UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 82
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 82
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: MALATHION 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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MALATHION FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary..........................................................................................1 

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

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

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

B. Other Scientifically Relevant Information (OSRI)......................................4 

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8 

1. Effects on Estrogen Pathway.....................................................8 

2. Effects on Androgen Pathway..................................................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-Estrogenic 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.............................64 

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MALATHION 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

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MALATHION FINAL 

iv 

Abbreviation Terminology 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

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v 

Abbreviation Terminology 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

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vi 

Abbreviation Terminology 

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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MALATHION FINAL 

Page 1 of 74 

Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) 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 pathways, the number and 

type of effects induced, the magnitude and pattern of responses observed across studies, taxa, 

and sexes were considered. Additionally, the conditions under which effects occur were 

considered, in particular, whether or not endocrine-related responses occurred at dose(s) that 

also resulted in general systemic toxicity or overt toxicity. 

On 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 pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

There is no convincing evidence for potential interaction with the estrogen pathway. The Tier 1 

ER binding and ERTA assays were negative. The Tier 1 steroidogenesis assay was negative, 

however, in a steroidogenesis assay considered as OSRI (Taxvig et al., 2013), a ≤1.5-fold 

induction in estradiol was reported. In the Tier 1 aromatase assay, malathion showed an 

equivocal response. The Tier 1 uterotrophic assay 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 observed 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 toxicity. In the mallard duck study, reductions in eggshell thickness (↓5%) 

and egg viability were observed. However, no other 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, and there are no other estrogen-related responses observed in the 

other in vivo studies. 

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There was no convincing evidence of an interaction with the androgen pathway. In the in vitro

Tier 1 assays, malathion was equivocal for Androgen Receptor (AR binding and negative in the 

steroidogenesis assay. 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, decreases in testosterone were observed at the 

lowest dose in the presence of systemic toxicity; overt toxicity was observed at the mid and high 

dose. In the fish short-term reproduction assay (FSTRA), 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 Part 158 mallard duck reproduction study; however, 

this endpoint in limited in its diagnostic capabilities. 

There was no convincing evidence for an interaction with the thyroid pathway. The only 

thyroid-related effects observed in the Tier 1 pubertal assays was a non-dose-dependent increase 

in T4 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 alterations in developmental endpoints. 

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

recommended since there was no evidence of potential interaction with the estrogen, androgen or 

thyroid pathways. 

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MALATHION FINAL 

Page 3 of 74 

I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) 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 pathways, the number and 

type of effects induced, the magnitude and pattern of responses observed across studies, taxa, 

and sexes were considered. Additionally, the conditions under which effects occur were 

considered, in particular, whether or not endocrine-related responses occurred at dose(s) that 

also resulted in general systemic toxicity or overt toxicity. 

On April 22, 2014, the 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 

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 more 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 octanol/water partition coefficient (log KOW) of 2.36, and 

bioconcentration has not been observed in select species [Pseudorasbora parva or in pinfish 

(Lagodon sp)]. Malathion is susceptible to photolysis and hydrolysis and it is not persistent in the 

environment, with 81 to 94% degradation occurring in various non-sterile soils within ten days. 

Volatilization is not expected to significantly contribute to the dissipation of malathion in the 

environment. 

The available information considered in determining 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 toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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MALATHION FINAL 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order for malathion are shown below in Table 1. Executive Summaries are 

presented in Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order recipients and/or the public. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

(e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team on Malathion (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the Tier 1 assays and the studies cited as OSRI are 

presented in Appendix 1 and 2, respectively. Additionally, literature/studies considered but not 

utilized for the WoE analysis are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria, and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A, or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

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

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

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

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

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

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

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

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

above. 

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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 include published literature studies as well as 

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

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

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

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

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

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

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

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

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

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

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

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

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization? Finally, under what 

conditions did the responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic 

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toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

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

This uncertainty in distinguishing whether the responses were endocrine-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, weight should be placed on signs of endocrine disruption in the 

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

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g.,

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

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

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

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

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MALATHION FINAL 

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The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether a substance has the potential to interact with the E, A, 

or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for 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) 

ERα 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) 

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B. Effects on Hypothalamic-Pituitary-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 assays are delineated so as to 

facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table 

also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the 

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

considered further in the WOE assessment. 

Malathion was negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA and 

steroidogenesis assays. Malathion weakly induced (113% of control) 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 inhibition 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 malathion 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 pubertal assay. Pituitary weights were reduced 29 to 

31% at the two doses tested in the female pubertal study. In the female pubertal rat assay, 

decreases in ChE activity (RBC and 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 respiration (gasping) and mortality 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 observed. 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 considered to be overtly toxic. 

Page 16 of 82
MALATHION FINAL 

Page 9 of 74 

In the Part 158 bobwhite quail study, a significant 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 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 hatchlings/normal 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 sexes and increased 

mortality. In the mallard duck, there were significant decreases in eggshell thickness (5%) and 

decreased embryo viability (25%). In a fish life-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. In an early life stage study with the rainbow trout there were significant reductions 

(p<0.05) in post hatch survival, mean length, fry survival, and mean weight at concentrations as 

low as 44 μg a.i/L. 

There was no treatment-related effect on the prevalence of cracked 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, 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 hatchlings/normal hatchlings). Eggshell thickness was 

also decreased (p<0.05; ↓15.5%) at the high dose. Hatchling and 14-d hatchling weights were not 

affected

Page 17 of 82
MALATHION FINAL 

Page 10 of 74 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Malathion

Lines 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) 

EDSP Tier 1 Assay 

ER Binding Assay satisfied by OSRI (Chen et al., 2002; MRID 48160705, see below) ERTA (MRID 48617505) N Steroidogenesis (MRID 48617509) N4Aromatase (MRID 48617504) E Uterotrophic (MRID 48617510 ) N Female Pubertal Rat (MRID 48685701) NE N N N ↓29% (M) N N FSTRA (MRID 48617506) NE N N N N N 

OSRI 

ER Binding (Chen et al., 2002; 

MRID 48160705) 

N 

ERTA (Kjeldsen et al., 

2013) 

 P 
MALATHION FINAL 

Page 11 of 74 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Malathion

Lines 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) 

ERTA (Kojima et al., 2004) N ERTA (Kojima et al., 2005) N Aromatase (Kjeldsen et al., 

2013) 

 N 

Steroidogenesis (Taxvig et al., 2013) E 21-Day Dermal (Rabbit; MRID 41054201) 

 NE N N NE 

13-Week Inhalation (Rat; MRID 43266601) 

N N N N 

Developmental toxicity (Rat; MRID 41160901) 

N N 

Developmental toxicity (Rat; Prabhakaran and Devi, 1993) 

NE N 

Developmental toxicity (Rabbit; MRID 00152569; 45626801) 

NE N 
MALATHION FINAL 

Page 12 of 74 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Malathion

Lines 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) 

Developmental toxicity (Rabbit; Machin and McBride, 1989) 

NE N 

Developmental Neurotoxicity (Rat; MRID 45646401) 

 N N 

ChE Inhibition — Developmental Toxicity (Rat; MRIDs 45566201 and 46005001) 

 NE NE NE NE NE 

Two-generation reproduction (Rat; MRID 41583401) 

