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 40
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 40
 

EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: ACETONE

OFFICE OF PESTICIDE PROGRAMS 

OFFICE OF SCIENCE COORDINATION AND POLICY 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

Page 3 of 40
ACETONE 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......................................................11 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis................................14 

IV. Committee’s Assessment of Weight of Evidence..............................................16 

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .........................16 

1. Tier 1 in vivo Assays.................................................................17 

2. OSRI...................................................................................17 

B. Estrogen Pathway...............................................................................18 

C. Androgen Pathway.............................................................................18 

D. Thyroid Pathway...............................................................................18 

E. Conclusions.....................................................................................18 

V. EDSP Tier 2 Testing Recommendations.........................................................18 

VI. References...........................................................................................19 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Acetone........................................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Acetone...........................................9 

 Table 3. Androgenic/Anti-Androgenic Pathway for Acetone......................................12 

 Table 4. Thyroid Pathway for Acetone................................................................15 

APPENDIX 1. EDSP Tier 1 Screening Assays...........................................................21 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................30 

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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 

F1 First filial generation 

Page 5 of 40
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iv 

Abbreviation Terminology 

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 Methods

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

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v 

Abbreviation Terminology 

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

T3 Triiodothyronine 

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vi 

Abbreviation Terminology 

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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Page 1 of 32

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-pituitarygonadal (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 acetone 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 February 5, 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 acetone with 

the E, A or T signaling pathways. The T1WoERC conclusions from the WoE evaluation in this 

report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro

assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for 

mammalian and wildlife studies (40 CFR Part 158 and literature).

For acetone, there was no convincing evidence of potential interaction with the estrogen pathway 

in mammals or wildlife. Acetone was negative in the Tier 1 in vitro and Tier 1 uterotrophic

assays. In the female pubertal assay, while increases in uterine weight were observed, there were 

no other effects observed in the study including histopathology. Additionally, it is noted that 

this isolated finding was observed at the limit dose (1000 mg/kg/day) and half the limit dose 

(500 mg/kg/day). Furthermore, there were no effects on female mammalian reproductive 

parameters observed in the mammalian OSRI. In the Fish Short Term Reproduction assay 

(FSTRA), while there was a significant decrease in fecundity at the intermediate concentration of 

6.2 mg a.i./L (↓51%), there was high variability in fecundity among the treatment groups in this 

study as well as a lack of complementary responses across other endpoints. 

There was no convincing evidence of potential interaction with the androgen pathway in mammals 

or wildlife. Acetone was negative in the Tier 1 in vitro and in vivo assays. In the male pubertal 

assay, while there was an increase in serum testosterone concentration at the limit dose (1000 

mg/kg/day) there were no effects on the other androgen-related endpoints including age of 

attainment of preputial separation, male reproductive organ weights and no corroborating 

microscopic findings. While alterations in the androgen-related tissue weights and effects on 

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Page 2 of 32

sperm were observed in the mammalian OSRI, these effect occurred at very high doses (2500-

6900 mg/kg/day). In the FSTRA, there were no androgen-related effects observed. 

For thyroid pathway, there were no thyroid-related effects observed in mammals or amphibians. 

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

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

androgen or thyroid pathways. 

 

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Page 3 of 32

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-pituitarygonadal (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 acetone 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 February 5, 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 acetone with 

the estrogen (E), androgen (A) or thyroid (T) signaling pathways. The T1WoERC conclusions from 

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

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

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

Acetone is an organic compound (also known as propanone and dimethyl ketone). The compound 

is miscible in water and has a low octanol/water partition coefficient (log Kow = -0.24). Acetone has 

a whole body bioconcentration factor value in fish of approximately 3, suggesting that the potential 

for bioconcentration in aquatic organisms is low. Acetone has a high vapor pressure (231 mm Hg at 

25°C), and, therefore, it volatilizes readily and should exist solely as a vapor in the atmosphere. 

Acetone is not degraded by hydrolysis, but does undergo photo-decomposition. 

The available information considered in determining the potential interaction of acetone with the E, 

A, T pathways include submitted EDSP Tier 1 assays and other scientifically relevant information 

(OSRI) such as general toxicity studies and/or other published articles. These data are summarized in 

Sections III.A through III.C. An analysis of the data submitted to the agency, using the WoE 

approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 

studies recommendations are presented in Section V. 

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Page 4 of 32

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

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

assays were submitted as OSRI by the Test Order recipients and/or the public, including studies 

published in the open literature. In addition, the Agency has conducted a more recent search of 

available scientific literature for any additional relevant information on acetone. Summaries of the 

available OSRI are presented in Appendix 2. 

III. Weight of Evidence (WoE) Evaluation 

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

to interact with endocrine-related processes (i.e., E, A or T hormonal pathways) were as described in 

the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA Scientific Advisory 

Panel (SAP) (USEPA 2013). The weight of evidence process identifies how the individual lines of 

evidence are assembled and integrated along two concepts (i.e., complementarity and redundancy) 

within the conceptual framework of an adverse outcome pathway). Broadly, there are four main 

steps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to 

evaluate the individual studies for their scientific quality and relevance in evaluating potential 

endocrine interaction(s). The second step is to integrate the data along different levels of biological 

organization while examining the extent of complementarity (i.e., the concordance of endpoints 

within an assay that measures multiple endpoints) and redundancy (i.e., the concordance of 

endpoints/responses across assays) in the observed responses across these different levels of 

biological organization. The third step is to characterize the main lines of evidence as well any 

conclusions. Finally, the last step is to evaluate whether additional testing is needed based on the 

evidence and conclusions described above. 

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

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

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

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

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Page 5 of 32

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 toxicity introduces uncertainty in 

the ability to distinguish effects specifically related to an endocrine-related effect from a nonendocrine 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, the SAP stated that , “In summary, the Panel agreed 

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

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

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

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Page 6 of 32

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., change 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 case-by-case analysis which took all 

available toxicity data into account 

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. 

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Page 7 of 32

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for acetone are shown below in 

Table 1. 

