreased to 50 ppm at 3 months due to RBC
ChE inhibition that was observed in females at the 3-month interi m sacrifice. After 104 weeks, all surviving rats in all groups were sacrificed for gross and histopathological evaluation. Select organs were weighed at each sa crifice interval (including liver, kidneys, testes, epididymides, ovaries, adrenals, and thyroid/parathyroids). Gross and microscopic pa thological examination included the liver, kidney, pituit ary, thyroid/parathyroids, adre nals, mammary gland, testes, epididymides, prostate, seminal vesicles, uterus, and ovaries.

The only clinical sign observed was yellow a nogenital staining among fe males in the high-dose group. Increased mortality was seen in females at the high dose only, but occurred in males at all but the low dose ( ≥ 500 ppm). All high-dose males died or were sacrificed moribund by Week 94. Treatment-related decrements in body weight gain were observed in males and females in the medium-high (3 to 13% and 4 to 5%, respectiv ely) and high-dose (12 to 32% and 10 to 15%, respectively) groups. Food consump tion was generally similar to co ntrol or elevated, especially in the medium-high and high-dose animals. Several hematology parameters were affected (p<0.05 or 001) in males or both sexes in th e medium-high or high-dose groups. Additionally, several clinical chemistry parameters (pri marily liver-related enzymes or compounds; e.g. , aspartate and alanine am inotransferases, alkaline phosphatase , gamma glutamyltransferase, and cholesterol) were generally aff ected (p<0.05 or 0.01) in the medium-high or high-dose animals.

Inhibition (p<0.01 or 0.05) of Ch E activity in a dose-dependent manner was observed in males and females in plasma, RBC, and brain at all sampling periods (3, 6, and 12 months, and at study termination excluding high-dose males) in the medium-high and high-dose groups. ChE activity at study termination in the medium-high males was decreased 64, 43, and 31% in plasma, RBC, and brain, respectively, and was decreased 61, 44 , and 18%, respectively, in the medium-high females. No samples were collected for high-dos e males at study termination (100% mortality), but ChE activity at study termination in the hi gh-dose females was decreased 89, 52, and 67% in plasma, RBC, and brain, respectively. Dose-dep endent inhibition (p<0.01 or 0.05) was also noted for plasma ChE in the medium-low dose males at study termination only ( ↓ 29%), and inMALATHION FINAL

Page 56 of 74 RBC ChE activity in females in the medium-l ow group at 3 and 12 months and at study termination ( ↓ 27%). Finally, ChE effects were noted in males and females in the low-dose group at 3 months (prior to decrease from 100 to 50 ppm).

Organ weight effects included increased (p<0.0 1) liver weights in the medium-high and highdose males ( ↑ 22 and 36%, respectively) at the interim s acrifice, and increased (p<0.01) liver weights of 34% in the medium-high males at termination. In females, liver weights were increased (p<0.01) in the high-dose animals ( ↑ 16%) at the interim sacrifice, with increases (p<0.01) in the medium-high and high-dose females ( ↑ 30 and 31%, respectively) at termination. Kidney weights were increased (p<0.01) in the medium-high and high-dose males ( ↑ 14 and 29%, respectively) at the interim sacrifice, with an increase (p<0.01) of 11% in the medium-high males at termination. In females, kidney weights were increased (p<0.01) in the medium-high and highdose animals ( ↑ 9 and 13%, respectively) at the interim sacrifice, with increases (p<0.01) in the same groups of females ( ↑ 22 and 37%, respectively) at termina tion. Increases (p<0.01) were also noted in thyroid/parathyr oid weight in the medium-high and high-dose males ( ↑ 15 and 25%, respectively) at the interim sacrifice, with an increase (p<0.01) of 19% in the medium-high males at termination. Finally, testes weights were incr eased (NS) by 11% at term ination in the mediumhigh males. Microscopic findings were varied, but did not demons trate any consistent effect on
endocrine-related e ndpoints or tissues.

Treatment-related increases in tumors included combined hepatocellular adenomas/carcinomas in females across the treatment groups. Othe r tumor types observed included increased
incidences of testicular interstitial cell tumors significant at all doses with possibly decreased latency; a significant trend in thyroid follicular cell adenomas and/or carcinomas in males; a significant trend and positive pairwise comparison at the medium-low dose for thyroid c-cell carcinoma in males; and a significant difference in pair-wise comparisons for pituitary pars
distalis carcinomas or adenomas and/or carcinomas combined in medium-low and medium-high dose females. Tumorigenic responses may have b een compromised by high mortality in males at the medium-high dose and in males and females at the high dose.

For inhibition of ChE activity, in males the ove rall NOAEL is 50 ppm (4 mg/kg/day) and the LOAEL is 500 ppm (29 mg/kg/da y) based on inhibition of plas ma activity at 24 months. The overall (beyond Month 3) NOAEL fo r ChE inhibition in females is 50 ppm (5 mg/kg/day) and
the LOAEL is 500 ppm (35 mg/kg/day) based on inhi bition of RBC activity. A NOAEL for a 3-month exposure period is not established in females.

No significant treatment-related cha nges were seen in absolute or relative weights of the testes, epididymides, ovaries, thyroid or adrenal glands, and no treatment-related lesions were seen in the epididymides, seminal vesicle, prosta te, uterus, ovaries, and mammary gland.MALATHION FINAL

Page 57 of 74 Carcinogenicity in Rodents (Mouse)

In an 18-month carcinogenicity study ( MRID 43407201 ), malathion (96.4% purity) was administered in the diet to groups of B6C3F1 BR strain mice (65/sex/dose) at doses of 0 (control), 100 ppm, 800 ppm, 8000 ppm or 16,000 ppm (equivalent to 0, 17.4, 143, 1476 or 2978 mg/kg/day in males and to 0, 20.8, 167, 1707 or 3448 mg/kg/day in females). The two highest doses used in this study both exceeded the standard limit dose of 1000 mg/kg/day, which is used by EPA for carcinogenicity studies in mi ce and rats. These doses were required by EPA to duplicate the doses used in a previously conducted carcin ogenicity study (NCI, 1978) in B6C3F1 mice that yielded equivocal results. A subset of study animal s (10 mice/sex/group) was sacrificed at 12 months, and the remaining survi vors were sacrificed at 18 months. Mortality, clinical signs of toxicity, body weights and food consumption were monitored at appropriate times during the study. Determinations of plasma , RBC, and brain ChE activities were made on 8 to 10 mice/sex/group at 9 (RBC ChE only), 12, and 18 months. Necropsy examinations were performed on all animals and organ weight dete rminations were conducted at the interim and terminal sacrifices. Histopathological exam inations were made on a complete set of organs/tissues from all animals that died or we re sacrificed in extrem is during the study, and on all control and high-dose animals at the interi m and terminal sacrifi ces. Histopathological
examinations were also conducted on a limited set of organs/tissues from the other dose groups
at the scheduled sacrifices.