 NE NE N NE N 

Chronic toxicity / carcinogenicity (Rat; MRID 43942901) 

NE N N NE 

Carcinogenicity (Mouse; MRID 43407201) 

NE N N NE 

Chronic toxicity (Dog; MRID 40188501) 

NE N N NE 
MALATHION FINAL 

Page 13 of 74 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Malathion

Lines 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) 

Avian reproduction (Duck; MRID 42782101) 

 P6 

Avian reproduction (Quail; MRID 43501501) 

 N 

Fish Life-Cycle (Flagfish; Hermanutz, 1972 

 N 

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

Arrows (↓ r ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not ap

LW = Liver weight; KW = Kidney weight; 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 OS

this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X 

details see Section IV. A 

4. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected. Although th

were not optimal to detect very weak activity, malathion responses were similar to those of the negative control corticosterone and responses of 17α-methyltestosterone, which was able to reach a maximum of 6.5-37.4% PC. Malathion was only able to reach a mwhen tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, malathi

negative 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 sig

feeding were suggestive of overt toxicity at the high concentration. 

6. In the mallard duck, there were significant decrease in eggshell thickness (5%) and decreased embryo viability (25%). 

E Equivocal P Positive findings N Negative findings (in vitro) / No effect (in vivo) NE Not examined 
MALATHION FINAL 

Page 14 of 74 

2. Effects on Androgen Pathway 

The potential for malathion to interact with the androgen pathway is summarized in Table 3. The 

various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to 

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

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

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

considered further in the WOE assessment. 

In an evaluation of androgenic/anti-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 [3H]-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 antagonist 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 inhibited at the high dose. Finally, 

liver weights were increased at all doses and kidney 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 reflex and decreased motor activity), dyspnea, tremors, and 

excessive salivation; all were observed at the mid and high doses. 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 histological 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 treated animals. Dose-dependent 

decreases (p<0.01) in one AST weight (LABC) were observed in the mid- and high-dose 

animals. 

Page 22 of 82
MALATHION FINAL 

Page 15 of 74 

The only androgen-related effect observed in the Part 158 mammalian 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 concentration (0.69 mg/L). In addition, effects on 

male gonadal histology was observed at the high dose. As noted previously, clinical signs of 

toxicity were apparent in 33% of the high-dose fish at his dose along with reduced feeding. 

Additionally, a non-significant reduction in male body weight (17.3%) was observed at the high 

dose. 

As previously indicated, avian reproductive toxicity studies are available for the northern 

bobwhite quail study and a mallard duck study. Egg viability was significantly decreased in both 

species. However, in the quail, this effect was observed in conjunction with overt toxicity, and 

decreases in male body weight gain were observed in the mallard duck study were reproductive 

effects were observed.. In a fish life-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.

Page 23 of 82
MALATHION FINAL 

Page 16 of 74 

Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / Literature Citation 

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 

EDSP Tier 1 Assay 

AR Binding (MRID 48617503) E Steroidogenesis (MRID 48617509) N Hershberger (MRID 48617507) NE N 
MALATHION FINAL 

Page 17 of 74 

Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / Literature Citation 

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 

Male Pubertal Rat (MRID 48685701) 

↓60%,

(L) N N N N N N N 

FSTRA (MRID 48617506) NE N N N N 

OSRI 

ARTA (Kojima et al., 2004) N ARTA (Kjeldsen et al., 2013) N Steroidogenesis (Taxvig et al., 2013) E 13-Week Inhalation (Rat; MRID 43266601) N N NE N N 21-Day Dermal (Rabbit; MRID 41054201) N N N N NE 
MALATHION FINAL 

Page 18 of 74 

Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / Literature Citation 

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 

Developmental toxicity (Rabbit; MRID 00152569; 45626801 

N N 

Developmental Neurotoxicity (Rat; MRID 45646401) 

N N 

ChE Inhibition — Developmental Toxicity (Rat; MRIDs 45566201 and 460050001) 

NE NE NE NE NE NE NE 

Two-generation reproduction (Rat; MRID 41583401) 

NE N NE N NE NE N 

Chronic toxicity / carcinogenicity (Rat; MRID 43942901) 

N N N N NE NE 

Carcinogenicity (Mouse; MRID 43407201) 

 N N NE N NE NE 

Chronic toxicity (Dog; MRID 40188501) N N NE NE NE NE Avian reproduction (Duck; MRID 42782101) 

P4 
MALATHION FINAL 

Page 19 of 74 

Table 3. Androgenic/Anti-Androgenic Pathway for MalathionLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Malathion1Study Type / Literature Citation 

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 

Avian reproduction (Quail; MRID 43501501) 

N 

Fish Life-Cycle (Flagfish; Hermanutz, 1972) 

N 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not a

Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Le

bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). LW = Liver weight; KW = Kidney weight; 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 OS

this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X 

details see Section IV. A 

4. In the mallard duck, there were significant decrease in embryo viability (25%). E Equivocal P Positive findings N Negative findings (in vitro) / No effect (in vivo) 

NE Not examined 
MALATHION FINAL 

Page 20 of 74 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

pubertal female and male rat assays, and the AMA (frog). 

The potential for malathion to interact with thyroid regulation are summarized in Table 4. The 

various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

In both the female and male pubertal assays, no treatment-related effects were noted for thyroid 

weight, histopathology, or serum TSH concentration. Concentrations of T4 were increased at all 

doses in males and females, and pituitary weight was decreased (p<0.01) by approximately 30% 

in the mid- and high-dose females only. Systemic toxicity was observed at all doses in the males 

and females and overt toxicity was 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 length (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 were 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 increased in the rat and dog chronic studies (and not 

examined in the mouse carcinogenicity study), but no histopathological findings were observed. 

 

Page 28 of 82
MALATHION FINAL 

Page 21 of 74 

Table 4. Thyroid Pathway for Malathion

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Malathion1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, BL, SVL) Systemic Toxicity Observed2

Overt Toxicity Observed3 

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 applicable 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. A 

Page 29 of 82
MALATHION 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 recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

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

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

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tire 1 assays stated that “In summary, 

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

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

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

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI 

studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. 

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

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

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

Page 30 of 82
MALATHION FINAL 

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1. Tier 1 in vivo Assays 

In the Hershberger assay, general systemic toxicity was noted in both the mid- and high-dose 

groups (300 and 1000 mg/kg/day) based on significant increases in absolute 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, and absolute and relative kidney weights were 

increased by 13 to 18%. At the mid dose, relative liver and kidney weights were increased 

(p≤0.05) by 9% and 7%, respectively. Although clinical signs of toxicity were not observed in 

the study, ChE activity was substantially 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, and 1000 mg/kg/day, respectively. 

In both the female and male pubertal rat studies, the initial high 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-dose females (2/20) during the remainder of the studies, along 

with clinical signs of cholinergic toxicity (excessive salivation, tremors, irregular gait, and 

irregular respiration). Low-dose males and low- and mid-dose females also showed sporadic 

signs of clinical toxicity. There 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 pituitary weights were also decreased by 29 to 

31% in both mid- and high-dose females. In the males, absolute and 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 activity 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 and 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 clinical sign of toxicity were observed over three days of treatment 

at doses up to 1000 mg/kg/day, but general systemic 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 inhibited 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%, respectively, at 1000 mg/kg/day. 