Table 1. Tier 1 Assay for Acetone

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay 

(Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied 

(MRID No. 48614604) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48614605) 

AR Binding Assay 

(Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied 

(MRID No. 48614602) 

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.155; 

OECD 456 

Requirement Satisfied 

(MRID No. 48614610) 

Aromatase Assay 

(Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied 

(MRID No. 48614603) 

Uterotrophic Assay (Rat) OCSPP 890.160; 

OECD 440 

Requirement Satisfied 

(MRID No. 48614611) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48614607) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 48614608) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

(MRID No. 48614609) 

Fish Short-term Reproduction Assay OCSPP 890.1350 

OECD 229 

Requirement Satisfied 

(MRID No. 48614606) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48614601) 

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Page 8 of 32

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

1. Effects on Estrogen Pathway 

The potential for acetone 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 the 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 estrogenic/anti-estrogenic activity from Tier 1 testing results, acetone was 

classified as negative (or not interactive) in the in vitro ER binding, ERTA, steroidogenesis and 

aromatase assays. In the uterotrophic assay, acetone did not induce uterine weight increases when 

administered at doses up to and including the limit dose (1000 mg/kg/day). 

In the female pubertal assay, there were no treatment-related effects on mean estrous cycle length, 

percent cycling or percent regularly cycling animals, ovarian weight or histopathology up to and 

including the limit dose of 1000 mg/kg/day. Mean absolute wet and blotted uterine weights were 

increased (p<0.05) in both the low, 500 mg/kg/day, (↑15% and 9%, respectively) and high, 1000 

mg/kg/day, (↑31% and 20%, respectively) treatment groups. There was no reported estrogen-related 

effects in the mammalian OSRI. 

In the FSTRA assay, there was a significant decrease (p<0.05) in fecundity seen at the intermediate 

concentration of 6.2 mg a.i./L (↓51%); however, there was high variability in the fecundity endpoint 

across treatment groups in this study. There were no other estrogen-related effects observed. Female 

body weight was significantly (p<0.05) increased by 11% in the high concentration. 

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Page 9 of 32 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Acetone

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Acetone1

Study 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 Assays

ER Binding (MRID 48614604) 

N

ERTA (MRID 48614605) N Aromatase (MRID 48614603) N Steroidogenesis (MRID 48614610) N Uterotrophic (MRID 48614611) 

N

Female Pubertal Rat (MRID 48614608) ↑9-15% (L) 

20-31% 

(H) 

N N N 

N

N

FSTRA (MRID 48614606) NE 

N

N

N ↓51% (M) 

N
ACETONE FINAL 

Page 10 of 32 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Acetone

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Acetone1Study 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 

OSRI

Oral Subchronic (14-Day) Rat (NTP, 1991; Dietz et al., 1991)

N

N N N 

Oral Subchronic 

(14-Day) Mouse (NTP, 1991; Dietz et al., 1991)

N

N N N 

Oral Subchronic 

(90-Day) Rat (NTP, 1991; Dietz et al., 1991)

N

N N N 

Oral Subchronic (90-Day) Mouse (NTP, 1991; Dietz et al., 1991)

N

N N N 

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

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

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for th

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 

For details see Section IV. A 

N Negative findings (in vitro)/No effect (in vivo) 
ACETONE FINAL 

Page 11 of 32 

2. Effects on Androgen Pathway 

The potential for acetone 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 the 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, acetone was 

classified as negative (non-binder) in the in vitro AR binding and the steroidogenesis assays. 

In the Hershberger assay, acetone did not induce significant increases in accessory sex tissue 

(AST) weights in the androgenic portion of the assay or significant decreases in the antiandrogenic portion of the assay when tested up to and including the limit dose of 1000 mg/kg/day. 

In the male pubertal assay, serum testosterone concentration was increased (140%; p<0.001) at 

the high-dose animals (1000 mg/kg/day). It is noted that mean serum testosterone value (4.84 ng/ 

mL) for the high-dose group was only slightly above the range of historical controls (1.62–4.20 

ng/mL). There were no other androgen-related effects observed in this study. In the FSTRA 

assay, there were no androgen-related effects observed. 

There was limited evidence of effects on any male mammalian reproductive parameters in the 

mammalian OSRI. Relative testes weights in F344/N rats were increased when administered via 

drinking water at very high doses (4300 and 6900 mg/kg/day) in a 14-day study. In the 90-day 

rat drinking water study at 2500 mg/kg/day, relative testes weights were increased as well as 

decreases in sperm motility, caudal weight, epididymal weight and an increased incidence of 

abnormal sperm (NTP, 1991; Dietz et al., 1991). 

Page 19 of 40
ACETONE FINAL 

Page 12 of 32

 Table 3. Androgenic/Anti-Androgenic Pathway for Acetone

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acetone1

Study Type/ Literature

Citation

AR Binding

Steroidogenesis

Sex Steroid 

Hormones

Testes

Weight/GSI

Gonad Staging 

and 

Histopathology

Epididymides

Weight

Epididymides 

Histopathology

Pituitary Weight

Accessory Sex

Organ Weights/

2° Sex

Characteristics

Fertility(Frt)

/Fecundity (Fcd)

Age and Weight at

PPS

Vitellogenin

EDSP Tier Assays

AR Binding (MRID 48614602)

N

Steroidogenesis (MRID 48614610)

N

Hershberger

(MRID 48614607)

N

N

Male Pubertal Rat 

(MRID 48614609)

N

N

N

N

N

N

N

N

FSTRA (MRID 4861606) NE

N

N

N

N

N
ACETONE FINAL 

Page 13 of 32

 Table 3. Androgenic/Anti-Androgenic Pathway for Acetone

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acetone1

Study Type/ Literature

Citation

AR Binding

Steroidogenesis

Testosterone

Testes

Weight/GSI

Gonad Staging 

and 

Histopathology

Epididymides

Weight

Epididymides 

Histopathology

Pituitary Weight

Accessory Sex

Organ Weights/

2° Sex

Characteristics

Fertility(Frt)

/Fecundity (Fcd)

Age and Weight at

PPS

Vitellogenin

OSRI

Oral Subchronic 

(14-Day) Rat (NTP, 1991; Dietz et al., 1991)

↑

(H)

N N N N 

N

Oral Subchronic 

(14-Day) Mouse (NTP, 1991; Dietz et al., 1991)

N 

N N N N 

N

Oral Subchronic 

(90-Day) Rat (NTP, 1991; Dietz et al., 1991)

↑

(H)

N ↓(H) N N 

N P4 

Oral Subchronic 

(90-Day) Mouse (NTP, 1991; Dietz et al., 1991)

N 

N

N N N 

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. 