Although four unscheduled deaths occurred in the high-dose males during Weeks 1 to 39 (compared to none in any other group), deaths in males in the control, low-, and medium-low dose groups surpassed those in the higher-dose groups during the remainder of the study. Few unscheduled deaths in the females in any group during the study. No treatment-related clinical signs, including no signs associated with ChE inhibition, were noted during the study. At the high- and medium-high dose in both males and females, treatment-related effects included decreased (p<0.01) absolute body weights thr oughout the entire duration of the study ( ↓ 14.3 and 20.0%, respectively, in males and ↓ 9.7 and 16.1% in females at 18 months, respectively); decreased food consumption ( ↓ 2.0 and 5.9% and ↓ 5.4 and 12.5% in males and females, respectively); decreased (p<0.01) plasma ChE activity at 12 and 18 months ( ↓ 86 to 96%); decreased (p<0.01 or p<0.05) RBC ChE activity at 9, 12 and 18 months ( ↓ 61 to 92%); and decreased brain ChE activity at 18 months (37 an d 43% in males and females, respectively) at the high dose only. Plasma ChE activity was also decreased in the medium-low dose males (NS; ↓ 23 and 24%) and females (p<0.05; ↓ 18 and 36%) and RBC ChE activity was also decreased in the medium-low dose males (NS or p<0.05; ↓ 37 to 44%) and females (NS, p<0.05, or p<0.01; ↓ 35 to 58%).

A treatment-related increased incidence of hepato cellular tumors was observed in both male and female mice in this study in the medium-high and high-dose groups. For male and female mice which died during the final 6 months of the study or were killed at the terminal sacrifice atMALATHION FINAL

Page 58 of 74 18 months, the combined hepatocellular adenom as/carcinomas (primarily adenomas) were 1.9%, 18.2%, 9.1%, 32.7% and 96.1%, and 1.8%, 1.8%, 3.7%, 18 .9% and 84.6%, for the control, low-, medium-low, medium-high, and high-dose groups, respectively. The dose -related increases in combined hepatocellular adenomas/carcinomas in the medium-high and high-dose animals were
attributed to treatment with malathion. Concu rrent hepatic observations included masses, nodules
and foci in these animals at the terminal sacrific e, as well as increased liver weights and steeply increased incidences of hepatocellular hypertro phy in the livers of the medium-high and highdose animals. Additional findings were not ed in the kidney and adrenal cortex.

The NOAEL for ChE inhibition fo r both sexes was estimated to be 17.4/20.8 mg/kg/day (LDT) for plasma and RBC ChE and 1476/1707 mg/kg/da y (medium-high dose) for brain ChE. Although there was some decrease in ChE activity at these doses, the data were considered to be
too variable to conclude that the inhibition observed was related to treatment. The NOAEL for systemic effects was 143/167 mg/kg/day, a nd the LOAEL was 1476/1707 mg/kg/day, based on decreased body weights and food consumption, increased liver weights, and increased hepatocellular hypertrophy in males and females.

Chronic Oral Toxicity/Carcinogenicity in Non-Rodents (Dog)

In a chronic one-year oral toxicity study in dogs ( MRID 40188501 ), malathion (95.0% purity) was administered at 0, 62.5, 125, or 250 mg/kg/day by capsule to Beagle dogs (6/sex/dose) for 12 months. ChE activity was determined in plas ma, RBC, and selected brain regions (cerebrum and cerebellum only). Organ weights determined included adrenals, kidneys, liver, ovaries, testes, and thyroid; histopatholog ical examinations were conducted on these tissues as well as the uterus and vagina. Several recommended tissues were not examined, including the pituitary.

All animals survived until terminal sacrifice, with no treatment-related clinical signs and no effects on body weight or food consumption. Seve ral clinical chemistry parameters were decreased (significance not reported) at all doses in males and/or females ( i.e. , albumin, creatinine, calcium, SGPT, and BUN). RBC and pl asma ChE activity was inhibited (p<0.05) in both sexes at all doses and sample times. Plasma ChE activity in males was decreased by 23 to 30% at all doses and sample times; plasma Ch E activity in females was decreased by 22% to 37% in the low- to high-dose groups. RBC ChE activ ity in both males and females was inhibited to approximately the same extent ( ↓ 25%) at all dose and all sample times. Brain ChE (cerebrum and cerebellum only) was not affected by treatmen t. Absolute kidney weights were increased in mid-and high-dose males ( ↑ 25 and 47%) and females ( ↑ 24 and 55%), and absolute liver weights were increased in low- and high-dose females only ( ↑ 40 and 41%). In females, thyroid/parathyroid combined weig ht exhibited a dose-related upwar d trend with absolute weight increased (p<0.05) at the high dose. This parameter was increased (N S) in males at all doses. No endocrine-related organ weight changes were observed at study termination. Gross and microscopic pathology were gene rally unremarkable, with no tr eatment-related findings inMALATHION FINAL

Page 59 of 74 endocrine-related organs. For ChE inhibition, the NOAEL is not determined and the LOAEL is 62.5 mg/kg/day based on decreased plasma and RBC ChE activity. For systemic toxicity, the NOAEL is 250 mg/kg/day due to the lack of histopathological effects supporting systemic effects, no systemic LOAEL is determined.

This chronic one-year oral toxicity study in dogs ( MRID 40188501 ) was previously considered unacceptable because NOAELs for plasma and RBC ChE inhibition were not determined. It was determined by HIARC (1998) that the defici ency could be addressed by conducting and submitting a 90-day subchronic study in dogs. The mo st recent re-registration eligibility decision (RED) for malathion (USEPA, 2006) lists the pr esent study and does not mention an additional requirement.