Page 31 of 82
MALATHION FINAL 

Page 24 of 74 

In the FSTRA study, at 0.69 mg/L, 8 of 24 fish exhibited 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 reduced on Days 5 through 16 and male body weights 

were reduced by 17.3% (NS) at study termination. No overt or systemic signs 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 endpoint across studies was ChE inhibition, with 

increases in liver and kidney weights noted in most in vivo studies as well. In the 21-day dermal 

toxicity study in rabbits, serum, RBC, and brain ChE activities were decreased (p<0.01) at study 

termination (↓41–75%) in all high-dose males and females. 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%, respectively. 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 developmental 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 control 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 treatment observed in the dams and 

offspring in the developmental neurotoxicity study occurred at doses at which ChE activity was, 

or had been, inhibited. 

In a two-generation, two-litter reproduction study, no clinical signs related to treatment were 

reported. In the F0 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 entire 

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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 F1 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 females 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 sign observed was yellow anogenital staining among 

females in the high-dose group. However, increased 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-high (3 to 13% and 4 to 5%, respectively) and 

high-dose (12 to 32% and 10 to 15%, respectively) groups. Inhibition (p<0.01 or 0.05) of ChE 

activity was observed in males and females in plasma, RBC and brain at all sample periods (3, 6, 

and 12 months, and at study termination) in the 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 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-dose 

males at study termination (100% mortality), but ChE activity at study termination in the highdose females was decreased 89, 52, and 67% in plasma, RBC and brain, respectively. Dosedependent 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 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 months (prior to decrease from 100 to 50 ppm). 

Organ weight effects included increased (p<0.01) liver weights in the medium-high and highdose males (↑22 and 36%, respectively) at the interim sacrifice, and increased (p<0.01) 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 high-dose 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 termination. 

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In the mouse carcinogenicity study, treatment-related 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 included 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 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%). 

In the chronic dog study, all animals survived until terminal sacrifice, with no treatment-related 

clinical signs and no effects 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 sample 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 approximately 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 increased 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 treatment 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 malathion concentrations of ≥25 μg/L, and body 

lengths were reduced by 11-18% at concentration ≥11 μg/L. 

B. Estrogen Pathway 

There is no convincing evidence for potential interaction with the estrogen pathway. The Tier 1 

ER binding and ERTA assays were negative. The Tier 1 steroidogenesis assay was negative, 

however, in a steroidogenesis assay considered as OSRI (Taxvig et al., 2013), a ≤1.5-fold 

induction in estradiol was reported. In the Tier 1 aromatase assay, malathion showed an 

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equivocal response. The Tier 1 uterotrophic assay 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 observed 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 toxicity. In the mallard duck study, reductions in eggshell thickness (↓5%) 

and egg viability were observed. However, no other 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, and there are no other estrogen-related responses observed in the 

other in vivo studies. 

C. Androgen Pathway 

There was no convincing evidence of an interaction with the androgen 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, decreases in testosterone were observed at the lowest dose in the 

presence of systemic toxicity; overt toxicity was observed at the mid and high dose. In the fish 

short-term reproduction assay (FSTRA), 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 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 interaction with the thyroid pathway. The only 

thyroid-related effects observed in the Tier 1 pubertal assays was a non-dose-dependent increase 

in T4 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. 

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V. EDSP Tier 2 Testing Recommendations 

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

recommended since there was no evidence of potential interaction with the estrogen, androgen 

thyroid pathways. 

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VI. References 

Barnett, J. F. (2011). Oral (gavage) uterotrophic bioassay of malathion in female rats. 

Unpublished study conducted by Charles River Laboratories, Preclinical Services, Horsham, PA. 

Laboratory Project ID: 20012263, November 3, 2011. (MRID 48617510). 

Barnett, J. F. (2011). Oral (gavage) Hershberger bioassay of malathion in male rats. 

Unpublished study prepared by Charles River Laboratories, Preclinical Services, Horsham, PA. 

Laboratory Project ID 20012264, November 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 Services, Horsham, PA. Laboratory Project ID: 20012266, June 18, 

2012. (MRID 48685701). 

Barnett, J.F. (2012). Oral (Gavage) Repeated 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. Laboratory 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., Xiao, H., and Wang, X (2002) Estrogenicity of 

organophosphorous and pyrethroid pesticides. J. of Tox and Environ. Health. Part A. 65, 1419-

1435. 

Cohle, P. (1989). Early Life Stage Toxicity 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/oncogenicity study of malathion in the rat via 

dietary administration. Laboratory report Project Number: 90-3641, submitted to Cheminova 

Agro A/S. (MRID 43942901). 

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Fulcher, S.M . (2001). Malathion: Effects on cholinesterase in the CD rat (adult and juvenile) by 

oral gavage administration. Unpublished study prepared by Huntingdon Life Sciences, Ltd., 

Huntingdon, Cambridgeshire, England. Doc. No. CHV067/012452. November 30, 2001. 

(MRID 45566201). 

Fulcher, S.M. (2002). Malathion developmental neurotoxicity 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 administration. Unpublished study prepared by 

Huntingdon Life Sciences, Ltd., Cambridgeshire, 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 activities in 200 pesticides by in vitro 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 using an in vitro reporter gene assay. Int. J. Environ. Health 

Res. 15:271-280. 

Krause, W., K. Hamm, and J. Weissmuller. (1976). Damage to spermatogenesis 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., and 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. 

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Lochry, E.A. (1989). A development toxicity study with AC6,601 in rats. Laboratory report 

Project Number: 101-005, submitted to American Cyanamid Company and A/S Cheminova. 

Unpublished study conducted by Argus Research Laboratories, 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 study 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 Krueger, H. (2011). Malathion: Amphibian 

Metamorphosis Assay Final Report. Unpublished 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 Krueger, H. (2011). Malathion: Fish Short-Term 

Reproduction Assay with the Fathead Minnow (Pimephales promelas). Unpublished study 

conducted by Wildlife International, Ltd., Easton, 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 Number 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-Lethal Potential of Malathion in the Rabbit. 

Unpublished study prepared by ClinTrials BioResearch, Ltd. 12 p. (MRID 45626801).

Schroeder, R.E. (1990). A two-generation (two-litters) reproduction 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). 

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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, submitted to American Cyanamid Company, New 

Jersey. Unpublished study conducted by Food and Drug Research Laboratories, 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 Development 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 toxicity study in purebred beagle dogs with AC 

6,60l. Laboratory report Project Number: 85010, submitted to American Cyanamid Company. 

Unpublished study conducted by Tegeris Laboratories, Inc., Maryland. (MRID 40188501). 

USEPA (2006). Reregistration Eligibility Decision (RED) for Malathion. Office of Chemical 

Safety and Pollution Prevention, Office of Pesticide Programs, U.S. Environmental Protection 

Agency, Washington, D.C. EPA 738-R-06-030, July, 2006.

USEPA (2010). Malathion — Report of the Endocrine Disruptor Review Team; Test Order# 

EDSP-057701-44. September 23, 2010. Office of Chemical Safety and Pollution Prevention. 

