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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

# &

Health Effects Divison

# ::~:E£'~:::e 

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

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL

INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID

PATHWAYS

CHEMICAL: ACETONE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYACETONE 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)....................................30ACETONE FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance

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

CG Cowper’s Gland

ChE Cholinesterase

ChEI Cholinesterase inhibition

CMC Carboxymethyl cellulose

CTA Comparative Thyroid Assay

CV Coefficient of Variation

CYP Cytochrome 450

DER Data Evaluation Recor d
DMSO Dimeth y l Sulfoxide
DNA Deox y ribonucleic Aci d
DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team
EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol
ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
ER Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase )
ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationACETONE FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act

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

K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level

LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylaseACETONE FINAL

v Abbreviation Terminology

MTC Maximum Tolerated Concentration

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d NF stage Nieuwkoo p and Faber’s Sta g in g Atlas
NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level
NS Not Statisticall y Si g nificant
NR Not Re p orte d
OCSPP Office of Chemical Safet y Pollution and Prevention

OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

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

PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose

RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel

SC Solvent Control

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

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th
SV Seminal Vesicles
T Th y roid ( hormonal p athwa y)
T1WoERC EDSP Tie r 1 Wei g ht of Evidence Review Committee
T3 TriiodothyronineACETONE FINAL

vi Abbreviation Terminology

T4 Thyroxine (tetraiodothyronine)

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control

VO Vaginal Opening

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

Page 1 of 32 Executive Summary

The Endocrine Disruptor Screening Pr ograms (EDSP) Tier 1 assay batt ery is designed to provide the necessary empirical data to evalua te the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. Th is interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitarygonadal (HPG) and hypothalamic-pitu itary thyroid (HPT) axes. In addi tion to the available Tier 1 assay data, other scientifically relevant informatio n (OSRI), including genera l toxicity data and open literature studies of sufficient quality were consider ed 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 occu r were considered, in particular, whether or not endocri ne-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 Weig ht of Evidence Revi ew Committee (T1WoERC)
of the Office of Pesticide Pr ograms (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a Weight-of-Evidence (WoE) analys is of the potential inte raction 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 as say, while increases in uterine weight were observed, there were
no other effects observed in the study including hi stopathology. Additional ly, it is noted that this isolated finding wa s observed at the limit dose (1000 mg/kg/day) and half th e limit dose (500 mg/kg/day). Furthermore, there were no effects on female mammalian reproductive parameters observed in the ma mmalian OSRI. In the Fish Short Term Reproduction assay (FSTRA), while there was a signific ant decrease in fecundity at th e intermediate concentration of 6.2 mg a.i./L ( ↓ 51%), there was high variability in fec undity among the treatment groups in this study as well as a lack of complementary respons es across other endpoints.

There was no convincing evidence of potential interacti on 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 te stosterone concentration at the limit dose (1000 mg/kg/day) there were no effects on the othe r androgen-related endpoi nts including age of attainment of preputial sepa ration, male reproductive organ weights and no corroborating microscopic findings. While alterations in the androgen-related tissue weights and effects onACETONE FINAL

Page 2 of 32 sperm were observed in the mamm alian OSRI, these effect occurr ed at very hi gh doses (2500- 6900 mg/kg/day). In the FSTRA, there we re 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, mamm alian or wildlife EDSP Tier 2 testing is NOT
recommended for acetone since there was no eviden ce of potential interaction with the estrogen, androgen or thyroid pathways.ACETONE FINAL

Page 3 of 32 I. Introduction

The Endocrine Disruptor Screening Pr ograms (EDSP) Tier 1 assay batt ery is designed to provide the necessary empirical data to evalua te the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. Th is interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitarygonadal (HPG) and hypothalamic-pitu itary thyroid (HPT) axes. In addi tion to the available Tier 1 assay data, other scientifically relevant informatio n (OSRI), including genera l toxicity data and open literature studies of sufficient quality were consider ed in this weight of evidence (WoE) assessment.

In determining whether acetone interacts with E, A or T hormone pathwa ys, 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 occu r were considered, in particular, whether or not endocri ne-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 Weig ht of Evidence Revi ew Committee (T1WoERC)
of the Office of Pesticide Pr ograms (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a Weight-of-Evidence (WoE) analys is of the potential inte raction 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 wi ldlife studies (40 CFR Pa rt 158 and literature).