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 

4. Decreased in sperm motility, caudal weight, epididymal weight, and an increased incidence of abnormal sperm. 

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

Page 14 of 32

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 

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

The potential for acetone to interact with the thyroid pathway is summarized in Table 4. The 

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

facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPTrelevant findings from data evaluated as OSRI. This table also includes HPG-relevant findings 

from the 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 the male and female pubertal assays, the only effects observed at 1000 mg/kg/day were 

statistically non-significant increases in thyroid weight in females (↑10%) and increases in TSH 

concentration in males (↑22%). There were no effects on T4 or histopathology in either sex. 

In the AMA, there were no significant alterations in developmental stage (including no 

asynchronous development) as well as no effects on thyroid histopathology. A significant 11% 

increase in day 21 normalized hind limb length (HLL) was observed at the high concentration 

(95.83 mg a.i./L). Additionally, Day 7 body wet weight was decreased in a dose-responsive 

manner, with a significant decrease of 11% in the high-concentration group. Since there were no 

effects on development or thyroid histopathology, the change in HLL was considered to likely not 

be thyroid-related. Clinical signs of tail curvature were observed in all treatment groups, with a 

higher incidence in the treated groups relative to the negative control. 

Thyroid effects were not observed in any OSRI toxicity studies conducted with acetone. 

Page 22 of 40
ACETONE FINAL 

Page 15 of 32

Table 4. Thyroid Pathway for Acetone

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Acetone1

Study Type / 

Literature Citation

Thyroid Weight

Thyroid: Gross and HistopathologySerum 

T4 Levels

Serum TSH Levels

Pituitary WeightDevelopmental stage (+ or asynchronous & HLL)

Growth (BW, SVL)

Systemic Toxicity Observed2Overt Toxicity Observed3

EDSP Tier 1 Assays

Female Pubertal Rat 

(MRID 48614608) N N N N N N N N

Male Pubertal Rat 

(MRID 48614609) N N N N N N N N

AMA (Frog; 

MRID 48614601) N ↑11% (H) ↓11% (H)

BW N

OSRI

Oral Subchronic 

(14-Day) Rat 

(NTP, 1991; 

Dietz et al., 1991)

N N N N N X

(M, HM, H) N

Oral Subchronic 

(14-Day) Mouse 

(NTP, 1991; 

Dietz et al., 1991) 

N N N N N

X

(M, MH, 

H) N

Oral Subchronic 

(90-Day) Rat 

(NTP, 1991; 

Dietz et al., 1991) 

N N N N N X 

(MH, 

H) 

N

Oral Subchronic 

(90-Day) Mouse 

(NTP, 1991; 

Dietz et al., 1991) 

N N N N N X 

(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. 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the 

OSRI is indicated by an X in this column. For details see Section IV. A 

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

an X in this column. For details see Section IV. A 

N Negative findings 

Page 23 of 40
ACETONE FINAL 

Page 16 of 32

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of acetone 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 case-by-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 elicit a number of phenotypic 

consequences other than those evaluated in the Tier 1 assays. 

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

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

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

this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of 

evidence evaluation of the results of the Tier 1 assays stated that “In summary, the Panel agreed 

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

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

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

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

A. Systemic/Overt toxicity in the in vivo Tier 1 Assay and OSRI 

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

studies are described below. In addition to the endocrine-related effects described above for the in vivo

Tier 1 assays and OSRI, other effects observed which may be considered overt/systemic toxicity are 

also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body 

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

instances, more than one parameter was needed to determine overt toxicity. Effects which were 

considered to be signs of systemic toxicity were generally less severe forms of toxicity (e.g., changes 

in organ weights or blood chemistry).

Page 24 of 40
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Page 17 of 32

1. Tier 1 in vivo Assays 

For acetone, no overt toxicity or signs of systemic toxicity was observed in the mammalian in vivo 

assays at dose up to and including 1000 mg/kg. 

No overt toxicity was observed in the FSTRA assay. In the AMA, tail curvature was observed in all 

treatment groups and the reviewer noted a higher incidence in the treated groups, relative to the 

negative control. Tail curvature was seen in 8% (5/60) of tadpoles in the negative control and in 

23% (14/60), 13% (8/60), and 23% (14/60) of the tadpoles in the 0.79, 8.45, and 95.83 mg a.i./L 

treatment levels, respectively. 

2. OSRI 

No part 158 studies are available for acetone. Data were available in the public literature. The most 

noteworthy findings in subchronic studies were increased liver and kidney weights, and decreased 

spleen weights. In mice administered acetone via drinking water, adverse effects (liver and kidney 

toxicity) were observed at doses ≥ 1,600 mg/kg/day. Rats treated with acetone via gavage for 90 

days exhibited decreased body weight and increased relative kidney and liver weights, 

hemosiderosis of the spleen and an increased incidence and severity of nephropathy at 1,700 

mg/kg/day. The NOAEL in rats was 900 mg/kg/day. In a subchronic toxicity study in rats via 

gavage, acetone resulted in kidney weight changes and lesions at 500 mg/kg/day. The NOAEL in 

this study was 100 mg/kg/day. Male Sprague-Dawley rats were exposed to acetone via inhalation at 

a concentration of 19,000 ppm (45,106 mg/m3) for 3 hours/day, 5 days/week, for 8 weeks. No 

treatment related effects were observed in this study at exposure concentrations of 19,000 ppm 

(equal to 11,703mg/kg/day). 

In a reproduction study with mice, acetone was administered via gavage at doses of 0 or 3,500 

mg/kg/day. Toxicity was manifested as decreased reproductive index, increased gestation length, 

reduced birth weights, decreased neonatal survival and increased neonatal weight gain at 3,500 

mg/kg/day. Two developmental toxicity studies in rodents via the inhalation route of exposure were 

also available for review. In mice, maternal (increased incidence of late resorptions) and fetal 

(reduced weight) toxicities were observed at the same dose, 6,600 ppm (approximately 4,066 

mg/kg/day). No teratogenic effects were observed in mice. The NOAEL was 2,200 ppm (equivalent 

to 1,348 mg/kg/day). In rats, maternal (reduction in body weight, uterine weight and extragestational weight gain) and fetal (malformations) toxicities were observed at the same dose, 11,000 

ppm (approximately 6,773 mg/kg/day). The NOAEL was 2,200 ppm (equivalent to 1,348 

mg/kg/day). These data reveal that acetone induces toxicity only at doses at very high doses (above 

the limit dose of 1000 mg/kg/day). 