U.S. Environmental Protection Agency, Washington, D.C. 

USEPA. (2011). Endocrine Disruptor Screening Program — Weight of Evidence: Evaluating 

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

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

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. 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 the protective role of vitamins C and E. Food Chem. Toxicol. 

47: 1903–1908. 

Wagner, H. (2011). Malathion: H295R steroidogenesis assay. Unpublished study conducted by 

CeeTox, Inc, Kalamazoo, MI. Laboratory Report No.: 9148V-100372STER, Nov 3, 2011. 

(MRID 48617509). 

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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., Kalamazoo, MI. Laboratory Study Number: 

9148V-100372ARB, November 4, 2011. (MRID 48617503). 

Willoughby, J.A. (2011). Malathion: Estrogen receptor transcriptional activation (human cell 

line (HeLa-9903)). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory 

Study No.: 9148V-100372ERTA, November 4, 2011. (MRID 48617505). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 48617501) of malathion on amphibian metamorphosis of South 

African clawed frogs (Xenopus laevis) was conducted under flow-through conditions. 

Amphibian larvae at Nieuwkoop-Faber (NF) Stage 51 (80 per control or treatment group, 

20/replicate) were exposed to malathion (96.0% purity) at nominal concentrations of 0 (negative 

control), 0.040, 0.13, and 0.40 mg a.i./L; mean-measured 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 survival 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, respectively. 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, respectively. At test termination, tail curvature was observed in 92% of 

tadpoles in the negative control and 86, 93, and 95% of tadpoles in the low, intermediate, and 

high concentration groups, respectively. The incidence of tail curvature did not appear to be 

treatment-related. 

There were no significant differences 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 development 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 control at the termination 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-concentration group relative to the negative control. 

However, there were no significant effects on growth 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 negative controls on Day 21 (effects on body weight were 

observed at 0.2 and 0.6 mg a.i./L in the rangefinder suggesting the high concentration in this 

study was sufficient). 

Late-stage (NF>60) tadpoles were excluded from analyses with respect to body weight, SVL, 

and HLL (including normalized HLL) by the reviewer; these included 4 (6.8%), 1 (1.7%), 4 

(6.7%) and 1 (1.7%) tadpoles in the negative control and low, intermediate, and high treatment 

groups, respectively 

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Androgen Receptor (AR) Binding 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 rat prostate cytosol was present in reasonable 

numbers and was functioning with appropriate affinity for the radiolabeled reference androgen 

(R1881). The competitive binding assay was conducted to measure the binding of a single 

concentration of [3H]-R1881 (1 nM) in the presence of increasing concentrations 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 provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The mean dissociation constant (Kd) for [3H]-R1881 was 

1.080±0.690 nM, and the estimated Bmax 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 Kd values was within 

the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values were 

below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for 

individual runs was acceptable (R2

 = 0.880-0.986), there was a large variation in Kd 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 reduced 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, respectively. 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 concentration 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 IC50s for R1881 and the weak positive control (dexamethasone) were −9.3 and 

−4.4 M, respectively. The mean RBA for the weak 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 likely due to the concentration differences used for 

R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the first run 

and therefore the calculation for the IC50 may be affected. The range of concentrations for 

R1881 in the first run fully defined the curve, but the concentration range was off by more than 

an order of magnitude. 

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The performance criteria were generally met; however, the bottom percent binding for 

dexamethasone was not achieved in the first run. The concentrations prepared for R1881 and 

dexamethasone were lower than the targeted (guideline) 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, and the resulting curve for dexamethasone fit 

only two of the three criteria (Hill slope and Top plateau). All performance criteria were met for 

the other two runs. 

Based on the results from the three runs, malathion 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 tritiated androstenedione ([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) for 

15 minutes at 37°C to assess the potential of malathion to inhibit 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 concentration of chemical. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three independent runs were conducted, 

and each run included a full activity 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 full 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 activity 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 ranges for the top of the 

curve, bottom curve, hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variations (%CVs) for replicates of each 

concentration of malathion within a run were within the 15% guideline. For 4-OH ASDN, the 

estimated log IC50 averaged –7.16 M, and the slope was –1.01. 

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For malathion, aromatase activity averaged 0.170 ± 0.066 nmol·mg-protein−1·min−1 at the lowest 

tested concentration of 10–9 M (the lowest concentrations tested from Run 1, 10–9 and 10–10, were 

excluded 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). Precipitation 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 best fit mean inhibition response curve was a straight line (essentially no 

inhibition was observed at other doses). The response for malathion was considered equivocal 

since the average lowest portion of the response curve across runs was between 50 and 75% 

activity. 

Based on the data from the average response curve, malathion is classified as Equivocal for 

aromatase inhibition in this assay. 

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

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

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

In an estrogen receptor transcriptional activation assay (MRID 48617505), hERα-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. Luciferase expression in response to activation of the estrogen 

receptor was measured upon addition of a luciferase substrate and detection with a luminometer. 

Malathion was tested up to the limit of solubility, which was 10-5 M. The mean RPCMax for 

malathion was 3.4% for the first run and 3.7% for the second run, and the associated PCMax was 

10-7 M for both runs. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, malathion responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach a maximum of 6.5-37.4% PC. Malathion was only able to reach a maximum of 3.4-3.7% 

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

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RPCMax < PC10 in both assay runs, malathion was considered negative for estrogen receptor 

transcriptional activation in this test system. 

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

The 21-day short-term reproduction assay (MRID 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 concentrations of 0 (control), 0.10, 0.32 and 1.0 mg a.i./L. 

The mean-measured concentrations, calculated 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 samples due to suspected handling or analysis 

error, however, no supporting data were provided. Therefore, the reviewer 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%, respectively. 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 effects are reported based on comparison to the 

negative (clean water) control. 

Male and female survival was 100% in the negative control and in the lowest treatment 

concentration (0.08 mg a.i/L). There was a single male and female mortality each at the mid 

treatment concentration and a single female mortality at the high treatment concentration. 

Observed clinical signs of toxicity included one fish at the mid concentration exhibiting a 

crooked spine, which was not attributed to treatment. However, in the high concentration group, 

8 of 24 fish (33%) exhibited clinical signs of toxicity that included 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 day of exposure and increased in incidence as the test continued. 

Excess feed was noted in the test chamber on Days 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 concentration groups, respectively; there was no effects 

on female body weight and length or male length. 

Spawning and fecundity in controls, respectively, were every 3 days and 26.3 eggs/female/day; 

fertilization success in controls was 99.3%. There was a statistically 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 trend, with an increase of 21% at the high 

concentration compared to the negative control (p<0.05; Jonckheere-Terpstra). At the high 

concentration, male tubercle scores were significantly reduced compared to the negative control 

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(10.5 vs. 25.5 in control) (p<0.01; Jonckheere-Terpstra). 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 endpoints involved with the development of the 

testes at the highest treatment level but did not appear to affect ovarian development to the same 

degree, with the exception of increased oocyte atresia. In the high-concentration group males, 

there were treatment-related effects (p<0.05; Jonckheere-Terpstra trend test) including increased 

spermatogonia, altered proportion of spermatocytes 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 reviewer’s observations were validated by the 

study authors’ statistical analysis. 