Acetone is an organic compound (a lso known as propanone and dimeth yl ketone). The compound

is miscible in water and has a low octanol/water partition coefficient (log K ow = -0.24). Acetone has a whole body bioconcentration factor value in fish of approximatel y 3, suggesting that the potential for bioconcentration in aqua tic organisms is low. Acetone has a high vapor pressure (231 mm Hg at 25°C), and, therefore, it volatilizes readily and shoul d exist solely as a vapor in the atmosphere. Acetone is not degraded by hydrolysis, but does undergo photo-decomposition.

The available information considered in determini ng 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 st udies and/or other published articles . These data are summarized in Sections III.A through III.C. An analysis of th e 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 pr esented in Section V.ACETONE FINAL

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 be low in Table 1. Executive Summaries are presented in Appendix 1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Or ders, 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 lite rature. 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 pres ented in Appendix 2.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and appro ach used in the WoE determinati on on the potential of a substance
to interact with endocri ne-related processes ( i.e., E, A or T hormonal pathways) were as described in the WoE guidance document (USEPA, 2011) and pr esented 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 inte grated 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 whic h provide the foundation for WoE eval uations. 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 al ong 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 acro ss these different levels of biological organization. The third st ep is to characterize the main lines of evidence as well any conclusions. Finally, the la st step is to evaluate whether addi tional 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 agon ist and antagonist), steroidogene sis, and other effects on the HPG and HPT axes. OSRI may include published li terature studies as we ll as studies conducted under USEPA (often referred to as Part 158 data ) or OECD guidelines submitted in support ofACETONE FINAL

Page 5 of 32 pesticide registrations. Each st udy is evaluated for scientific qu ality and relevance for informing interactions with the E, A or T pathway. Add itionally, the consistency of the responses in the individual study is eval uated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a specific endpoint for discerning wh ether or not the chemical has interacted with the endocrine system, are consid ered. The collective resp onse of the individual endpoints, as well as the condi tions under which they were e xpressed, are considered when evaluating an overall indication of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data support a hypothesis that a chemical has the potential to interact E, A or T pathways ar e the concepts of comple mentarity and redundancy. These two concepts provide a basis for consider ing the plausibility, c oherence, 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 c onsisting of different levels of biological organization from a molecular initiating event such as receptor bindi ng through potential adverse effects in apical endpoint s such as sexual development and fecundity at the whole organism level. The extent of expression of responses at hi gher levels of biological or ganization can indirectly provide information on potential compensatory capabilities of an i ndividual organism.

After the data have been assembled and integrated, th e third step is to charact erize the main lines of evidence along with the conclusi ons; this characterization involv es three components. The first component is whether the data provide relevant , robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finall y, under what conditions did the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity? The pr esence of overt and/or systemic t oxicity introduces uncertainty in the ability to distinguish effect s specifically related to an endo crine-related effect from a nonendocrine toxic response.

This uncertainty in distinguishing whether the responses were endocri ne-related was discussed at the FIFRA SAP meeting that evaluated scientific issu es 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. second ary effects related to toxicity in non-endocrineACETONE FINAL

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 asse ssments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mortality; clinical sign s such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibian s); and body weight decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and ot her clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, espe cially 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 t oxic. 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 system ic toxicity were also ca ptured and these effects were generally considered as le ss severe forms of toxicity ( e.g ., change in organ weights or blood chemistry). The circumstances for which a dose/c oncentration was considered overtly toxic for a particular study are desc ribed 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 ch emical’s physical/chemical properties and its known
modes of action in its overall charac terization of a chemical’s potential to interact with the E, A or T pathway. Adequately addressing the three main co mponents 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) uncerta inties and their potential impact to conclusions; 2) discussion of key studies; 3) desc ription of inconsistent or conf licting 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 biol ogically 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 th e 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.ACETONE FINAL

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)

ER α 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)ACETONE FINAL

Page 8 of 32 B. Effects on Hypothalamic-Pit uitary-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-estrogeni c or HPG axis effects. Th is 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 ta ble 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 uterotr ophic assay, acetone did not induc e uterine weight increases when administered at doses up to and in cluding the limit dose (1000 mg/kg/day).

In the female pubertal assay, there were no treatme nt-related effects on mean estrous cycle length, percent cycling or percent regularly cycling anim als, ovarian weight or histopathology up to and including the limit dose of 1000 mg/kg/day. Mean ab solute 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 gr oups. There was no reported estrogen-related
effects in the mammalian OSRI.