Mechanistic Study: Ovarian Toxicity (Rodent)

In a published non-Guideline study examining reproductive system function in rats ( Ojha et al.
, 1992 ), malathion (source and purity not reported) was administered to female Wistar at 0, 10, 50, 100, 1000, 3000, or 5000 ppm in the diet for 7 days. Actual malathion intake was not reported, but may have been as high as 100, 300, and 500 mg/ kg/day in the three highest dose groups (the lack of purity and age/initial weight information, and potential for use of a formulated material impedes this determination). Histopathological examinations were conducted on the ovary, oviduct, uterus and vagina

. No effects on mortality, food intake or body weight were noted in the 10, 50, and 100 ppm dose groups, and only slight va riations were noted in food intake and body weight in the 1000 ppm dose group. Increased mortality ( ↑ 20 and 40%), decreased food intake ( ↓ 20 and 40%), and decreased body weight ( ↓ 10 to 15 g and ↓ 15 to 30 g) were seen in the 3000 and 5000 ppm dose groups, respectively, along with in creased clinical sign s of ChE inhibition. No histopathological changes were seen in rats at the 10 to 100 ppm diets. At 1000 ppm, minor changes in the uterus (epithe lium was irregularly distorted) were seen. At 3000 ppm, marked changes were seen in the corpora lutea, oviduct, uterus, uterine glands, and the vagina. Severe changes were seen in the 5000 ppm animals, with a complete absence of corpora lutea.

Mechanistic Studies: Testicular Toxicity (Rodent)

In a published study examining acute effects of malathion on testicular function in rats ( Piramanayagam et al. , 1996 ), a single gavage dose of malathion (95% purity) at 1950 mg/kg (median LD 50 ) was administered to male Wistar rats (18 control and 32 treated). Animals were sacrificed on Days 1, 2, 3, 6 and 12 post-injec tion. Sperm count and morphology, and testicular histopathology, were evaluated. No gross changes were seen in the testes, but congestion was observed in the liver and kidneys. Mortality apparently occurred, but was not quantified. Histopathology showed a reduction in the number of germinal layers, and degeneration and necrosis of gonocytes in the seminiferous t ubules through Day 3. These changes were mild on Day 6 and the spermatogenic activity returned to normal in almost all the tubules by Day 12.MALATHION FINAL

Page 60 of 74 In a published study examining malathion effects on spermatogenesis in rats ( Krause et al.
, 1976 ), malathion (purity not reported) in olive oil was administered to juvenile Wistar rats (16 males/treatment) as 40 mg/kg/day on PND 4 and 5 or 20 mg/kg/day on PND 4 to 23. Two
males/treatment were sacrificed on PND 6, 12, 18, 26, 34, and 50 to allow histological
examination of the testes. Body and testes weights were not a ffected by treatment. Quantitative
evaluation showed alterations in the number of Sertoli cells on PND 6; the number of type A spermatogonia/tubule on PND 6, 12 and 18; the number of pac hytene sperm on PND 18; and the number of Leydig cells on PND 6. Recovery was observed by PND 50. It was proposed that testosterone synthesis was redu ced due to malathion treatment, with subsequent damage to spermatogenetic cells. In a published follow-up study ( Krause, 1977 ) examining effects on spermatogenesis in rats, malath ion (purity not reported) in pe anut oil was administered by gavage to adult Wistar rats at 20 mg/kg every s econd day for two weeks to 7 males. Three of the seven animals died during treatment. Animals we re sacrificed at the end of treatment, blood was collected, and the testes were prepared for testosterone determination and histological
examination. Body, testes, and seminal vesicles weights were not affected by treatment. Serum
LH and testosterone concentrations also were unaffected, but FSH concentration was increased (p<0.05) approximately two-fold. No histopath ological changes in the testes were noted.

In another published study ( Uzun et al. , 2009 ) examining protective effects against malathioninduced testicular toxicity in rats, malathion (purity not reported) was administered by gavage to male Wistar rats (6/treatment ) at 27 mg/kg/day for 28 days. The malathion-treated group had decreased (p<0.05) concentrations of LH ( ↓ 32%), FSH ( ↓ 17%), and testosterone ( ↓ 27%) compared to control. The malathion-treated group also exhibited decreased (p<0.05) sperm counts ( ↓ 22%) and sperm motility ( ↓ 36%) with an increased per centage of abnormal sperm ( ↑ 59%). In addition, there were fewer spermatogeni c cells, with necrosis in some seminiferous
tubules and interstitial edema. These results demonstrate that a low dose of malathion can produce testicular toxicity.

In another published study examining repr oductive system function in male rats ( Ojha et al.
, 1992 ), malathion (purity and source not reported) was administered to male Wistar at 0, 10, 50, 100, 1000, 3000, or 5000 ppm in the diet for 7 days. Actual malathion intake was not reported, but may have been as high as 100, 300, and 500 mg/kg/day in the three highest dose groups. Histopathological examinations were conducted on the testes

. No effects on m ortality, food intake or body weight were noted in th e 10, 50, and 100 ppm dose groups, and only slight variations were noted in food intake and body weight in the 1000 pp m dose group. Increased mortality ( ↑ 20 and 40%), decreased food intake ( ↓ 20 and 40%), and decreased body weight ( ↓ 10 to 15 g and ↓ 15 to 30 g) were seen in the 3000 and 5000 ppm dose groups, respectively. Clinical signs of toxicity consistent with ChE inhibition also were noted in these two highest dose groups. No histopathological changes were seen in rats at the 10 to 100 ppm diets. At 1000 ppm, minor changes in the seminiferous tubul es (loss of normal pattern of th e Sertoli cells) were seen. AtMALATHION FINAL

Page 61 of 74 3000 ppm, marked changes were seen in the semi niferous tubules, with severe changes observed
in the seminiferous tubules and vas deferens at 5000 ppm.

Mechanistic Study: Hormone (Rodent)

In a published study ( Ozmen and Akay, 1993 ), the effect of malathion treatment on hormone concentrations and hormone-secreting tissues was examined. Malathion (94% purity) in sunflower oil was administered by gavage to Sw iss albino rats (13/sex/ dose) at doses of 0 (negative control), 0 (vehicle contro l), 10, or 100 mg/kg/day for 15 weeks

. The parameters
evaluated were: T 3 , T 4 , aldosterone, cortisol, te stosterone (male), and 17 β -estradiol (female) concentrations, and histopathology of the test es, ovaries, adrenal, and thyroid glands.

No difference was observed between negative and vehicle control animals. No treatment-related changes were seen in T 3 , T 4 (NS; ↑ 14% at high dose), testosterone (NS; ↓ 10 and 20% at the low and high doses, respectively), and 17 β -estradiol concentrations at either dose. Significant
decreases in aldosterone and cortisol concentratio ns were seen at the low dose, but not at the high dose. No histopathological changes were se en in the thyroid or ovaries. Histopathology revealed hyperemia of the veins and slight degene ration of the seminiferous tubules at both doses in the testes, and adrenals exhibited conges tion of the zona reticularis at both doses.