Page 25 of 40
ACETONE FINAL 

Page 18 of 32

B. Estrogen Pathway 

There was no convincing evidence of potential interaction with the estrogen pathway in mammals 

or wildlife. Acetone was negative in the Tier 1 in vitro and Tier 1 uterotrophic assays. In the female 

pubertal assay, while increases in uterine weight were observed, there were no other effects 

observed in the study including histopathology. Additionally, it is noted that this isolated finding 

was observed at the limit dose (1000 mg/kg/day) and half the limit dose (500 mg/kg/day). 

Furthermore, there were no effects on female mammalian reproductive parameters observed in the 

mammalian OSRI. In the FSTRA assay, while there was a significant decrease in fecundity at the 

intermediate concentration of 6.2 mg a.i./L (↓51%), there was high variability in fecundity among 

the treatment groups in this study as well as a lack of complementary responses across other 

endpoints. 

C. Androgen pathway 

There was no convincing evidence of potential interaction with the androgen pathway in mammals 

or wildlife. Acetone was negative in the Tier 1 in vitro and in vivo assays. In the male pubertal 

assay, while there was an increase in serum testosterone concentration at the limit dose (1000 

mg/kg/day) there were no effects on the other androgen-related endpoints including age of 

attainment of PPS, male reproductive organ weights, and no corroborating microscopic findings. 

While alterations in the androgen-related tissue weights and effects on sperm were observed in 

the mammalian OSRI, these effect occurred at very high doses (2500-6900 mg/kg/day). In the 

FSTRA, there were no androgen-related effects observed. 

D. Thyroid pathway 

There were no thyroid-related effects observed in mammals or amphibians. 

E. Conclusions 

There were no potential interactions with estrogen, androgen or thyroid pathways or mammals or 

wildlife 

V. EDSP Tier 2 Testing Recommendations 

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

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

thyroid pathways. 

Page 26 of 40
ACETONE FINAL 

Page 19 of 32

VI. References 

Dietz, DD; Leininger, JR; Rauckman, EJ; et al. (1991) Toxicity studies of acetone administered in 

the drinking water of rodents. Fund. Appl. Toxicol. 17:347–360. 

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

(Xenopus laevis). Unpublished study performed by Smithers Viscient, Inc., Laboratory report 

number 1781-6765.Study sponsored by BASF Corporation, NC. Study completed February 11, 

2013. (MRID 48614601) 

Mast, T., Evanoff, J., Rommereim, R., et al. (1988) Inhalation developmental toxicity studies: 

teratology study of acetone in mice and rats. Pacific Northwest Laboratory, Richland, WA, NTIS 

No. DE89005671. 

NTP (National Toxicology Program). (1991) Toxicity studies of acetone (CAS No. 67-64-1) in 

F344/N rats and B6C3F1 mice (drinking water studies). NTP, Research Triangle Park, NC, NTP 

TOX 3, NIH Publication No. 91-3122. 

Stump, D.G. (2013) A Hershberger Assay of Acetone Administered Orally in Peripubertal 

Orchidoepidymectomized Rats. Unpublished study conducted by WIL Research, Ashland, OH. 

Laboratory Project No.: WIL-186063, February 6, 2013. (MRID 48614607) 

Stump, D.G. (2013) A Pubertal Development and Thyroid Function Assay of Acetone Administered 

Orally in Intact Juvenile/Peripubertal Female Rats. Unpublished study conducted by WIL Research, 

Ashland, OH. Laboratory Project No.: WIL-186065, February 7, 2013. (MRID 48614608). 

Stump, D.G. (2013) A Pubertal Development and Thyroid Function Assay of Acetone 

Administered Orally in Intact Juvenile/Peripubertal Male Rats. Unpublished study conducted by 

WIL Research, Ashland, OH. Laboratory Report No.: WIL-186064, February 7, 2013. (MRID 

48614609) 

Stump, D.G. (2013) A Uterotrophic Assay of Acetone Administered Orally in Ovariectomized 

Rats. Unpublished study conducted by WIL Research, Ashland, OH. Laboratory Project No.: WIL186062, February 6, 2013. (MRID 48614611) 

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. 

Page 27 of 40
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Page 20 of 32

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. 

Wagner, H. (2013) Acetone: H2958R Steroidogenesis Assay. Unpublished study conducted by 

CeeTox, Inc, Kalamazoo, MI. Laboratory Report No.: 9186V-100381STER, January 28, 2013. 

(MRID 48614610). 

Willoughby, J.A. (2013) Acetone: Androgen Receptor Binding (Rat Prostate Cytosol). Unpublished 

study conducted by CeeTox, Inc, Kalamazoo, MI. Laboratory Report No.: 9186V-100381ARB, 

January 23, 2013. (MRID 48614602). 

Willoughby, J.A. (2013) Acetone: Human Recombinant Aromatase Assay. Unpublished study 

conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.:9186V-100381AROM, 

January 23, 2013. 

(MRID 48614603). 

Willoughby, J.A. (2013) Acetone: Estrogen Receptor Binding (Rat Uterine Cytosol). Unpublished 

study conducted by CeeTox, Inc, Kalamazoo, MI. Laboratory Report No.: 9186V-100381ERB, 

January 23, 2013. (MRID 48614604) 

Willoughby, J.A. (2013) Acetone: Estrogen Receptor Transcriptional Activation (Human Cell Line 

(HeLA-9903)). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Report 

No.: 9186V-100381ERTA, January 23, 2013. (MRID 48614605). 

York, D.O. (2013). Acetone- Short-Term Reproduction Assay with Fathead Minnow (Pimephales 

promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished study performed 

by Smithers Viscient, MA. Laboratory report number 14036.6106. Study sponsored by American 

Chemistry Council, Acetone EDSP Consortium, Washington, D.C. 20002. February 12, 2013. 

(MRID 4861606) 

Page 28 of 40
ACETONE FINAL 

Page 21 of 32

APPENDIX 1. EDSP T1 Screening Assays

Amphibian Metamorphosis Assay; (OCSPP 890.1100)

The 21-day assay (MRID 48614601) of acetone (99.9% purity) on amphibian metamorphosis of African 

clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to negative control 

and nominal concentrations of 1.0, 10, and 100 mg a.i./L; the %CV of measured concentrations exceeded 

20% over the course of the study (i.e., 21-39%). The time-weighted average (TWA) concentrations 

(calculated by the reviewer) were <0.525 (<LOQ; control), 0.79, 8.45, and 95.83 mg a.i./L. The test 

system was maintained at 20.8 to 23.3oC and a pH of 7.0 to 8.2. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) 

control. No treatment-related mortalities were observed during the study. One tadpole in the mid 

treatment group was inadvertently killed during one of the daily cleanings. Tail curvature was observed 

in all treatment groups and the reviewer noted a higher incidence in the treated groups, relative to the 

negative control; tail curvature was seen in 8% (5/60) of tadpoles in the negative control and in 23% 

(14/60), 13% (8/60), and 23% (14/60) of the tadpoles in the low, mid, and high treatment groups, 

respectively. 