In the FSTRA assay, there was a si gnificant decrease (p<0.05) in fec undity seen at the intermediate concentration of 6.2 mg a.i./L ( ↓ 51%); however, there was high vari ability in the fecundity endpoint across treatment groups in this study. There were no other estrogen-related e ffects observed. Female body weight was significantly (p<0.05) increased by 11% in the high concentration.ACETONE FINALPage 9 of 32Table 2. Estrogenic/Anti-Estrogenic Pathway for AcetoneLines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway for Acetone1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity observed 2 Overt Toxicity Observed 3EDSP Tier1 AssaysER Binding(MRID 48614604)NERTA(MRID 48614605)NAromatase(MRID 48614603)NSteroidogenesis(MRID 48614610)NUterotrophic(MRID 48614611)NNNFemale Pubertal Rat(MRID 48614608)↑9-15%(L)20-31%(H)NNNNNNNFSTRA(MRID 48614606)NENNN↓51%(M)NNACETONE FINALPage 10 of 32Table 2. Estrogenic/Anti-Estrogenic Pathway for AcetoneLines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway 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 C h a r acte ri s t i cs Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity observed 2 Overt Toxicity Observed 3OSRIOral Subchronic(14-Day) Rat(NTP, 1991;Dietzet al., 1991)NNN NX(M, MH,H)NOral Subchronic(14-Day) Mouse(NTP, 1991;Dietzet al., 1991)NNN NX(M, MH,H)NOral Subchronic(90-Day) Rat(NTP, 1991;Dietzet al., 1991)NNN NX(MH,H)NOral Subchronic(90-Day) Mouse(NTP, 1991;Dietzet al., 1991)NNN NX (H)N1. Key to responses: L= Low treatment, ML= Medium-low treatment,, M= Medium treatment, MH= Medium-high, 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 thisassay.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 indicatedby an X in this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column.For details see Section IV. AN 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 pa thway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androg enic, anti-androgenic or HPG axis effects. This
table also includes HPG-relevant findi ngs 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 test ing results, acetone was classified as negative (non-binder) in the in vitro AR binding and the ster oidogenesis assays.

In the Hershberger assay, acetone did not induce significant incr eases in accessory sex tissue (AST) weights in the an drogenic portion of the assay or si gnificant decreases in the antiandrogenic portion of th e assay when tested up to and includi ng 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/da y). It is noted that mean se rum testosterone value (4.84 ng/ mL) for the high-dose group was only slightly ab ove the range of histor ical 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 reproductiv e 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, relati ve 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).ACETONE FINALPage12of32Table 3. Androgenic/Anti-Androgenic Pathway for AcetoneLines of Evidence Indicating Potential Interaction with theAndrogenic/Anti-AndrogenicPathway for Acetone1Study Type/LiteratureCitation 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP TierAssaysAR Binding(MRID 48614602)NSteroidogenesis(MRID 48614610)NHershberger(MRID 48614607)NNNNMale Pubertal Rat(MRID 48614609)NNNNNNNNNNFSTRA(MRID 4861606)NENNNNNNACETONE FINALPage13of32Table 3. Androgenic/Anti-Androgenic Pathway for AcetoneLines of Evidence Indicating Potential Interaction with theAndrogenic/Anti-AndrogenicPathway for Acetone1Study Type/LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides His t o p a t holo gy Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed3OSRIOral Subchronic(14-Day) Rat(NTP, 1991;Dietzet al., 1991)↑(H)NN N NNX(M, MH,H)NOral Subchronic(14-Day) Mouse(NTP, 1991;Dietzet al., 1991)NNN N NNX(M, MH,H)NOral Subchronic(90-Day) Rat(NTP, 1991;Dietzet al., 1991)↑(H)N↓(H)N NNP4X(MH,H)NOral Subchronic(90-Day) Mouse(NTP, 1991;Dietzet al., 1991)NNNN NNNN1. 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 notroutinely 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 forthe OSRI is indicated by an X in this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated byan X in this column. For details see Section IV. A4. Decreased in sperm motility, caudal weight, epididymalweight, and an increased incidence of abnormal sperm.P Positive findingsN 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 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroi d hormone production, but it does
include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal 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 th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPTrelevant findings from data evaluated as OSRI . This table also include s HPG-relevant findings
from the data evaluated as OSRI. Effects that occurred in the pr esence 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, th e 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 T 4 or histopathology in either sex.