Avian Reproductive Toxicity (OCSPP 850.2300)

In an avian reproduction study ( MRID 42782101 ), 23-week old Mallard ducks ( Anas platyrhynchos ) were administered malathion (94.0% purit y) in the feed at concentrations of 0 (acetone; vehicle control) , 240, 1200, and 2400 ppm for 20 weeks (includes an 8-week administration with a decreased photoperiod. Each dose group consisted of 16 ducks/sex, with a single drake and hen being paired in 16 pens /dose group. The photo period was 7 hours/day for the first 8 weeks, increased to 17 hours/day fo r the final 12 weeks of the study. Temperature was maintained at 16°C with a relative humidity of 88%. Body weight determinations were conducted at study initiation and at Weeks 2, 4, 6, and 8, and at study termination. Eggs were collected daily, and stored at 15°C and 65% re lative humidity. Eggs were incubated at 37.7°C and 61% humidity, candled on Days 14 (viability ) and 21 (survival), and placed in a hatcher on Day 23. Feed consumption was measured bi weekly. The average minimum and maximum temperatures in the hatchling study room ra nged from 22 to 27°C and relative humidity ranged from 43 to 87%. Hatchling body weights were meas ured and recorded at hatch and on Day 14.
ChE determinations were not conducted during this study.

Two males died (Weeks 7 and 19) in the high-d ose group with neither death attributed to treatment; no overt signs of toxicity or clinical signs were observ ed in any dose group. No effects were seen at any test dose on female body weight or average feed consumption in males and females. Male body weight was decreased (p <0.01, 10%) in the high- dose group at Week 20, with a time-related trend to decrease (NS, ↓ 3 to 6%) observed throughout the study. ApparentMALATHION FINAL

Page 62 of 74 treatment-related decreases (NS) on the total number of eggs laid and set ( ↓ 20%), and viable embryos (NS; ↓ 7%), were observed in the high-dose group. Mean eggshell thickness also was decreased (p<0.01; 2%) at the high dose, and the nu mber of infertile eggs was increased at the high dose. Occasional differences in duckling body weight (Day 1 or 14) and Day 14 viability
were not considered to be related to treatment.

The NOAEC is 1200 ppm, and the LOAEC is 2400 ppm (HDT) based on increased infertility and decreased reproductive parameters.

In another avian reproduction study ( MRID 43501501 ), 18-week old Bobwhite Quail ( Colinus virginianus ) were administered malathion (96.4% purity) in the feed at nominal concentrations of 0 (corn oil; vehicle control), 110, 350, or 1200 ppm for 21 weeks (includes 11-week administration prior to the initiation of egg laying). Mean-measure d malathion concentrations in the diet were not indicated. Each dose group cons isted of 32 quails, with a single cock and hen paired in 16 pens/dose group. Th e photo period was 8 hours/day for the first 8 weeks, increased to 17 hours/day for the final 13 weeks of the st udy. Temperature was maintained at 22.9±3.4°C with a relative humidity of 70±19%. Body weight determinations were conducted at study initiation and at Weeks 2, 4, 6, and 8, and at stu dy termination. Eggs were collected daily, and
stored at 12.8°C and 84% relati ve humidity. Eggs were candled on Days 11 (viability) and 21 (survival), and eggs were placed in a hatche r on Day 21 (37.2°C and 76% humidity). Hatchling housing details were not provide d; hatchlings were weighed on Day 14. ChE determinations were not conducted during this study.

Mortality occurred in all treatm ent groups (one each in the co ntrol, low- and mid-dose groups, and nine in the high-dose group). Necropsy reveal ed regressing ovaries and enlarged/flaccid gizzards in mid- and high-dose adults. Adult body we ights gains were decrea sed (p<0.01) at test termination in high-dose males ( ↓ 53%) and females ( ↓ 55%). Feed consumption in the treated groups generally was comparable to control, although variable due to wastage. There was no treatment-related effect on the prevalence of crack ed eggs; the number of eggs not cracked/eggs laid in the vehicle-control group was generally greater compared to treatment groups. Decreases (p<0.01) in eggs laid ( ↓ 76%), viable embryos/egg set ( ↓ 75%) were observed in the high-dose group. Subsequently, live embryos, hatchlings, a nd 14 d hatchlings per egg set were also significant at the high treatment, but these e ndpoints were not signifi cantly different when compared when using the preceding stage in th e ratio calculation (i.e., live embryos/viable embryos, hatchlings/live embryos, 14 d hatchlin gs/normal hatchlings). Eggshell thickness was also decreased (p<0.05; ↓ 15.5%) at the high dose. Hatchling a nd 14-d hatchling weights were not affected. The overall NOAEC is 110 ppm and the LOAEC is 350 ppm based on regressing ovaries and enlarged/flaccid gizzards.

In a published study ( Lillie, 1973 ), avian reproduction was assessed in caged White Leghorn pullets. Twenty-week old Single Comb White Leghorn pullets were administered malathionMALATHION FINAL

Page 63 of 74 (95% purity) in the feed at nominal concen trations of 0 (vehicle NR), 250, or 500 ppm. Treatment was initiated at Week 32 (n=20 /treatment) and malathion administration was
conducted for 36 weeks. Mean-measured malathion c oncentrations in the diet were not indicated. Insemination was accomplished with a c o mpo s ite sample of undiluted semen obtained from caged White Leghorn male s fed the same diet as the control pullets. Pullets were inseminated
once a month with a volume of 0

. 05 mL/ bird. T wo days after in se mination

, eggs were saved for a 5-day period and set according to hen and group number. All embryos that did not hatch after 21 days were e x amined for evidence of abnormalities that might be attributed to malathion

treatment

. The photoperiod was 14 hours/day and temperature was maintained at 10°C. Body weight determinations were conducted at st udy initiation and throughout the study (terminal body weight was reported). In four bi-monthly progeny studies, chicks from the control and high-dose groups were housed in chick brooder batte ries and fed their resp ective diets for 4-week periods to assess growth and mortality. Finally, at the termina t ion of the reproduction/progeny assessments, male s were retained for a 4

- week trial to determine the effect of malathion
administration on fertilizin g capacity. Groups (5/treatment) were fe d the same breeder diet

sup p lement ed with 0 or 500 ppm. Females u s ed in this stud y were those that had been f e d either 0 or 500 ppm of malathion in the previous experiment, and were con t inued on their re spective
diets in the fertilizing assessment . One week after the initiation of malathion administration in
the male diet , a composite sample of undiluted semen was obtained f rom each group of males
and u s ed to inseminate both non- s upplemented a nd malathion-treated females at the rate of

0.05 mL/bird. This procedure was carried out a total of three times on a weekly basis.