 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48617507) screening 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 control group consisted of eight castrated rats 

exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-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 in conjunction 

with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The 

anti-androgenic positive control 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 anti-androgenic negative control. 

For both components of the assay, body weights and food consumption were determined daily. 

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

final dose. At necropsy, the five androgen-dependent tissues, liver, and kidneys were collected 

and weighed. Brain and red blood cell (RBC) samples were also collected from rats in the 

androgen agonist assay for determination of cholinesterase (ChE) activity.

There were no treatment-related clinical signs of toxicity or gross pathological findings in either 

assay. 

In the androgen agonist assay, there were no treatment-related effects of malathion on accessory 

sex organ weights. At 1000 mg/kg/day, absolute and relative (to body weight) liver weights 

were increased (p≤0.01) by 29% and 32%, respectively, compared to controls; absolute and 

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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, brain 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 performance criteria in the Guidelines. The %CV values were less than 

the maximum permissible values for all accessory 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; compared to maximum %CV = 40). The study 

author noted that the same prosector dissected these organs for every rat in the androgen agonist 

assay. 

In the anti-androgen assay, there were no treatment-related effects for malathion on the weights 

of accessory sex organs. At 1000 mg/kg/day, absolute 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 relative liver weights were increased 

(p≤0.05) by 14% and 10%, respectively, at 100 mg/kg/day. The performance 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 level was decreased to 750 mg/kg/day on PND 24 

due to mortality and clinical observations at the high-dose. Animals were examined for vaginal 

opening (VO) daily beginning on PND 22, and age and weight on the day of attainment of VO 

was recorded. After sacrifice on PND 42, blood was collected for clinical chemistry analyses, 

including total thyroxine (T4) and thyroid stimulating hormone (TSH), which were determined 

using radioimmunoassays. Kidney, liver, adrenal gland, thyroid, pituitary, uterus, and ovary 

weights were recorded, and microscopic examinations 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. 

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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 were found dead between 

PNDs 23-25. All other rats survived until scheduled sacrifice. Treatment-related clinical signs 

observed in the high-dose group included effects on gait (decreased motor activity and 

prostration), irregular respiration (dypsnea), nervous system (whole body tremors, tremors in the 

head, slight excess salivation), and general toxicity (urine stained abdominal fur, 

chromodacryorrhea, laceration on the neck and scab 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 significant effects on age or weight at VO, mean age at first vaginal 

estrous, mean cycle length, number of females cycling, number of females regularly cycling, or 

cycle status at necropsy. However, there were apparent dose-related trends toward decreased 

percentages in the number of animals cycling and the number of animals regularly cycling in the 

malathion-treated rats. 

Serum T4 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 changes in thyroid weight or histology. Therefore, the biological 

significance of the changes in T4 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 relative kidney weights were increased 

(p≤0.01) by 7% and 11%, respectively. Given that the weight changes in liver and kidneys were 

minor in magnitude and there were no adverse histopathological 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 effects on organ weights, 

including the uterus and ovaries, and there were no microscopic changes 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 treatmentPage 49 of 82
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related changes were also noted in cholesterol, creatinine, phosphorus, calcium, potassium, and 

urea nitrogen; however, the relevance 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/day) was considered adequate based on the 

mortality, clinical signs and cholinesterase inhibition observed at 1000 mg/kg/day. 

Male Pubertal Assay; (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 malathion 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 sacrifice on PND 53, blood was collected for clinical 

chemistry analyses, including total thyroxine (T4), testosterone, and thyroid stimulating hormone 

(TSH). Testosterone, T4, and TSH were analyzed using radioimmunoassay kits. Kidney, liver, 

adrenals, pituitary, thyroid, and urogenital organ weights were recorded, and microscopic 

examinations were performed on the thyroid, kidney, testis and epididymis. In addition to the 

main study groups, additional groups of 8 male rats/dose were used in a cohort that examined 

blood and brain cholinesterase (ChE) activity at 2 hours after the last dose on PND 53. 

In the main study, 14 rats from the high-dose group 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, the observed clinical signs included effects on gait 

(e.g., ataxia, lost righting reflex, and decreased motor activity), irregular 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 chromorhinorrhea). 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 final 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 first day of PPS in the high-dose group. 

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Compared to the control group, absolute 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, respectively. The liver and 

kidney weight effects are treatment related and are 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 changes in weights of accessory sex tissues. 

There were no other significant effects on organ weights. 

Serum T4 concentrations in the low-, mid-, and high-dose groups were increased (p≤0.01) by 41, 

35, and 32%, respectively, but these changes were not dose-dependent, the T4 levels were within 

the range of historical control values, and there were no related changes in TSH levels. In 

addition, there were no changes in thyroid weight or any microscopic findings in the thyroids of 

malathion-treated rats. Therefore, the biological relevance of the changes in T4 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 bilirubin 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 were 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 evaluated in the high-dose group. 

Based on the ChE data, and the mortality and clinical signs, both the mid- and high-doses (500 

and 750 mg/kg/day) of malathion were clearly excessive for immature male 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 T4 were increased to a similar (non-dose dependent) extent at all doses. 

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Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48617509), H295R cells cultured in vitro in 24-well plates 

were incubated with malathion (96% purity, 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 levels were measured using HPLC/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 testosterone and estradiol concentrations 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 concurrently with each independent run of the test 

chemical plate to demonstrate that the assay responded properly to positive control agents at two 

concentrations; positive controls included the known inhibitor (prochloraz) and inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV), and appropriate induction and inhibition with positive controls. It was stated that all 

assays were performed at CeeTox by personnel that have demonstrated proficiency performing 

this assay, however, proficiency records were not provided. 

The only significant changes noted in hormone production of malathion treated cells were a 

decrease in testosterone production 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 malathion in Run 2 (p<0.05; 1.12-fold 

change). The validity of the observed decrease in testosterone is questionable as precipitation of 

malathion was observed at 100 μM in Runs 2 and 3, and there was no other concentration 

dependent effects noted in testosterone production. Similarly for the observed increase in 

estradiol, the effect was observed at 10 μM in only one of three runs, and no concentration 

dependent changes in estradiol production were observed. 

Based on hormone response in at least three independent runs, malathion treatment did not result 

in statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a uterotrophic assay (MRID 48617510) conducted to screen for potential estrogenic activity, 

malathion (96.0% a.i., Batch/lot 9010501) in corn oil was administered daily via oral gavage to 

groups of eight ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 100, 

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300, or 1000 (limit dose) mg/kg/day on post-natal days (PND) 68-70. A positive control group 

was treated with a daily dose of 17α-ethynyl estradiol (EE) at 5 μg/kg/day by subcutaneous (s.c.) 

injections. Body weights and food consumption were determined daily. All animals were 

terminated and necropsied on PND 71 approximately 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 control 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 rats 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 relative 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 duration 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 weights were in the expected range. 