In the AMA, there were no significant altera tions in developmental stage (including no asynchronous development) as well as no effect s on thyroid histopathol ogy. A significant 11% increase in day 21 normalized hind limb length (HLL) was obser ved 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 decrea se of 11% in the high-concentrat ion group. Since there were no effects on development or thyroid histopathology, the change in H LL was considered to likely not be thyroid-related. Clinical sign s of tail curvature were observed in all treatment groups, with a higher incidence in the treated group s relative to the negative control.

Thyroid effects were not observed in any OS RI toxicity studies conducted with acetone.ACETONE FINAL

Page 15 of 32 Table 4. Thyroid Pa thway for Acetone

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Acetone 1

Study Type / Literature CitationThyroid WeightThyroid: Grossand HistopathologySerumT4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (+ orasynchronous &HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48614608) N N N N N N N N

Male Pu b e r tal Rat (MRID 48614609) N N N N N N N N

A MA (F r og; MRID 48614601) N ↑ 11% (H) ↓ 11% (H)
BW N

OSRI
O r al Su b chronic (14-Day) Rat (NTP, 1991; Dietz et a l

. , 1991 ) N N N N N X (M, HM, H) N

O r al Su b chronic (14-Day) Mouse (NTP, 1991; Dietz et al. , 1991) N N N N N X (M, MH, H) N

O r al Su b chronic (90-Day) Rat (NTP, 1991; Dietz et al. , 1991) N N N N N X (MH, H) N

O r al Su b chronic (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 findingsACETONE FINAL

Page 16 of 32 IV. Committee’s Assessment of Weight of Evidence

This section of the document de scribes the weight of evidence (WoE) determination on the potential of acetone to interact w ith 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 scient ifically 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 (U SEPA 2011) and is conduc ted on a case-by-case basis by first assessing the di fferent lines of evidence ( i.e. specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.

The WoE evaluation includes consid erations of biological plausi bility of the findings from the different lines of evidence by examining the cons istency, coherence, and interrelationships among the measured endpoints within and across studies. The available findi ngs from standard toxicology studies on the substance may contribu te to the WoE evaluation in he lping elucidate if effects seen in the Tier 1 assay are related to pe rturbations of the endocrine system per se or alternatively a sequelae of systemic effects. Endocrine modes of action ma y elicit a number of phenotypic consequences other than those ev aluated in the Tier 1 assays.

Endocrine-related findings in the pr esence of overt toxicity may resu lt in uncertainty as to whether or not the responses are relate d through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight chan ges) 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 endocr ine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. sec ondary effects related to to xicity 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 whic h 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).ACETONE FINAL

Page 17 of 32 1. Tier 1 in vivo Assays

For acetone, no overt toxicity or signs of syst emic 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 wa s seen in 8% (5/60) of tadpoles in the ne gative 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 fo r acetone. Data were available in the public literature. The most noteworthy findings in subchronic studies were incr eased liver and kidney weights, and decreased spleen weights. In mice admini stered 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 in creased relative kidne y and liver weights, hemosiderosis of the spleen a nd an increased inci dence 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 change s 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 expo sure concentra tions of 19,000 ppm (equal to 11,703mg/kg/day).

In a reproduction study with mi ce, acetone was administered via gavage at doses of 0 or 3,500 mg/kg/day. Toxicity was manifested as decreased reproductive index, incr eased gestation length, reduced birth weights, decreased neonatal survival and increase d 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 mi ce, maternal (increased incidenc e 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 obser ved in mice. The NOAEL was 2,200 ppm (equivalent to 1,348 mg/kg/day). In rats, maternal (reduc tion 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).ACETONE FINAL

Page 18 of 32 B. Estrogen Pathway

There was no convincing evidence of potential interacti on 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 observ ed, there were no other effects observed in the study including histopathology . Additionally, it is noted that this isolated finding
was observed at the lim it dose (1000 mg/kg/day) and half the limit dose (500 mg/kg/day). Furthermore, there were no effects on female mammalian reproductive parame ters observed in the
mammalian OSRI. In the FSTRA assay, while there was a signifi cant decrease in fecundity at the intermediate concentration of 6.2 mg a.i./L ( ↓ 51%), there was high variab ility in fecundity among the treatment groups in this stud y as well as a lack of comple mentary responses across other endpoints.