No treatment-related effects due to malath ion administration were observed on pullet body weight gain, egg production, egg wei ghts, specific gravity of eggs, and feed consumption; eggs set, fertility, and hatchability of fertile eggs; or progeny body weights and mortality. In the fertilization capacity assessment, fertility was not affected and hatchability of fertile eggs was slightly decreased (NS) by 8 and 28% with unt reated and treated pulle ts, respectively. The NOAECs for reproductive performance, progeny pe rformance, and male fertilization capability are 500 ppm (HDT); a LOAEC is no t determined in this study.

Fish Early Life-Stage Test (Freshwater) (OPPTS 850.1400)

In a freshwater fish early life-stage test ( MRID 41422401 ), Rainbow trout ( Oncorhynchus mykiss; previously Salmo gairdneri ) embryos were exposed to malathion (94% purity) at nominal concentrations of 0 [negative contro l and vehicle control (solvent and solvent concentration not identified)], 5, 10, 20, 40, or 80 μg/L. Achieved exposure concentrations were 5.1, 9.9, 21, 44, and 84 μg/L for the treated groups. E ggs were exposed from Day 1 (fertilization) until hatch ( ≥ 99% by Day 37); exposure was continued fo r 60 days post-hatch until termination on Day 96. Four replicates of 35 eggs/replicate were included in each treatment group on Day 1. On Day 39, unhatched eggs were removed, and the number of fry was reduced to 15/replicate. Fry survival, and length and weight, were determined on Day 37 and 60 post-hatch.MALATHION FINAL

Page 64 of 74 Mean egg viability was determined to be 99%. The percent hatch was ≥ 99% complete in all replicates by Day 37; no treatmen t-related effect on time to hatc h was noted. Notable sub-lethal physical and behavioral effects in the two highest treatment groups were persistent, and included fish resting on the bottom of th e chambers, discoloration, spinal curvature and quiescence. Fry survival on post-hatch Day 35 was decreased (significance not repor ted) in the medium-high and high-dose concentration groups by 40 and 100%, respectively. Mean length on post-hatch Day 37 was decreased (p<0.05) in the medium-high group (data not reported). Fry survival on post-hatch Day 60 was decreased (significance not reported) at the medium-high and high concentrations by 48 and 100%, re spectively. Mean length was d ecreased (p<0.01) on post-hatch Day 60 (data not reported). Mean weight on post-hatch Day 60 also was decreased (significance not reported) in the medium-high group. The maximum acceptable toxicant concentration (MATC) limits for malathion were estimated as the NOAEC and LOAEC of 21 and 44 μg/L.

Fish Life-cycle (Freshwater)

In a published study ( Hermanutz, 1978 ), life-cycle (growt h, survival, and re productive capacity) were assessed in flagfish ( Jordanella floridae ) exposed to malathion (95% purity) at nominal concentrations of 0 (water only; nega tive control), 6.4, 8.5, 11.5, 15.2, 20.3, 27.0, and 36.0 μg/L (mean-measured concentrations of 0, 5.8, 8.6, 10.9, 15.0, 19.3, 24.7, and 31.5 μg/L). Acetone was used as a solvent (maximum of 1.4 mg/L at the high dose, ≤ 0.00014%), but a solvent control was not included. Forty 1- to 2-day old larvae were randomly placed into one of the larvae chambers in each of the spawning chambers. Growth and survival were determined on Day 30 of exposure, after which a random sample of 15 fish were transferred to their respective spawning chambers. Survival was also determined during Day 30 to 65 and Day 65 to 110 exposure periods. During Week 6, the number of fish was re duced to two males and five females by random sampling from those fish that displayed secondary sex characteri stics. At that time, spawning substrates composed of yarn-covered st ainless steel screen we re placed in each spawning chamber. Excess fish were held in corresponding larvae chambers until Day 65 to
ensure establishment of a final 2:5 sex ratio. Fo ur to seven groups of twenty 1-day old secondgeneration larvae were reared for 30 days in ea ch concentration to determine survival and growth. Total lengths and weights of each surviving fish were recorded.

First-generation growth (length) was decreased (p=0.05) at ≥ 10.9 μg/L concentration ( ↓ 11 to 18%) during the first 30 days. Survival du ring the same period was reduced (p=0.05) at the two highest concentr ations, 24.7 and 31.5 μg/L ( ↓ 31 and 33%, respectively), with a decrease of 13% (NS) in the 19.3 μg/L c oncentration group. However, no additional adverse survival effects were detected in any of the concen trations during the Da y 30 to 65 and 65 to 110 intervals. Also, the mean number of eggs produced per female was not affected at any malathion
concentration. None of the malathion test c oncentrations demonstrat ed adverse effects on survival through hatching, and s ubsequent survival and growth, of the second generation larvae.MALATHION FINAL

Page 65 of 74 APPENDIX 3: References Not Utiliz ed in the Malathion WoE Analysis

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

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

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

Akbarsha M. et al. (2000). Retention of cytoplasmi c droplet by rat cauda epididymal spermatozoa after treatment with cytotoxic and xenobiotic agents. J. Reprod. Fertil . 120 (2):

385-390.

Akhtar N

. et al. (1996). Insecticide-induced changes in secretory activit y of the thyroid gland in

rats. Journ. Appl. Toxicol . 16 (5): 397-400.

Alam, M.K., and O.E. Maughan. (1992). The effect of malathion, diazinon, and various concentrations of zinc, copper, nick el, lead, iron, and mercury on fish. Bio. Trace Elem. Res

. 34:225-236.MALATHION FINAL

Page 66 of 74 Alfaro-Lira, S., Pizarro-Ortiz, M., and Calaf, G. M. (2012). Malignant transformation of rat kidney induced by environmental substances and estrogen. Int J Environ Res Public Health

. 9(5):1630-1648.

Anguiano, O.L., A. Caballero de Castro, A.M. Pechen de D'Angelo. (2000). The role of glutathione conjugation in the regulation of early toad embr yos' tolerance to pesticides. Comp. Biochem. Physiol , Part C 128:35-43.

Areechon, N., and J.A. Plumb. (1990). Subletha l effects of malathi on on channel catfish, Ictaluruspunctatus

. Bull. Environ. Contam. Toxicol . 44:435-442.

Awasthi, M., P. Shah, M.S. Dubale, and P. Gadhia. (1984). Metabolic changes induced by organophosphates in the piscine organs. Environ. Res . 35:320-325.

Blasiak J. et al. ( 1999). In vitro studies on the genotoxicity of the organophosphorous insecticide malathion and its two analogues. Mutat. Res . 445(2): 275-283.