Acetone reduced Day 7 wet weight in a concentration-responsive manner, with a significant reduction of 

11.5% (p<0.05; Jonckheere-Terpstra) at the high treatment, relative to the negative control. An increase 

in Day 21 normalized hind limb length (HLL) was observed, with a significant increase of 11.8% at the 

high treatment (p<0.05; Jonckheere-Terpstra). There were no other growth parameters affected by 

treatment at Day 7 or Day 21. No significant acceleration or delay of median NF developmental stage 

was observed after 7 or 21 days of exposure at any treatment level. Further, no asynchronous 

development was observed. 

One incidence of follicular cell hyperplasia was observed in each of the low and mid treatment groups. 

No histopathological observations were found to be significant. 

Androgen Receptor (AR) Binding; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48614602), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and 

a competitive binding experiment. The saturation binding experiment was conducted to 

demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radiolabeled reference androgen (R1881) prior to 

conducting AR competitive binding experiment. The competitive binding assay was conducted to 

Page 29 of 40
ACETONE FINAL 

Page 22 of 32

measure the binding of a single concentration of [3H]-R1881 (1 nM) in the presence of increasing 

concentrations (logarithmic increase from 10−10 to 10−3 M) of acetone (99.9% purity, Lot # 

114630). No solvent was necessary as acetone was completely miscible in the assay solution. 

Dimethyl sulfoxide (DMSO) was used as the solvent vehicle for the strong and weak positive 

controls at a final assay concentration of 3.2%. A total of three runs were performed, and each run 

included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. 

In the saturation binding experiment, the dissociation constant (Kd) for [3

H]-R1881 was 0.591 ± 

0.168 nM, and the estimated Bmax was 0.007 ± 0.002 for the single batch of prostate cytosol that 

was prepared. Confidence in these numbers is high according to the goodness of fit (R2

 = 0.930-

0.989) and the small variation among runs. 

In the competitive binding experiment, the estimated average log IC50 was −9.0 M for R1881 and 

−4.4 M for the weak positive control (dexamethasone). Compared to R1881, the relative binding 

affinity (RBA) for the weak positive control averaged 0.0029%. No precipitation of the test 

compound was observed. 

The percent [3

H]-ligand binding was >75% at all concentrations of acetone tested in all three 

competitive binding runs. A log IC50 and RBA for acetone could not be determined since 

displacement did not exceed 50% at any concentration. 

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

Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48614603), acetone (99.9% purity, Lot # 114630) 

was incubated with human recombinant aromatase and tritiated androstenedione (1-β [3

H(N)]-

androst-4-ene-3,17-dione; [3H]ASDN) at log concentrations from 10−10 to 10−3 M for 15 minutes to 

assess the potential of acetone to inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the end 

of a 15-minute incubation for each concentration of test chemical. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three runs were conducted and each run included a full 

activity control, a background activity control, a positive control series (10−10 to 10−5

 M) with a 

known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the acetone series (10−10 to 10−3

M) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.409 to 0.579 nmol·mg-protein−1·min−1

for the 3 test runs, with a mean and standard deviation of 0.474 ± 0.064 nmol·mg-protein−1

·min−1. 

Activity in the background controls averaged 0.51% of the full control activity. The response of 

Page 30 of 40
ACETONE FINAL 

Page 23 of 32

the full activity controls and background controls were acceptable for each run. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.453 ± 0.027 

nmol·mg-protein−1·min−1 at the lowest tested concentration, 10−10 M, and 0.0033 ± 0.0005 

nmol·mg-protein−1

·min−1 at the highest tested concentration, 10−5 M. 

For acetone, aromatase activity averaged 0.481 ± 0.053 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration, 10−10 M, and 0.458 ± 0.044 nmol·mg-protein−1·min−1 at the highest tested 

concentration, 10−3

 M. The average dose-response curve indicated that the aromatase activity of 

acetone at all concentrations tested was essentially equivalent to the activity observed in the full 

activity control. These data could not be fit to the 4-parameter nonlinear regression model. 

Based on the data from the average response curve, acetone is classified as a Non-inhibitor of 

aromatase activity in this assay. 

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

In an estrogen receptor (ER) binding assay (MRID 48614604) for acetone (99.9% purity, Lot 

#114630 ), uterine cytosol from Sprague Dawley rats was used as the source of estrogen receptors 

(ER) to conduct saturation and competitive binding experiments. The saturation binding 

experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in 

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

estrogen prior to routinely conducting competitive binding experiment. The competitive binding 

experiment was conducted to measure the binding of a single concentration of [3

H]-17β-estradiol 

(1 nM) in the presence of increasing concentrations (logarithmic increase from 10−10 to 10−3 M) of 

acetone. A solvent was not required for acetone in this study. Dimethyl sulfoxide (DMSO) was 

selected as a suitable vehicle for the positive control, weak positive control and negative control, at 

a final assay concentration of 4%. A total of three assay runs were performed, and each run 

included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, 

and 17β-estradiol as the natural ligand reference material. 

For the saturation binding experiment, the Kd for [3

H]-17β-estradiol was 0.046 ± 0.012 nM, and the 

estimated Bmax was 0.008 ± 0.002 nM for the prepared rat uterine cytosol. The Kd for each run was 

within the expected range of 0.03 to 1.5 nM. The data produced a linear Scatchard plot. 

For the competitive binding experiment, the mean log IC50 was −9.0 M for 17β-estradiol and −5.5 

M for the weak positive control. Compared to the natural ligand, the relative binding affinity 

(RBA) averaged 0.0322% for the weak positive control. Acetone did not competitively displace the 

radiolabeled estradiol from the ER at any test concentration in the three runs. 