C. Androgen pathway

There was no convincing evidence of potential interacti on 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 te stosterone concentration at the limit dose (1000 mg/kg/day) there were no effects on the othe r androgen-related endpoi nts including age of attainment of PPS, male reproductive organ weight s, and no corroborating microscopic findings. While alterations in the androgen -related tissue weights and effect s 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 androge n-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 estroge n, androgen or thyroid pa thways or mammals or
wildlife

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, mamm alian and wildlife EDSP Tier 2 testing are not recommended since there was no evidence of potentia l interaction with the estrogen, androgen or thyroid pathways.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 - Am phibian 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. (1 988) Inhalation developm ental toxicity studies: teratology study of acetone in mice and rats. Pacific Northwes t Laboratory, Richland, WA, NTIS

No. DE89005671.

NTP (National Toxicology Progra m). (1991) Toxicity studies of acetone (CAS No. 67-64-1) in F344/N rats and B6C3F1 mice (d rinking water studies). NTP, Re search Triangle Park, NC, NTP TOX 3, NIH Publication No. 91-3122.

Stump, D.G. (2013) A Hershberger Assay of Acetone Administer ed Orally in Peripubertal Orchidoepidymectomized Rats. Unpublished stud y conducted by WIL Research, Ashland, OH. Laboratory Project No.: WIL-186063, February 6, 2013. (MRID 48614607)

Stump, D.G. (2013) A P ubertal Development and Thyroid Func tion Assay of Acet one Administered Orally in Intact Juvenile/Peripube rtal Female Rats. Unpublished study conducted by WIL Research, Ashland, OH. Laboratory Project No.: WI L-186065, February 7, 2013. (MRID 48614608).

Stump, D.G. (2013) A Pubert al Development and Thyroid Function Assay of Acetone Administered Orally in Intact Juvenile/Peripubertal Male Rats. Unpublishe d study conducted by WIL Research, Ashland, OH. La boratory Report No.: WIL-186064, February 7, 2013. (MRID 48614609)

Stump, D.G. (2013) A Uterotrophi c Assay of Acetone Administered Orally in Ovariectomized Rats. Unpublished study conducted by WIL Researc h, Ashland, OH. Laboratory Project No.: WIL186062, February 6, 2013. (MRID 48614611)

USEPA. (2011) Endocrine Disruptor Screening Program – Weig ht of Evidence: Evaluating Results of EDSP Tier 1 Screening to Iden tify the Need for Tier 2 Testing. Office of Chemical Safety and
Pollution Prevention. U.S. Environmenta l Protection Agency, Washington, DC.ACETONE FINAL

Page 20 of 32 USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

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

Wagner, H. (2013) Ac etone: H2958R Steroido genesis Assay. Unpublis hed study conducted by CeeTox, Inc, Kalamazoo, MI. Laboratory Report No.: 9186V-100381STER, January 28, 2013. (MRID 48614610).

Willoughby, J.A. (2013) Acetone: Androgen Re ceptor Binding (Rat Prosta te Cytosol). Unpublished study conducted by CeeTox, Inc, Kalamaz oo, MI. Laboratory Report No.: 9186V-100381ARB, January 23, 2013. (MRID 48614602).

Willoughby, J.A. (2013) Acetone: Human Re combinant 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 Rece ptor Binding (Rat Uterin e Cytosol). Unpublished study conducted by CeeTox, Inc, Kalamazoo, MI. Laboratory Repor t No.: 9186V-100381ERB, January 23, 2013. (MRID 48614604)

Willoughby, J.A. (2013) Acetone: Estrogen Rece ptor Transcriptional Acti vation (Human Cell Line (HeLA-9903)). Unpublished study conducted by CeeT ox, Inc., Kalamazoo, MI. Laboratory Report No.: 9186V-100381ERTA, Janua ry 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 Guid elines. Unpublished study performed by Smithers Viscient, MA. Laboratory repor t number 14036.6106. Study sponsored by American Chemistry Council, Acetone ED SP Consortium, Washington, D.C. 20002. February 12, 2013. (MRID 4861606)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 fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and tr eatment 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%). Th e 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 23.3 o C and a pH of 7.0 to 8.2.

Unless otherwise indicated, all eff ects 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 ki lled during one of the daily cleanings. Tail curvature was observed in all treatment groups and the reviewer noted a highe r incidence in the treated groups, relative to the negative control; tail curvature was seen in 8% (5/ 60) of tadpoles in the ne gative control and in 23% (14/60), 13% (8/60), and 23% (14/60) of the tad poles 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 observe d, with a significant in crease 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 accelera tion 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 satura tion binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affi nity for the radiolabeled refe rence androgen (R1881) prior to conducting AR competitive binding experiment. Th e competitive binding assay was conducted toACETONE FINAL

Page 22 of 32 measure the binding of a si ngle concentration of [ 3 H]-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 concentr ation of 3.2%. A total of three runs were performed, and each run included dexamethasone as a weak positive contro l, and R1881 as the ligand reference standard.