Bonilla E. et al. ( 2008). Effects of the insecticides malathion and diazinon on the early oogenesis in mice in vitro. Environ. Toxicol . 23:240-245.

Boone, M

. D. (2008). Examining the single and intera ctive effects of th ree insecticides on amphibian metamorphosis. Environ. Toxicol. Chem . 27(7):1561-1568.

Bossi, R., Vinggaard, A.M., Taxvig, C., et al. (2013). Levels of Pes ticides and their Metabolites
in Wistar Rat Amniotic Fluids and Ma ternal Urine upon Gestational Exposure. Int. J. Environ.
Res. Public Health . 10, 2271-2281;

Bustos-Obregon E. and Gonzalez-Hormazabal P. (2003). Effect of a single dose of malathion on spermatogenesis in mice

. Asian J. Androl

. 5 (2): 105-107.

Caballero de Castro, A.C., E.A. Rosenbaum, and A.M. Pechen de D'Angelo. (1991). Effect of
Malathion on Bufo arenarum hensel development -1. Esterase inhibition and recovery.
Biochem. Pharmacol 41(4)491-495.

Calaf, G. M., Echiburu-Chau, C., and Roy, D. (2009). Organophosphorous pesticides and estrogen induce transformation of breast ce lls affecting p53 and c-Ha-ras genes. Int J Oncol

. 35(5):1061-1068.MALATHION FINAL

Page 67 of 74 Calaf, G. M. and Echiburú-Chau, C. (2012). S ynergistic effect of ma lathion and estrogen on mammary gland carcinogenesis. Oncol Rep . 28(2):640-646.

Casas, E., Bonilla, E., Ducolomb, Y. et al . (2010). Differential effects of herbicides atrazine and fenoxaprop-ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro. Toxicology in Vitro 24, 224-230

Chauhan, U.P.S., C.B. Jaggi, and V.K. Rastogi. (1974). Hypertrophy of adrenals and ascorbic
acid status in various tissues of malathion treated rats. Indian J Med. Res . 62, 987-989.

Chou, K. C., and R.M. Cook. (1994 ). Paraoxon inhib its fertilization of mouse gametes in vitro.
Bull. Environ. Contam. Toxicol 53, 864-868.

Contreras H. and Boustos-Obregon E. (1999). Morphological alteration in mouse testis by a single dose of malathion. J. Exp. Zool . 284: 355-359.

Cook, LW., C.H. Paradise, and B. Lorn. The pestic ide malathion reduces survival and growth in developing zebrafish. Environ. Toxicol. Chem . 24(7): 1745-1750

Costa L. et al. ( 2005). Modulation of paraoxonase (PON1) activity. Biochem. Pharmacol . 69:

541-550.

Crawford, R.B., and A.M. Guarino. (1985). Eff ects of environmental toxicants on development of a teleost embryo. J. Environ. Pathol. Toxicol. Oncol . 6(2):185-194.

Degraeve, N., M.C. Chollet, and J. Moutschem. Cytogenetic and geneti c effects of subchronic treatments with organophosphorus insecticides. Arch. Toxicol. 56, 66-67. Male guinea pigs after repeated dermal application. " Vet. Hum. Toxicol . 29 (2):138-143.

del-Rahman, Ali , Dechkovskaia, Anjelika M. , Go ldstein, Larry B. , Bullman, Sara H. , Khan, Wasiuddin , El-Masry, Eman M. andAbou-Donia, Mohamed B.(2004). Neur ological Deficits Induced by Malathion, Deet, and Permethrin, Al one or in Combination in Adult Rats. Journal of Toxicology and Environmental Health , Part A, 67: 4, 331-356

Dikshith T. et al. (1987). Interaction of hexachlorocycloh exane and malathion in Environmental Protection Agency. 2009. Reregistration El igibility Decision (RED) for Malathion.

Ducolomb, Y., Casas. E., Valdez, A . et al . (2009). In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine. Cell Biol Toxicol 25: 623-633.MALATHION FINAL

Page 68 of 74 Dutta H. et al. ( 1994). Sublethal malathion induced changes in the ovary of an airbreathing fish, Heteropneustes fossilis: a histological study. Hydrobiologia . 294 (3): 215-218.

El-Gawad, E.A.A., et al. (2011). Impact of Some Organophos phorus Insecticides on Growth Performance, Fecundity and Semen Ch aracteristics in Nile Tilapia ( Oreochromis niloticus ).

Lucr ă ri Ş tiin ţ ifice Med. Vet . 54(1): 150-160.

Espinoza-Navarro O. and Bustos-Obregon E. (2 005). Effect of malathion on the male reproductive organs of earthworms, Eisenia fo etida. Asian J. Androl.7 (1): 97-101. 15.

Falugi C. et al . (1991). Possible functions for the em bryonic cholinergic system present in gametes at fertilization

. in: L. Scaleraliaci e, C. Canica tt’ 1 (Eds.), Echinoderm Research, Balkema Press, Rotterdam, 1991: 161-164.

Fletcher, D.W. and Pedersen, C.A. (1989). AC 6,601 Technical: 28 Day Dietary Toxicity and Reproduction Pilot Study in Bobwhite Quail. BLAL Study #88 QRP 32. Bio-Life Associates, Ltd. Route Box 156 Neillsville, WI 54456. S ponsored by American Cyanamid Company.

Fraysse B. et al. ( 2006). Development of a zebrafish 4-da y embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicol. Environ. Saf . 63: 253-267.

Freuer G. and Kardish R. (1975). Hormonal regulation of drug metabolism during pregnancy.
Int. J. Clin. Pharmacal. 11: 366.

Galloway, T. and Handy R. (2003). Immunot oxicity of Organophosphorous Pesticides. Ecotoxicology , 12, 345-363, 2003

Garcia A. et al. ( 1998). Paternal exposure to pesticides and congenital malformations. Scand. J.
Work. Environ. Health . 24 (6): 473-480.

Gilbertson M. et al. ( 2003). Immunosuppression in the northern leopard frog ( Rana pipiens ) induced by pesticide exposure. Environ. Toxicol. Chem . 22:101-110. 27.

Giri, S. et al. (2002). Genotoxic effects of malathion in chick in vivo micronucleus assay. Cytologia 67: 53-39

Giri, S. et al. (2002). Genotoxic effects of malathion: an organophosphorus insecticide, using three mammalian bioassays in vivo. Mutation R esearch 514: 223-231MALATHION FINAL

Page 69 of 74 Grether K. et al. ( 1987). Exposure to aerial malathion a pplication and the occurrence of congenital anomalies and low birthweight. AJPH. 77 (8): 1009-1010.