No statistically significant changes were seen in uterine weight in this assay. Malathion is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of malathion with the endocrine system are summarized below. Given the 

breadth of toxicity data available on malathion, 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 (E2) 

binding to the receptor. The general procedures used were clearly described and were similar to 

the procedures specified in the test Guideline, including the 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 (hERα and 

hERβ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster 

ovary cells (CHO-K1 cells) was investigated. For detection of hERα or hERβ activity, cells were 

transfected with pcDNAERα or pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of 

hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and 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 17β-estradiol (E2; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) for 

the ER or AR assays, respectively. For measurement of antagonistic activity to hERα, hERβ and 

hAR, either 10−11 or 10−10 M E2 or 10−10 M DHT was added to the cell cultures along with the 

test compound, respectively. Agonistic activities were evaluated by the relative activity 

expressed as 20% relative effective concentration (REC20), the concentration of the compound 

demonstrating 20% of the activity of 10−10 M E2, 10−9 M E2, or 10−9

 M DHT for ERα, ERβ, and 

AR, respectively. Antagonistic activities were evaluated similarly by the relative activity 

expressed as 20% relative inhibitory concentration (RIC20), the concentration of the compound 

showing 20% inhibition of the activity of E2 or DHT against ERα, ERβ, and AR, respectively. 

Malathion did not show agonist or antagonist activity for ERα, ERβ, 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 after exposure to thirty two pesticides 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. 

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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 promoter, confer estrogen responsiveness to the 

viral promoter and reporter gene. The transfected cells are made stable by an antibiotic resistance 

gene resulting in the cell line used in the assay (BG1Luc4E2). Compounds that interact with 

estrogen-responsive DNA are identified by the luciferase gene response and quantified in terms 

of Relative Light Units (RLU). 

The BG1Luc4E2 cells were grown in medium for four days, then the assay medium was 

replaced, and cells were transferred (2 × 105 cells/mL) into 96-well plates and incubated at 37 ̊C 

with 5% CO2 for 24 hours. The medium was removed, and the test compound or estrogen 

standard (E2) was added. After incubation at 37 ̊C with 5% CO2 for another 24-hour period, the 

medium was removed and the cells were lysed. Luciferase activity was measured with a 

luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes, 

the EC10 (Effective Concentration at which 10% of the maximum response was observed) was 

used. The data were not sufficient to allow calculation of an EC50. 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 aromatase activity were investigated. The 

androgenic, anti-androgenic, estrogenic, and anti-estrogenic properties were determined from in 

vitro tests including an ER transactivation assay using MCF-7 derived MVLN cells and an AR 

transactivation assay using the Chinese hamster 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 concentrations within the range of 1×10-10 to 1×10-4 M. In the ER 

transactivation assay with MVLN cells, malathion weakly (113% of control) induced luciferase 

activity. Co-exposure with 25 pM E2 (E2-EC65; 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, malathion was not cytotoxic at the highest 

concentration tested (1×10-5 M) and did not elicit an AR agonist or antagonist response in this 

assay. In the aromatase activity 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 

125I-labeled T3 to Japanese quail (Coturnix japonica) thyroid transthyretin (TTR) and the ligandbinding domain of the quail thyroid receptor (TR) were examined in an in vitro cell-free assay. 

Blood was collected from quail, serum was separated, and the quail TTR was isolated with a 

column chromatographic method. A recombinant plasmid containing the ligand binding domain 

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(LBD) of the quail TR was cloned into Escherichia coli to generate a GST-fusion protein 

containing the LBD of TR. The binding of T3 to TTR and TR was measured by using 

radiolabeled T3 in the presence and absence of increasing concentrations (range not reported) of 

malathion. Malathion inhibited binding of T3 to quail TTR with an IC50 of 1,400±370 nM. The 

IC50 for T3 in this system was 9.7±0.9 nM. Malathion had no effect on T3 binding to the quail 

TR. 

21-Day (Subchronic) Dermal Toxicity 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 days/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 treatment (serum and RBC) and at study termination 

[serum, RBC, and brain regions (i.e., cerebrum and cerebellum)]. Liver, kidneys, ovaries, 

testes/epididymides, and adrenals 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 chemistry, organ weights (except for adrenals in 

males), macro- and microscopic lesions were comparable between the treated 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 females. Statistical re-analysis demonstrated 

significance (with or without this value included), but the pertinent treatment comparisons were 

not reported. The only consistent effects observed were on inhibition of ChE activity. Serum, 

RBC, and brain ChE activities were decreased (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/day, 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. 

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13-Week (Subchronic) Inhalation Toxicity in Rodents (Rat) 

In a 13-week subchronic inhalation 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 concentrations 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 signs of toxicity, consistent with decreased ChE 

activity, were observed in most animals at the high dose and sporadically in a few animals at the 

intermediate and low doses. These signs, which occurred in both males and females, 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). 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 variability impacted statistical 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%, respectively. For males, the NOAEL and LOAEL, 

respectively, are 0.45 mg/L and 2.01 mg/L for plasma and brain ChE inhibition and 0.1 mg/L 

and 0.45 mg/L for RBC ChE inhibition. For females, the LOAEL for plasma and RBC ChE 

inhibition is 0.1 mg/L and a NOAEL is not determined. The 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 males and females. Non-significant increases in 

liver and kidney weights were occasionally observed at the intermediate dose. No treatmentrelated effects on organ weights or histopathology were observed in any endocrine-related 

tissues. The only treatment-related histopathological lesions noted in the study were lesions in 

the nasal cavity and larynx observed in nearly all animals (10 to 15/15 at the low dose and 14 to 

15/15 at the intermediate and high 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 inhibition 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 systemic effects, a NOAEL is not determined in 

either males or females (<0.1 mg/L). The LOAEL 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 studies in rats with both malathion and malaoxon, it 

is not clear whether these lesions are local or systemic in nature. 

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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 concentrations 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 fetuses in each litter were examined for visceral soft 

tissue alterations using a variation of Wilson’s sectioning technique. The remaining one-half of 

the fetuses in each litter were examined for skeletal alterations. 

There were no mortalities; clinical signs of toxicity observed 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, respectively, 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, implantations, 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 (400 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 examining 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 peanut 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 developmental 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 control and low-protein diet groups (8 and 18%, respectively) with 

malathion treatment. In rats fed the control diets plus malathion, significant (p<0.0001) decreases 

were seen in fetal weights, crown-to-rump lengths, tail lengths, umbilical cord lengths, placental 

weights, and amniotic fluid weights. In rats fed the protein-deficient diets plus malathion, 

significant (p<0.0001) decreases were seen only the tail lengths and placental and amniotic fluid 

weights. No skeletal anomalies were seen except for poor ossification in the protein-deficient 

dams, which were exacerbated by malathion exposure. ChE activity in the brain was decreased 

by 24 and 28%, respectively, in the control and protein-deficient animals treated with malathion. 

The fetal effects showed that protein deficiency intensified malathion-induced toxicity. 

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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 dose 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 euthanized 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 intermediate dose; causes of death were not reported. 

Two deaths in high-dose animals were attributed to pulmonary intubation during dosing. 