In the saturation binding experime nt, the dissociation constant (K d ) for [ 3 H]-R1881 was 0.591 ± 0.168 nM, and the estimated Bmax was 0.007 ± 0.002 for the single batch of pr ostate cytosol that was prepared. Confidence in these number s is high according to the goodness of fit (R 2 = 0.930- 0.989) and the small variation among runs.

In the competitive binding experiment, the estimated average log IC 50 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 pos itive control averaged 0.0029%. No precipitation of the test compound was observed.

The percent [ 3 H]-ligand binding was >75% at all concentr ations of acetone tested in all three competitive binding runs. A log IC 50 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 arom atase and tritiated androstenedione (1- β [ 3 H(N)]- androst-4-ene-3,17-dione; [ 3 H]ASDN) at log conc entrations 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 th e 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). Thr ee runs were conducted and each run included a full activity control, a background activity c ontrol, 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 repetiti ons 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 sta ndard deviation of 0.474 ± 0.064 nmol·mg-protein

− 1 ·min

− 1

. Activity in the background controls averaged 0.51% of the full co ntrol activity. The response ofACETONE FINAL

Page 23 of 32 the full activity controls and background c ontrols 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 test ed 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 i ndicated that the aromatase activity of
acetone at all concentrations tested was essentiall y equivalent to the activity observed in the full activity control. These data could not be f it 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) Bi nding Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding a ssay (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 bi nding 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 appropr iate 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]-17 β -estradiol (1 nM) in the presence of increasing con centrations (logarithmic increase from 10

− 10 to 10

− 3 M) of acetone. A solvent was not requir ed for acetone in this study. Di methyl sulfoxide (DMSO) was selected as a suitable vehicle for the positive contro l, 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 17 β -estradiol as the natural li gand reference material.

For the saturation binding experiment, the K d for [ 3 H]-17 β -estradiol was 0.046 ± 0.012 nM, and the
estimated B max was 0.008 ± 0.002 nM for the prepared rat uterine cytosol. The K d for each run was within the expected range of 0.03 to 1.5 nM. Th e data produced a linear Scatchard plot.

For the competitive binding experiment, the mean log IC 50 was

− 9.0 M for 17 β -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 cont rol. Acetone did not competitively displace the radiolabeled estradiol from the ER at any test concentration in the three runs.ACETONE FINAL

Page 24 of 32 Based on the results of the three runs, acetone is classified as Not Interactive in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48614605), hER α -HeLa-9903 cells
cultured in vitro were exposed to acetone (99.9% pur ity, 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 con centration 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 (luciferas e) gene products. Luciferase expression in response to activation of the estrogen receptor by acetone was measured upon addition of a luciferase substrate and de tection with a lumino meter with acceptable sensitivity.

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

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. In addition, cyto toxicity was observed at the highest tested
concentrations of corticosterone in Run 1. Resu lts from cytotoxic concen trations were excluded from the analysis. Although the conditions of this assay were not optimal to detect very weak activity, acetone responses were si milar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 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 PC Max was 10

− 6 M for both runs. Because the RPC Max <
PC 10 in both assay runs, acetone was considered ne gative 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 (M RID 48614606) of acetone with fathead minnow ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (24/group, ~23 weeks old) were exposed to ace tone (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 review er-calculated time-weighted average (TWA) concentrations were 0.66, 6.2, and 71.4 mg a.i./L. Th e 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 lo w- and mid-concentration treatment groups and 96% in the high-concentration tr eatment group, with one male fish dying. Acetone exposure was
not associated with an increase or decrease in b ody weight or length in male fish, or length inACETONE FINAL

Page 25 of 32 female fish at any treatment level. However, female body weight was si gnificantly increased by 10.6% at the high treatment level compared to the control (p=0.051; Jonckheere-Terpstra). No other effects on secondary sex char acteristics 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 repl icates and 22.8 eggs/female/day/ replicate (replicate range: 17-33 eggs/female/day); fertilization success in cont rols was 100%. Acetone significantly decreased fecundity by 51% at the mid treatment leve l (p<0.05; Dunnett’s); however, there was high variability in the fecundity e ndpoint across treatment groups in this study. Fertility was not significantly different in the treat ment groups compared to the control. Plasma vitellogenin (VTG) levels were not significantly di fferent between control and acetone treatment groups for both males and females. No significant increase or decreas e in gonado-somatic index (GSI) was observed in male or female fish at any treatment level. Nuptia l tubercle score was not si gnificantly increased or decreased in males at any treatment level; no nuptia l tubercles were observed in surviving females.