G ű ven M. et al. ( 1999). Endocrine changes in patients with acute organophosphate poisoning. Hum. Exp. Toxicol . 18 (10): 598-601.

Gwinn M. et al. ( 2005). Differential gene expression in normal human mammary epithelial cells treated with malathion monitored by DNA microarrays. Environ. Health Perspect . 113(8):

1046-1051.

Haider

, S. and N. Upadhyaya (1985). Effect of commercial formulation of four organophosphorus insecticides on ovar ies of a freshwater teleost, Mystus vittatus (Bloch)-a histological and hi stochemical study. J. Environ. Sci Health, B20(3):321-340.

Haider, S. and N. Upadhyaya (1986). Eff ect of Commercial Formulation of Four Organophosphorus Insecticides on the LH-Induced Germinal Vesicle Breakdown in the Oocytes of a Freshwater Teleost, Mystus vittatus (Bioch)-A Preliminary in Vitro Study. Ecotoxicology and Environmental Safety 12,161-165

Haney, A.F., S.F. Hughes, and C.L. Hughes, Jr. (1984). Screening of potential reproductive toxicants by use of porcine granulosa cell cultures. Toxicology 30:227-241.

Hoffman, D.J., and W.C. Eastin, Jr. (1981). E ffects of malathion, diazinon, and parathion on mallard embryo development and cholinesterase activity. Environ. Res . 26:472-485.

Kalow W. and Marton A. (1961). Second gene ration toxicity of ma lathion in rats. Nature . 192:

464-465.

Karczmar A. et al. (1970). Investigations, particularly by means of the anticholinesterase agents, of the multiple peripheral and central cholinergic mechanisms and of their behavioral implications. Acta Vitaminol. Enzymol . 24 (4); 131-189.

Kaur S. et al. (2005). Biochemical effects of some or ganophosphorous pesticides on the ovaries
of albino rats. Indian J. Physiol. Pharmacol . 49: 148-152.

Kitamura S. et al. (2003). Antiandrogenic activity a nd metabolism of the organophosphorus pesticide fenthion and related compounds. Environ. Health Persp . 111 (4): 503-508.MALATHION FINAL

Page 70 of 74 Koc, N.D., F. E. Kayhan, C. Sesal, and M.N. Muslu. (2009). Dose-dependent effects of
endosulfan and malathion on adult Wistar albino rat ovaries. Pakistan Journal of Biological

Sciences 12(6):498-503.

Komori T. et al. (1991). A case of delayed myeloneuropath y due to malathion intoxication. No To Shinkei. 43 (10): 969-974.

Koutros, S., Beane, F. L. E., Lubin, J. H., Helts he, S. L., Andreotti, G., Barry, K. H., DellaValle, C. T., Hoppin, J. A., Sandler, D. P., Lynch, C. F., Blair, A., and Alavanja, M. C. (2013). Risk of total and aggressive prostate cancer and pesticide use in the Agricultural Health Study. Am J Epidemiol. 177(1):59-74

Lai, B.,.and T.P. Singh. (1986). In vivo modificati on of fatty acids and glycerides metabolism in response to 1,2,3,4,5,6-hexachlorocyclohexane and cythion exposure in the catfish, Clarias
batrachus

. Ecotoxicol. Environ . So/. 11:295-307.

Lai, B., and T.P. Singh. (1987). v-BHC and Cythion-induced alterations in lipid metabolism in a freshwater catfish, Glorias batrachus , during different phases of its annual reproductive cycle.
Ecotoxicol. Environ. So /. 14:38-47.

Lai, B., and T.P. Singh. (1987). Impact of pesticides on lipid metabolism in the freshwater catfish, Clarias batrachus, duri ng the vitellogenic phase of its annual reproductive cycle.
Ecotoxicol. Environ. So /. 13:13-23.

Lai, B., and T.P. Singh. (1987). Effect of ma lathion and v-BHC on the lipid metabolism in relation to reproduction in the tropical teleost, Clarias batrachus

. Environ. Pollut . 48:37-47.

Lal, B., Sarang, M.K., and Kumar, P. (2013). Malathion exposure induces the endocrine disruption and growth reta rdation in the catfish, Clarias batrachus (Linn.). General and Comparative Endocrinology 181: 139-145.

Lu, Xian T., Yun Ma, Cui Wang, Xiao F. Zh ang, Da Q. Jin a Chang J. Huang (2012). Cytotoxicity and DNA damage of five organophos phorus pesticides related by oxidative stress in PC12 cells and protection by vitamin E, Journal of Environmental Science and Health , Part B: Pesticides, Food Contaminants, a nd Agricultural Wastes, 47:5, 445-454

Mackey, M.J., and M.D. Boone. (2009). Singl e and interactive effects of malathion, overwintered green frog tadpoles, and cyanobacteria on gray treefrog tadpoles. Environ. Toxicol.
Chem . 28(3):637-643.MALATHION FINAL

Page 71 of 74 Maitra S. and Sarkar R. (1996). Influen ce of methyl parathion on gametogenic and acetylcholinesterase activity in th e testis of whitethroated munia (Lonchuramalabarica). Arch.
Environ. Contam. Toxicol . 30: 384-389.

Mathews M. and Devi K. (1994). Effect of ch ronic exposure of pregnant rats to malathion and/or estrogen and/or progesterone on xenobiotic metabolizing enzymes. Pesti. Biochem Physiol. 48: 110-122.

Meulenberg E. (2002). A new te st to identify endocrine disruptors using sex hormone-binding globulins from human serum. Eur. J. Lipid Sci. Technol . 104:131-136.

Nataraj, M.B. and Krishnamurthy, S.V. (2012). Effects of combinations of malathion and cypermethrin on survivability and time of meta morphosis of tadpoles of Indian cricket frog (Fejervarya limnocharis). Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes , 47:2, 67-73

Nishihara, T. J. Nishikawa, T. Kanayama, F Da keyama, K. Saito, M. Imagawa, S. Takatori, Y. Kitagawa, S. Hori, and H. Utsumi. (2000). Es trogenic activities of 517 chemicals by yeast twohybrid assay. Journal of, Health Science 46(4):282-298.

Pedersen, C.A. (1989). AC 6,601 Technical: Toxicity and Reproduction Study on Bobwhite Quail. Lab Report No. BLAL study #88 QR 32, performed by Bi o-Life Associates, Ltd. Route 3, Box 156, Neillsville, WI. Sponsored by Am erican Cyanamid Company. MRID No.