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Based on the results of the three runs, acetone is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID 48614605), hERα-HeLa-9903 cells 

cultured in vitro were exposed to acetone (99.9% purity, Lot # 114630) at logarithmically 

increasing concentrations from 10−3 to 10−10 M in two independent runs. The experiments were 

performed using 96-well plates, and each acetone concentration was tested in 6 wells per plate in 

each run. No solvent was utilized as acetone was completely soluble in the assay media. Cells 

were exposed to the acetone for 22-23 hours to induce reporter (luciferase) gene products. 

Luciferase expression in response to activation of the estrogen receptor by acetone was measured 

upon addition of a luciferase substrate and detection with a luminometer with acceptable 

sensitivity. 

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. In addition, cytotoxicity was observed at the highest tested 

concentrations of corticosterone in Run 1. Results from cytotoxic concentrations were excluded 

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

activity, acetone 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 

22.8-34.5% PC. Acetone was only able to reach a maximum of 1.0-1.5% PC when tested up to the 

limit dose of 10−3 M, and the associated PCMax was 10−6 M for both runs. Because the RPCMax < 

PC10 in both assay runs, acetone 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 48614606) of acetone with fathead minnow 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (24/group, ~23 

weeks old) were exposed to acetone (99.9% purity) at nominal concentrations of 0 (negative 

control), 1.0, 10, and 100 mg a.i./L. Mean-measured concentrations were <0.051 (<LOQ; negative 

control), 0.68, 6.3, and 78 mg a.i./L. The reviewer-calculated time-weighted average (TWA) 

concentrations were 0.66, 6.2, and 71.4 mg a.i./L. The test system was maintained at 24 to 26°C 

and a pH of 6.8 to 7.8. Unless otherwise indicated, all effects are reported based on comparison to 

the negative (clean water) control. 

Overall survival was 100% in the control and low- and mid-concentration treatment groups and 

96% in the high-concentration treatment group, with one male fish dying. Acetone exposure was 

not associated with an increase or decrease in body weight or length in male fish, or length in 

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female fish at any treatment level. However, female body weight was significantly increased by 

10.6% at the high treatment level compared to the control (p=0.051; Jonckheere-Terpstra). No 

other effects on secondary sex characteristics or clinical signs (i.e., behavioral or other sublethal 

effects) were observed in any surviving fish. 

Spawning frequency and mean fecundity in controls, respectively, were at least every 4 days in 

three of the four control replicates and 22.8 eggs/female/day/replicate (replicate range: 17-33 

eggs/female/day); fertilization success in controls was 100%. Acetone significantly decreased 

fecundity by 51% at the mid treatment level (p<0.05; Dunnett’s); however, there was high 

variability in the fecundity endpoint across treatment groups in this study. Fertility was not 

significantly different in the treatment groups compared to the control. Plasma vitellogenin (VTG) 

levels were not significantly different between control and acetone treatment groups for both males 

and females. No significant increase or decrease in gonado-somatic index (GSI) was observed in 

male or female fish at any treatment level. Nuptial tubercle score was not significantly increased or 

decreased in males at any treatment level; no nuptial tubercles were observed in surviving females. 

There were no reported treatment-related effects on gonadal histopathology reported for male and 

female fish. 

Microbial growth was observed in the test chambers and an aggressive cleaning procedure was 

employed; fish were periodically not exposed to the test chemical during the cleaning process. The 

time duration for which the fish were not exposed was not reported, but rather it was stated that the 

time was kept to an absolute minimum. A concentration-dependent decrease in dissolved oxygen 

(DO) was observed. The mid and high concentration DO levels were significantly lower than the 

control; DO levels did not drop below 60%. 

Hershberger Assay; (OCSPP 890.140) 

In a Hershberger assay (MRID 48614607) screening for androgenic activity, acetone (99.9% 

purity, Batch# 114630) in deionized water was administered daily via oral gavage to groups of 54-

day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 100, 300, or 

1000 (limit dose) mg/kg/day. An androgenic positive control group was included consisting of six 

castrated rats exposed to 0.2 mg/kg/day testosterone propionate (TP) in corn oil by subcutaneous 

(s.c.) injection. 

To screen for potential anti-androgenic activity, acetone in deionized water was administered daily 

via oral gavage to 54-day old, castrated male Sprague Dawley rats (6/group) at dose levels of 100, 

300, and 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.2 

mg/kg/day by s.c. injection. The positive control group for androgenic activity served as the 

control group for the anti-androgenic activity. An anti-androgenic positive control group consisted 

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of six castrated rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjunction with 

a daily s.c. dose of TP (0.2 mg/kg/day). 

For both components of the assay, the animals were dosed for 10 consecutive days and sacrificed 

approximately 24 hours after the final dose administration to determine weights of the five 

androgen-dependent tissues (Cowper’s gland, glans penis, levator ani and bulbocavernosus 

[LABC] muscle group, seminal vesicles with coagulating glands, and ventral prostate), and the 

adrenal glands, kidneys, and liver. 

All animals survived until scheduled termination. No treatment-related clinical signs were 

observed in the androgen agonist or anti-androgen assays. 

In the androgen agonist assay, body weights and overall body weight gains for all acetone 

treatment groups were comparable to vehicle controls throughout the duration of the assay. No 

treatment-related effects were observed in any of the accessory sex organs of acetone-dosed 

animals. Animals in the positive control group had increases (p≤0.05) in accessory sex organ 

weights as follows: 356% in seminal vesicles; 519% in ventral prostate; 149% in LABC; 238% in 

Cowper’s gland; and 66% in glans penis. The maximum guideline-specified %CV was exceeded 

for the glans penis at 300 mg/kg/day (22.8% vs. 17%). All other %CV values were less than the 

maximum permissible values. No effects were observed for adrenal glands, kidneys or liver 

weights in the 100, 300, or 1000 mg/kg/day groups. 

In the anti-androgen assay, body weights and overall body weight gains for all acetone treatment 

groups were comparable to TP-control throughout the duration of the assay. No treatment-related 

effects were observed in any of the accessory sex organs of acetone-dosed animals. Animals dosed 

with the positive control had decreased (p≤0.05) accessory sex organ weights as follows: 75% in 

seminal vesicles; 75% in ventral prostate; 53% in LABC; 65% in Cowper’s gland; and 29% in the 

glans penis. All %CV values were below the maximum permissible values. No effects were 

observed for adrenal glands, kidneys or liver weights in the 100, 300, or 1000 mg/kg/day 

acetone+TP groups. 

Statistically significant changes were not seen in two or more of the five androgen sensitive tissues. 

Acetone was negative for androgenicity and anti-androgenicity in the Hershberger assay. 