There were no reported treatment -related effects on gonadal histopa thology reported for male and
female fish.

Microbial growth was observed in the test chambers and an ag gressive 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 con centration-dependent decr ease in dissolved oxygen (DO) was observed. The mid and high concentrati on 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) screen ing for androgenic activity, acetone (99.9% purity, Batch# 114630) in deionized wa ter was administered daily via oral gavage to groups of 54- day old, castrated male Sprague Dawley rats (6/g roup) at dose levels of 0 (vehicle), 100, 300, or 1000 (limit dose) mg/kg/day. An androgenic positiv e control group was included consisting of six castrated rats exposed to 0.2 mg/kg/day testostero ne propionate (TP) in corn oil by subcutaneous (s.c.) injection.

To screen for potential anti-androge nic activity, acetone in deionized water was administered daily via oral gavage to 54-day old, castrated male Spra gue Dawley rats (6/group) at dose levels of 100, 300, and 1000 mg/kg/day in conjunction with a da ily dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The positive cont rol group for androgenic activity served as the control group for the anti-androgenic activity. An anti-androgenic positive control group consistedACETONE FINAL

Page 26 of 32 of six castrated rats dose d 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 admini stration to determine weights of the five androgen-dependent tissues (Cowper’s gland, glans penis, le vator ani and bulbocavernosus [LABC] muscle group, seminal vesicles with coa gulating glands, and ventral prostate), and the adrenal glands, kidneys, and liver.

All animals survived until sche duled termination. No treatment -related clinical signs were observed in the androgen agoni st or anti-androgen assays.

In the androgen agonist assa y, body weights and overall body we ight gains for all acetone treatment groups were comparable to vehicle cont rols 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; 5 19% in ventral prostate; 149% in LABC; 238% in Cowper’s gland; and 66% in glans penis. Th e 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 gl ands, kidneys or liver weights in the 100, 300, or 1000 mg/kg/day groups.

In the anti-androgen assay, body weights and overal l body weight gains for all acetone treatment groups were comparable to TP-control throughout th e 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 Co wper’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 liv er weights in the 100, 300, or 1000 mg/kg/day acetone+TP groups.

Statistically significant changes were not seen in tw o 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 Sp rague Dawley [Crl:CD(SD)] rats/dose group were treated daily by oral gavage with acetone (9 9.9%; lot # 114630) in deionized water at doses of 0 (vehicle), 500, or 1000 (limit dose) mg/kg/day fr om post-natal day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age a nd weight at day of
attainment were recorded. After sacrifice on PND 42/43, blood was collected for clinicalACETONE FINAL

Page 27 of 32 chemistry analyses, and total thyroxine (T 4 ) and thyroid stimulating hor mone (TSH) concentrations
were determined with an elec trochemiluminescent immunoassay (T 4 ) or a radioimmunoassay (TSH). Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic
examinations were conducted on the ovaries, uterus, t hyroid, and kidneys.

There were no signs of systemic toxicity. All an imals survived until scheduled termination. There were no effects of treatment on body weight, bod y weight gain, organ weight, or gross or microscopic pathology. Creatinine was increased (p<0.05) by 13 a nd 25% and urea nitrogen was decreased (p<0.05) by 12 and 20%, in both lo w- and high-dose groups , respectively. No corroborating microscopic findings were noted. Minimal mineraliza tion, minimal to mild chronic progressive nephropathy, and pyelo nephritis were observed in the kidneys, but occurred at similar frequencies across all dose groups.

There were no effects of treatmen t on age or body weight at attain ment 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 tre nd to increase (p<0.05) at 500 and 1000 mg/kg/day; however, there were no microsc opic findings to suggest a treatment-related
effect. The uterus and ovaries of all animals we re 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 treatmen t on thyroid hormone concentrati ons. There were no differences in follicular cell height or colloid area obs erved between the control and treatment groups.