Treatment-related clinical signs observed in rabbits administered the high dose consisted of 

anorexia and soft stools. Although no treatment-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 reproductive and gestation indices was an increase 

(significance not reported) in the number of resorptions per doe (mean percent resorptions) in the 

mid- and high-dose does (↑87 and 82%, respectively). No treatment-related abortions were 

observed. Live fetal weights were not affected by malathion treatment. No treatment-related 

skeletal retardations were noted during the fetal examinations, and there were no treatmentrelated external, visceral, or skeletal 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 NOAEL 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 malathion) in corn oil from GD 7 through 12. Does 

were sacrificed on GD 27, and reproductive 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 gavage at doses of 0, 5.0, 50, 

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or 150 mg/kg/day in corn oil from GD 6 through 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 evaluated as follows: age-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 response 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 males 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). There were no other treatment-related effects on 

cholinergic signs, and there were no effects on maternal body weight, food consumption, or 

reproductive indices. The maternal LOAEL for malathion in rats is 150 mg/kg/day based on an 

increased incidence of post-dosing salivation, and the maternal NOAEL is 50 mg/kg/day. 

There were no apparent treatment-related clinical signs noted in offspring prior to direct 

treatment starting on PND 11. In addition, no effects 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 ambulatory (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 surface righting reflex in PND 11 female 

pups, and increased thickness of the corpus callosum in PND 63–67 males 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 treated on PND 11. Repeated exposure to malathion 

resulted in decreases in ChE activity in the blood and/or brain in dams, fetuses, weanling pups, 

and adult male and female SD rats. In pups, effects on RBC ChE activity were noted at 

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5 mg/kg/day in both sexes on PND 21 after 11 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 11 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 

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 LOAEL 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 Reproduction in Rodents (Rat) 

In a two-generation, two-litter reproduction study (MRID 41583401), male and female SD rats 

(25/sex/dose) were administered malathion (94.0% purity) in the diet to the F0 and F1 parental 

generations at dosages of 0, 550, 1700, 5000, or 7500 ppm. Mean-measured intake during the 

premating period in the F0 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-measured intake during the premating period 

in the F1 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 was 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 F0

generation was treated for 63 days prior to the initial mating and the subsequent mating to 

produce the second litter was initiated after a 2-week resting period following weaning of the 

first litter. The F1 generation was treated for 79 days (100 days of age) prior to mating, with the 

same schedule described previously to produce the second litter. 

After weaning of the second litters and selection of the F1 parental animals, the F0 parents were 

sacrificed and subjected to a gross examination. Tissues collected for possible histopathological 

examination included: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, 

and pituitary. The F1 parental animals were sacrificed according to the same regimen after 

weaning and sacrifice of the F2 litters. The tissues listed above were examined microscopically 

from all F0 and F1 control and high-dose animals. 

There was no evidence of a treatment-related effect on survival; five animals in each parental 

generation died or were sacrificed moribund in the control and treatment groups. Clinical signs 

reported for all animals (unrelated to treatment) included chromodacryorrhea, excess lacrimation, 

and alopecia (females only). In the F0 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 F1 generation, body weight was decreased (p≤0.01 or 0.05) at the high dose during 

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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 gestational period, and at the medium-low and low 

doses on GD 14 and 20, respectively, during the 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 F1 generation, the lactation 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 F2 generation, fertility indices in the control and treated animals for F2b litters 

were slightly reduced (range: 70 to 83%) compared to the other litters (range: 88 to 100%). 

Viability indices were decreased (p≤0.05) at the medium-high dose 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 weights were not conducted in this study. No 

treatment-related histopathological 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 LOAEL is 7500 ppm (612 mg/kg/day in males and 

703 mg/kg/day in females) based on decreased body weights in F0 females during gestation and 

lactation and on decreased body weights in F1 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 the lactation period in 

F1A and F2B pups. The reproductive toxicity NOAEL is >7500 ppm (612 mg/kg/day in males and 

703 mg/kg/day in females). The reproductive toxicity LOAEL is >7500 ppm (612 mg/kg/day in 

males and 703 mg/kg/day in females). No reproductive 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 malathion (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 unclear as it is reported as a negative effect, instead 

of a decrease from control (100%). Dose-related decreases (p<0.001) in testicular and 

epididymidal sperm density were also observed. Serum testosterone concentrations were 

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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 intermediate 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 [equivalent 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 each period) and at 12 months 

(15/sex/dose). The initial low dose (100 ppm) was decreased to 50 ppm at 3 months due to RBC 

ChE inhibition that was observed in females at the 3-month interim sacrifice. After 104 weeks, 

all surviving rats in all groups were sacrificed for gross and histopathological evaluation. Select 

organs were weighed at each sacrifice interval (including liver, kidneys, testes, epididymides, 

ovaries, adrenals, and thyroid/parathyroids). Gross and microscopic pathological examination 

included the liver, kidney, pituitary, thyroid/parathyroids, adrenals, mammary gland, testes, 

epididymides, prostate, seminal vesicles, uterus, and ovaries. 

The only clinical sign observed was yellow anogenital staining among females 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%, respectively) and high-dose (12 to 32% and 10 to 15%, 

respectively) groups. Food consumption was generally similar to control 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 the medium-high or high-dose groups. Additionally, 

several clinical chemistry parameters (primarily liver-related enzymes or compounds; e.g., 

aspartate and alanine aminotransferases, alkaline phosphatase, gamma glutamyltransferase, and 

cholesterol) were generally affected (p<0.05 or 0.01) in the medium-high or high-dose animals. 

Inhibition (p<0.01 or 0.05) of ChE 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-dose males at study termination (100% mortality), 

but ChE activity at study termination in the high-dose females was decreased 89, 52, and 67% in 

plasma, RBC, and brain, respectively. Dose-dependent 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 in 

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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 months (prior to decrease from 100 to 50 ppm). 

Organ weight effects included increased (p<0.01) liver weights in the medium-high and highdose males (↑22 and 36%, respectively) at the interim sacrifice, 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 termination. Increases (p<0.01) were also 

noted in thyroid/parathyroid 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 increased (NS) by 11% at termination in the mediumhigh males. Microscopic findings were varied, but did not demonstrate any consistent effect on 

endocrine-related endpoints or tissues. 

Treatment-related increases in tumors included combined hepatocellular adenomas/carcinomas 

in females across the treatment groups. Other 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 been 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 overall NOAEL is 50 ppm (4 mg/kg/day) and the 

LOAEL is 500 ppm (29 mg/kg/day) based on inhibition of plasma activity at 24 months. The 

overall (beyond Month 3) NOAEL for ChE inhibition in females is 50 ppm (5 mg/kg/day) and 

the LOAEL is 500 ppm (35 mg/kg/day) based on inhibition of RBC activity. A NOAEL for a 

3-month exposure period is not established in females. 

No significant treatment-related changes 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, prostate, uterus, ovaries, and mammary gland. 

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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 mice and rats. These doses were required by EPA 

to duplicate the doses used in a previously conducted carcinogenicity study (NCI, 1978) in 

B6C3F1 mice that yielded equivocal results. A subset of study animals (10 mice/sex/group) was 

sacrificed at 12 months, and the remaining survivors 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 determinations were conducted at the interim and 

terminal sacrifices. Histopathological examinations were made on a complete set of 

organs/tissues from all animals that died or were sacrificed in extremis during the study, and on 

all control and high-dose animals at the interim and terminal sacrifices. 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 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 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 and 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 hepatocellular 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 at 

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18 months, the combined hepatocellular adenomas/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. Concurrent hepatic observations included masses, nodules 

and foci in these animals at the terminal sacrifice, as well as increased liver weights and steeply 

increased incidences of hepatocellular hypertrophy in the livers of the medium-high and highdose animals. Additional findings were noted in the kidney and adrenal cortex. 