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48614608), 15 Sprague Dawley [Crl:CD(SD)] rats/dose group 

were treated daily by oral gavage with acetone (99.9%; lot # 114630) in deionized water at doses of 

0 (vehicle), 500, or 1000 (limit dose) mg/kg/day from post-natal day (PND) 22 to 42/43. Animals 

were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of 

attainment were recorded. After sacrifice on PND 42/43, blood was collected for clinical 

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chemistry analyses, and total thyroxine (T4) and thyroid stimulating hormone (TSH) concentrations 

were determined with an electrochemiluminescent immunoassay (T4) or a radioimmunoassay 

(TSH). Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic 

examinations were conducted on the ovaries, uterus, thyroid, and kidneys. 

There were no signs of systemic toxicity. All animals survived until scheduled termination. There 

were no effects of treatment on body weight, body weight gain, organ weight, or gross or 

microscopic pathology. Creatinine was increased (p<0.05) by 13 and 25% and urea nitrogen was 

decreased (p<0.05) by 12 and 20%, in both low- and high-dose groups, respectively. No 

corroborating microscopic findings were noted. Minimal mineralization, minimal to mild chronic 

progressive nephropathy, and pyelonephritis were observed in the kidneys, but occurred at similar 

frequencies across all dose groups. 

There were no effects of treatment on age or body weight at attainment of VO, mean age at first 

vaginal estrus, mean cycle length, % cycling or regularly cycling, or cycle status at necropsy. 

Mean absolute wet and blotted uterus weights displayed a trend to increase (p<0.05) at 500 and 

1000 mg/kg/day; however, there were no microscopic findings to suggest a treatment-related 

effect. The uterus and ovaries of all animals were found to be unremarkable when examined. The 

grades for the corpora lutea, and primordial, secondary, and tertiary follicles examined in the 

ovaries were identical across all groups. 

There were no effects of treatment on thyroid hormone concentrations. There were no differences 

in follicular cell height or colloid area observed between the control and treatment groups. 

The dose levels tested were adequate since the high dose was the Limit Dose (1000 mg/kg/day). 

Male Pubertal Assay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48614609), 15 Sprague Dawley [Crl:(CD)SD] rats/dose group 

were treated daily via oral gavage with acetone (99.9%; lot # 114630) in deionized water at doses 

of 0 (vehicle), 500, or 1000 mg/kg/day from post-natal day (PND) 23 to 53/54. Animals were 

examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at day of 

attainment were recorded. Following sacrifice on PND 53/54, blood was collected for clinical 

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

(TSH) levels were determined using an electrochemiluminescent immunoassay (testosterone and 

T4) or a radioimmunoassay (TSH). Adrenal, liver, pituitary, thyroid, and urogenital organs were 

weighed, and microscopic examinations were performed on the testes, epididymides, thyroid, and 

kidneys. 

There were no signs of systemic toxicity. All animals survived until scheduled termination, and no 

clinical signs of toxicity were observed. There were no effects of treatment on final body weights, 

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overall (PND 23-53) body weight gains, organ weights, clinical chemistry, or gross or microscopic 

pathology. 

There were no effects of treatment on age and body weight at attainment of complete PPS. Serum 

testosterone levels were increased (p<0.001) by 140% at 1000 mg/kg/day. However, there were no 

effects on age of attainment of PPS or male reproductive organ weights, and no corroborating 

microscopic findings. Additionally, the mean serum testosterone value (4.84 ng/mL) at 1000 

mg/kg/day was only slightly above the range of historical controls (1.62-4.20 ng/mL). Therefore, 

this finding was considered incidental to treatment. 

There were no effects or treatment on serum T4 or TSH levels, and there were no differences in 

thyroid follicular cell height or colloid area observed between control and treatment groups. 

No toxicity was observed at the limit dose of 1000 mg/kg/day. 

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (MRID 48614610), H295R cells cultured in vitro in 24-well plates were 

incubated with acetone (99.9% purity. Lot #114630) at log concentrations from 0.0001 to 100 μM 

in triplicate for 48 hours. No solvent was necessary for the test chemical; it was added directly to 

the growth medium. 

Testosterone and estradiol levels were measured by using a HPLC/MS/MS method that included 

internal standards. Eight independent experiments were conducted to achieve three acceptable 

assay runs. A Quality Control (QC) plate was run concurrently with each independent run of a test 

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

concentration levels; positive controls included a known inhibitor (prochloraz) and known inducer 

(forskolin) of estradiol and testosterone production. 

The between-plate %CVs for the solvent control (SC) on the test chemical plates were 38% and 

24%, respectively, for testosterone and estradiol, and 38% and 22%, respectively, on the QC plates. 

The between-plate %CV for testosterone for the test chemical and QC plates was above the 

Guideline limit of ≤30%. The %CVs within the test chemical plates for the SC ranged from 1.87 to 

6.05% for testosterone and 2.55 to 6.40% for estradiol. The %CVs within each run for the QC 

plates ranged from 4.02 to 6.19% for testosterone and 5.50 to 6.98% for estradiol. All within-plate 

%CVs met Guideline recommendations. 

Precipitation was not observed at any tested acetone concentration, and acetone was not overtly 

cytotoxic to the H295R cells. Viability for acetone on the test plates was ≥95.2% 

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Based on the hormone responses in at least three independent runs, acetone 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 48614611) conducted to screen for potential estrogenic activity, 

acetone (99.9% purity, Lot # 114630) in deionized water was administered daily via oral gavage to 

groups of 6 ovariectomized Sprague-Dawley rats at dose levels of 0 (vehicle), 100, 300 and 1000 

(limit dose) mg/kg/day on post-natal days (PND) 60-62. A positive control group was treated with 

17α-ethynyl estradiol (EE) by daily subcutaneous (s.c.) injection at a dose level of 0.001 

mg/kg/day. All animals were terminated and necropsied, approximately 24 hours after the final 

dose, on PND 63 to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

animals for any acetone-treated groups. Body weights and body weight gains in the acetonetreated groups were comparable to control throughout the study, as were uterine weights in the 

treatment groups. Absolute wet and blotted uterus weights for the EE group were increased 

(p<0.0001) by 487% and 174%, respectively, as expected. 

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

in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and general toxicity studies considered relevant for determining the potential 

interaction of acetone with the endocrine system are summarized below. The only toxicity studies 

discussed in this appendix are those that are directly relevant to assessing potential endocrine 

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

studies, etc.). 