41367801

Pluth J. et al. (1998). Molecular bases of hprt muta tions in malathion-treated human Tlymphocytes. Mutat. Res . Feb. 2; 397 (2): 137-148.

Prabhakaran, S., and K.S. Devi. (1993). Im pact of protein deficiency and exposure to hexachlorocyclohexane or malathion on lipid metabolism in pregnant rats. Indian journal of Biochemistry and Biophysics 30:234-238.

Ram, R.N., K.P.Joy, and A.G. Sathyanesan. (1989). Cythion-induced histophysiological changes in thyroid and thyrot rophs of the teleost fish, Channa punctatus (Bloch). Ecotoxicol

. Environ. So /. 17:272-278.

Relyea, R.A. and N. Diecks. (2008). An unforeseen chain of events: Lethal effects of pesticides on frogs at subletha l concentrations. Ecol. Appl . 18:1728-1742.MALATHION FINAL

Page 72 of 74 Relyea, R.A. (2009). A cocktail of contamin ants: how mixtures of pesticides at low concentrations affect aquatic communities. Oecologia 159(2):363–376.

Saadi, L., N. Lebaili, and M. Benyoussi. (2008). Exploration of cytotoxic effect of malathion on
some rat organs structure. Comm. Appl. Bioi. Sci . 73(4):875-881.

Salazar Z. et al . (2007). Gene expression analysis on the early development of pig embryos exposed to malathion. Int. Joum. Toxicol . 26: 143-149.

Sánchez-Acevedo, Z. C., Riu, J., and Rius, F. X. (2009). Fast picomolar selective detection of bisphenol A in water using a car bon nanotube field effect transist or functionalized with estrogen receptor-alpha. Biosens Bioelectron . 24(9):2842-2846.

Simionescu, L., M. Oprescu, V. Sahleanu, V. Dimitriu, and E. Ghinea. (1977). The serum and pituitary prolactin variations under the influence of a pesticide substance in the male rat. Endocrinologie 15(3):181-188.

Singh, H., and T.P. Singh. (1980a). Short-term eff ect oftwo pesticides on the survival, ovarian

32 P uptake and gonadotrophic potency in a freshwater catfish Heteropneustes fossilis (Bloch). J.
Endocrinol . 85:193-199.

Singh, H., and T.P. Singh. (1980b). Effect s of two pesticides on testicular 32 P uptake, gonadotrophic potency, lipid and cholesterol content of testis , liver and blood serum during spawning phase in Heteropneustes fossilis (Bloch). Endokrinologie 76(3):288-96.

Singh, S., and T.P. Singh. (1987). Evaluation of toxicity limit and se x hormone production in response to cythion and BHC in the vitellogenic catfish Clarias batrachus

. Environ. Res.
42:482-488.

Singh, P.B. Impact of malathion and y-BHC on lipid metabolism in the freshwater female catfish, Heteropneustes fossilis

. Ecotoxicol. Environ . So/. 23:22-32.

Sinha, N., B. Lai, and T.P. Singh. (1991). Pestic ides induced changes in circulating thyroid
hormones in the freshwater catfish Clarias batrachus

. Comp. Biochem. Physiol . 100C(l/2): 107-

110.

Sinha, N., B. Lai, and T.P. Singh. (1992). Thyroid physiology impairment by malathion in the
freshwater catfish Clarias batrachus

. Ecotoxicol. Environ. Saf. 24:17-25.MALATHION FINAL

Page 73 of 74 Smith, G.R., Krishnamurthy, S.V. Burger, A.C. et al. (2011). Differential Effects of Malathion and Nitrate Exposure on American Toad and Wood Frog Tadpoles. Arch Environ Contam
Toxicol 60:327–335.

Snawder, J.E., and J.E. Chambers. (1993). Osteolathyrogenic effects of malathion in Xenopus embryos. Toxicol. Appl. Pharmacol . 121:210-216.

Soto, A.M., C. Sonnenschein, K.L. Chung, M.F. Fe rnandez, N. Olea, and P.O. Serrano. (1995). The ESCREEN assay as a tool to identify estr ogens: An update on estrogenic environmental pollutants. Environmental Health Perspectives 1 03(Suppl 7): 113-122.

Sun, L., Xu, W., He. J., et al. (2010). In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expre ssion and interrenal ster oidogenesis in POMC: EGFP transgenic zebrafish. Toxicology and Applied Pharmacology 248: 217-225

TaylorS. et al. (1999). Effects of malath ion on disease susceptibility in Woodhouse's toads. J.
Wildl. Dis . 35:536-541.

Uluitu, M., A. Boca, G.H. Petec, R. Chis, and G. Catrinescu. (1981). The influence of malathion on the brain serotonin and repr oductive function in rats. Rev. Roum. Morphol. Embryol. Physiol., 18(3):167-174.

Uzunhisarcikli M. et al. (2007) Acute, subacute and sucb chronic administration of methyl parathion-induced testicul ar damage in male rats and protective role of vitamins C and E . Pestic. Biochem. Phys . 87: 115-122.

Vidair C. (2004) Age dependence of organophos phate and carbamate neurotoxicity in the postnatal rat: extrapolation to the human. Toxicol. Appl. Pharmacol . 196 (2): 287-302

.

Windham G

. et al

. ( 1998) Genetic monitoring of malathi on-exposed agricultural workers. Am.
J. Ind. Med . 33 (2): 164-174.

Wyttenbach, C.R., and S.C.Thompson. (1985) . The effects of the organophosphate insecticidemalathion on very young chick embryos : Malformations detected by histological
examination . Am. J.Anat , 174:187-202.

Yadav, A.K., and T.P. Singh. (1986). Effect of pe sticide on circulating thyroid hormone levels in the freshwater catfish, Heteropneustesfossilis (Bloch). Environ. Res . 39:136

- 142.MALATHION FINAL

Page 74 of 74 Yadav, A.K., and T.P. Singh. (1987). Pesticide-i nduced changes in periph eral thyroid hormone levels during different reproductive phases in Heteropneustesfossilis. Ecotoxicol. Environ. So /.

13:97-103.

Yu, Y., Yang, A., Zhang, J. et al. (2013). Maternal Expos ure to the Mixture of Organophosphorus Pesticides Induces Repr oductive Dysfunction in the Offspring. Environmental Toxicology 28(9):507-515.

Yanai J. et al. (2004). Convergent effects on cell signali ng mechanisms mediate the actions of different neurobehavioral teratoge ns: alterations in cholinergic re gulation of protein kinase C in
chick and avian models. Ann. N.Y. Acad. Sci. 1025: 595-601.