The NOAEL for ChE inhibition for both sexes was estimated to be 17.4/20.8 mg/kg/day (LDT) 

for plasma and RBC ChE and 1476/1707 mg/kg/day (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, and 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 plasma, RBC, and selected brain regions (cerebrum 

and cerebellum only). Organ weights determined included adrenals, kidneys, liver, ovaries, 

testes, and thyroid; histopathological 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. Several 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 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 sample 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 approximately 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 increased in low- and high-dose females only (↑40 and 41%). In females, 

thyroid/parathyroid combined weight exhibited a dose-related upward trend with absolute weight 

increased (p<0.05) at the high dose. This parameter was increased (NS) in males at all doses. No 

endocrine-related organ weight changes were observed at study termination. Gross and 

microscopic pathology were generally unremarkable, with no treatment-related findings in 

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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 deficiency could be addressed by conducting and 

submitting a 90-day subchronic study in dogs. The most recent re-registration eligibility decision 

(RED) for malathion (USEPA, 2006) lists the present 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 variations 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 increased clinical signs 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 (epithelium 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 LD50) was administered to male Wistar rats (18 control and 32 treated). Animals were 

sacrificed on Days 1, 2, 3, 6 and 12 post-injection. 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 tubules 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. 

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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 affected 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 pachytene 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 reduced due to malathion treatment, with subsequent damage to 

spermatogenetic cells. In a published follow-up study (Krause, 1977) examining effects on 

spermatogenesis in rats, malathion (purity not reported) in peanut oil was administered by 

gavage to adult Wistar rats at 20 mg/kg every second day for two weeks to 7 males. Three of the 

seven animals died during treatment. Animals were 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 histopathological 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 percentage of abnormal sperm 

(↑59%). In addition, there were fewer spermatogenic 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 reproductive 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 mortality, food 

intake or body weight were noted in the 10, 50, and 100 ppm dose groups, and only slight 

variations 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. 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 tubules (loss of normal pattern of the Sertoli cells) were seen. At 

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3000 ppm, marked changes were seen in the seminiferous 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 Swiss albino rats (13/sex/dose) at doses of 0 

(negative control), 0 (vehicle control), 10, or 100 mg/kg/day for 15 weeks. The parameters 

evaluated were: T3, T4, aldosterone, cortisol, testosterone (male), and 17β-estradiol (female) 

concentrations, and histopathology of the testes, ovaries, adrenal, and thyroid glands. 

No difference was observed between negative and vehicle control animals. No treatment-related 

changes were seen in T3, T4 (NS; ↑14% at high dose), testosterone (NS; ↓10 and 20% at the low 

and high doses, respectively), and 17β-estradiol concentrations at either dose. Significant 

decreases in aldosterone and cortisol concentrations were seen at the low dose, but not at the 

high dose. No histopathological changes were seen in the thyroid or ovaries. Histopathology 

revealed hyperemia of the veins and slight degeneration of the seminiferous tubules at both doses 

in the testes, and adrenals exhibited congestion 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% purity) 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 for 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% relative 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 biweekly. The average minimum and maximum 

temperatures in the hatchling study room ranged from 22 to 27°C and relative humidity ranged 

from 43 to 87%. Hatchling body weights were measured 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-dose group with neither death attributed to 

treatment; no overt signs of toxicity or clinical signs were observed 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. Apparent 

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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 number 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-measured malathion concentrations in 

the diet were not indicated. Each dose group consisted of 32 quails, with a single cock and hen 

paired in 16 pens/dose group. The photo period was 8 hours/day for the first 8 weeks, increased 

to 17 hours/day for the final 13 weeks of the study. 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 study termination. Eggs were collected daily, and 

stored at 12.8°C and 84% relative humidity. Eggs were candled on Days 11 (viability) and 21 

(survival), and eggs were placed in a hatcher on Day 21 (37.2°C and 76% humidity). Hatchling 

housing details were not provided; hatchlings were weighed on Day 14. ChE determinations 

were not conducted during this study. 

Mortality occurred in all treatment groups (one each in the control, low- and mid-dose groups, 

and nine in the high-dose group). Necropsy revealed regressing ovaries and enlarged/flaccid 

gizzards in mid- and high-dose adults. Adult body weights gains were decreased (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 cracked 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, 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 hatchlings/normal hatchlings). Eggshell thickness was 

also decreased (p<0.05; ↓15.5%) at the high dose. Hatchling and 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 malathion 

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(95% purity) in the feed at nominal concentrations 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 concentrations in the diet were not indicated.

Insemination was accomplished with a composite sample of undiluted semen obtained from 

caged White Leghorn males fed the same diet as the control pullets. Pullets were inseminated 

once a month with a volume of 0.05 mL/bird. Two days after insemination, 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 examined 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 study 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 batteries and fed their respective diets for 4-week 

periods to assess growth and mortality. Finally, at the termination of the reproduction/progeny 

assessments, males were retained for a 4-week trial to determine the effect of malathion 

administration on fertilizing capacity. Groups (5/treatment) were fed the same breeder diet 

supplemented with 0 or 500 ppm. Females used in this study were those that had been fed either 

0 or 500 ppm of malathion in the previous experiment, and were continued on their respective 

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 from each group of males 

and used to inseminate both non-supplemented and 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 malathion administration were observed on pullet body 

weight gain, egg production, egg weights, 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 untreated and treated pullets, respectively. The 

NOAECs for reproductive performance, progeny performance, and male fertilization capability 

are 500 ppm (HDT); a LOAEC is not 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 control 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. Eggs were exposed from Day 1 (fertilization) 

until hatch (≥99% by Day 37); exposure was continued for 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. 

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Mean egg viability was determined to be 99%. The percent hatch was ≥99% complete in all 

replicates by Day 37; no treatment-related effect on time to hatch 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 the chambers, discoloration, spinal curvature and quiescence. Fry 

survival on post-hatch Day 35 was decreased (significance not reported) 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%, respectively. Mean length was decreased (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 (growth, survival, and reproductive capacity) 

were assessed in flagfish (Jordanella floridae) exposed to malathion (95% purity) at nominal 

concentrations of 0 (water only; negative 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 reduced to two males and five females by 

random sampling from those fish that displayed secondary sex characteristics. At that time, 

spawning substrates composed of yarn-covered stainless steel screen were 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. Four to seven groups of twenty 1-day old secondgeneration larvae were reared for 30 days in each 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 during the same period was reduced (p=0.05) at 

the two highest concentrations, 24.7 and 31.5 μg/L (↓31 and 33%, respectively), with a decrease 

of 13% (NS) in the 19.3 μg/L concentration group. However, no additional adverse survival 

effects were detected in any of the concentrations during the Day 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 concentrations demonstrated adverse effects on 

survival through hatching, and subsequent survival and growth, of the second generation larvae. 

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APPENDIX 3: References Not Utilized in the Malathion WoE Analysis 

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

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

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

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

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

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

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

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

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

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

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Malathion but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

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