14-Day (Subchronic) Oral Toxicity in Rodents (Rat)

Groups of F344/N rats (5/sex/dose) were administered acetone (99% purity, Lot/Batch #AN228) in 

drinking water at concentrations of 0 (control), 5000, 10,000, 20,000, 50,000, or 100,000 ppm for 

14 days (NTP, 1991; Dietz et al., 1991). Time-weighted average (TWA) doses for males were 0, 

700, 1600, 2600, 4300, and 6900 mg/kg/day, respectively, and for females were 0, 750, 1500, 2300, 

4350, and 8600 mg/kg/day, respectively. All animals survived to termination. Water consumption 

was reduced in both sexes in the high-mid- and high-dose groups. Mean final body weights of 

males in the high-mid- and high-dose groups were 87 and 63%, respectively, of control, and the 

mean final body weight of high-dose females was 87% of control. Increases in relative liver and 

kidney weights were observed in both sexes at the intermediate dose and higher, relative testes 

weights were increased in the high-mid- and high-dose animals, and thymus weights were 

decreased ( up to 77%) in all treated animals; further details of organ weight data were not 

included. Bone marrow hypoplasia was noted in all high-dose males, but not in the control animals. 

More detailed hematology and clinical chemistry studies were not included. 

14-Day (Subchronic) Oral Toxicity in Rodents (Mouse)

Groups of B6C3F1 mice (5/sex/dose) were administered acetone (99% purity, Lot/Batch #AN228) 

in drinking water at concentrations of 0 (control), 5000, 10,000, 20,000, 50,000, or 100,000 ppm for 

14 days (NTP, 1991; Dietz et al., 1991). TWA doses for males were 0, 960, 1600, 3900, 6300, and 

10,000 mg/kg/day, respectively, and for females were 0, 1600, 3000, 5500, 8800, and 13,000 

mg/kg/day, respectively. Water consumption was reduced in both sexes in the high-mid- and highdose groups, and mean final body weights of high-dose males and females were decreased (81 and 

93%, respectively) relative to control. All animals survived to termination. Kidney weights were 

increased in the high-mid- and high- dose groups for males and females, and liver weights were 

increased in all treated males and at the intermediate dose and higher in females. Dose-related 

increases in the incidence and severity of centrilobular hepatocellular hypertrophy were observed in 

males at the intermediate dose and higher and in females in the high-mid- and high-dose groups. 

Hematology and clinical chemistry evaluations were not conducted. 

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90-Day (Subchronic) Oral Toxicity in Rodents (Rat)

Groups of F344/N rats (10/sex/dose) were administered acetone (99% purity, Lot/Batch #AN228) 

in the drinking water at concentrations of 0 (control), 2500, 5000, 10,000, 20,000, or 50,000 ppm 

for 13 weeks (NTP, 1991; Dietz et al., 1991). TWA doses for males were 0, 200, 400, 900, 1700, 

and 3400 mg/kg/day, respectively, and for females were 0, 300, 600, 1200, 1600, and 3100 

mg/kg/day, respectively. No deaths occurred in any group. Water consumption was decreased in 

high-dose males and in the high-mid- and high-dose females. Mean final body weight of the highdose males was 81% of control; body weight in females was unaffected by treatment. No clinical 

signs of toxicity were observed in any group. At necropsy, increases (p≤0.01 or 0.05) in the 

following relative organ weights were noted: kidney weights were 114% of control for high-middose females and 126 and 123% of control for high-dose males and females, respectively; liver 

weights were 110 and 112% of control for high-mid-dose males and females, respectively, and 115 

and 105% of control for high-dose males and females, respectively; and testis weights were 119% 

of control at the high dose. Liver weight increases were not associated with histopathological 

lesions and were considered to be the result of enzyme induction. In high-dose males, decreased 

sperm motility, caudal weight, epididymal weight, and an increased incidence of abnormal sperm 

were observed. 

Nephropathy was observed in males in all treatment groups including control animals; therefore, the 

incidence of nephropathy rated as mild was considered an indicator of toxicity. Males given the 

two highest concentrations of acetone had increases in the incidence and severity of mild 

nephropathy (9/10), indicating early onset and enhanced progression of the disease. Nephropathy 

was not observed in females. Clinical chemistry parameters were not measured. In summary, the 

testis, kidney, and hematologic system were identified as target organs for male rats, with a LOAEL 

of 1700 mg/kg/day (high-mid-dose) and a NOAEL of 900 mg/kg/day (mid-dose). A LOAEL for 

female rats was not identified. 

90-Day (Subchronic) Oral Toxicity in Rodents (Mouse)

Groups of B6C3F1 mice (10/sex/dose) were administered acetone (99% purity, Lot/Batch #AN228) 

in the drinking water at concentrations of 0, 1250 (males only), 2500, 5000, 10,000, 20,000, or 

50,000 (females only) ppm for 13 weeks (NTP, 1991; Dietz et al., 1991). TWA doses for males 

were 0, 380, 600, 1400, 2300, and 4900 mg/kg/day, respectively, and for females 0, 900, 2000, 

4200, 5900, and 11,000 mg/kg/day, respectively. No deaths occurred, and no clinical signs of 

toxicity were observed in any group. Water consumption was not affected in males; however, doserelated decreases in water consumption were seen in all treated females (statistical significance not 

reported; ↓9 to 52%). Body weight and growth of the treated animals, and hematology parameters, 

sperm morphology, and vaginal cytology, were not affected in either sex. Organ weights in treated 

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males were similar to control; however, increases (p≤0.01 or 0.05) in high- dose female absolute 

and relative liver weights were 113 and 110% of control, respectively. The only microscopic lesion 

seen in mice was centrilobular hepatocellular hypertrophy, observed in two high-dose females, 

considered to be due to enzyme induction. Mild hepatic changes (observed in males exposed to 

≥20,000 ppm for 14 days) did not persist after 13 weeks of exposure, suggesting the development of 

tolerance toward acetone. In summary, the liver was identified as the target organ in male and 

female mice, but is considered an adaptive, and not toxic, response. Effects noted after acetone 

administration in the rat, particularly males, were not evident in mice. The LOAELs for male and 

female mice were 4900 and 11,000 mg/kg-day, respectively (high dose), and the NOAELs were 

2300 and 5900 mg/kg-day, respectively (high- mid dose). The LOAEL for male mice was selected 

by the study authors on the basis of the transient findings in the 14-day study. 

 

 

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