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

Male Pubertal Assay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48614609), 15 Sp rague Dawley [Crl:(CD)SD] rats/dose group were treated daily via oral gavage with acetone (99.9%; lot # 114630) in de ionized 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 be ginning on PND 30, and age and weight at day of
attainment were recorded. Following sacrifice on PND 53/54, bl ood was collected for clinical chemistry analyses, and total se rum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were determined using an electr ochemiluminescent immuno assay (testosterone and

T 4 ) or a radioimmunoassay (TSH). Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were perfo rmed 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 toxici ty were observed. There were no effect s of treatment on final body weights,ACETONE FINAL

Page 28 of 32 overall (PND 23-53) body weight gains, organ weights, clinical chem istry, or gross or microscopic pathology.

There were no effects of treatmen t on age and body weight at attainment of complete PPS. Serum
testosterone levels were increas ed (p<0.001) by 140% at 1000 mg/kg/da y. 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 contro ls (1.62-4.20 ng/mL). Therefore, this finding was considered incidental to treatment.

There were no effects or treatment on serum T 4 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 th e limit dose of 1000 mg/kg/day.

Steroidogenesis A ssay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 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 μ M in triplicate for 48 hours. No solvent was necessary for the test chemical; it was added directly to the growth medium.

Testosterone and estradiol leve ls were measured by using a HPLC/MS/MS method that included internal standards. Eight inde pendent experiments were conduc ted 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 resp onded properly to positive control agents at two concentration levels; positive controls included a known inhibitor (prochloraz) and known inducer (forskolin) of estradiol a nd testosterone production.

The between-plate %CVs for the solvent control (S C) 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 estrad iol. 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. A ll within-plate
%CVs met Guideline recommendations.

Precipitation was not observed at any tested aceto ne concentration, and acetone was not overtly cytotoxic to the H295R cells. Viabi lity for acetone on the test plates was ≥ 95.2%ACETONE FINAL

Page 29 of 32 Based on the hormone responses in at least three independent runs, acetone treatment did not result in statistically significant and re producible alterations in testosterone or estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

In a uterotrophic assay (MRID 48614611) conducted to screen for potenti al estrogenic activity, acetone (99.9% purity, Lot # 114630) in deionized wate r was administered daily via oral gavage to groups of 6 ovariectomized Sprague-Dawley rats at dose levels of 0 (v ehicle), 100, 300 and 1000 (limit dose) mg/kg/day on post-natal days (PND) 60-62. A positive control group was treated with

17 α -ethynyl estradiol (EE) by daily subcutaneous (s.c.) inject ion at a dose level of 0.001 mg/kg/day. All animals were terminated and n ecropsied, 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 ut erus weights for the EE group were increased (p<0.0001) by 487% and 174%, resp ectively, as expected.

No statistically significant changes were seen in uterine weight in this as say. Acetone is negative in the uterotrophic assay.ACETONE FINAL

Page 30 of 32 APPENDIX 2. Other Scientifica lly Relevant Info rmation (OSRI)

Published literature and general toxicity studies c onsidered relevant for de termining the potential interaction of acetone with the endocrine system ar e summarized below. The only toxicity studies discussed in this append ix are those that are directly rele vant to assessing potential endocrine effects ( i.e. , multiple- generation rodent reproductive studi es, development studies, chronic toxicity studies, etc.).

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

Groups of F344/N rats (5/s ex/dose) were administered aceton e (99% purity, Lot/Batch #AN228) in drinking water at concentr ations of 0 (control), 50 00, 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. Increase s in relative liver and kidney weights were observed in bo th 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; fu rther details of organ weight data were not included. Bone marrow hypoplasia was noted in all high-dose males, but not in th e 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 admini stered acetone (99% pu rity, 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 ma les 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 we re decreased (81 and 93%, respectively) relative to control. All animal s survived to terminati on. 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 centrilo bular hepatocellular hypert rophy were observed in
males at the intermediate dose a nd higher and in females in the high-mid- and high-dose groups. Hematology and clinical chemistry evaluations were not conducted.ACETONE FINAL

Page 31 of 32 90-Day (Subchronic) Oral Toxicity in Rodents (Rat)

Groups of F344/N rats (10/sex/dos e) were administered acetone (99% purity, Lot/Batch #AN228) in the drinking water at concentrations of 0 (control), 25 00, 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 fo r females were 0, 300, 600, 1200, 1600, and 3100 mg/kg/day, respectively. No deaths occurred in any group. Water consum ption was decreased in high-dose males and in the high-m id- and high-dose females. Mean final body weightof the highdose males was 81% of control; bod y 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 we re noted: kidney weights were 114% of control for high-middose females and 126 and 123% of c ontrol for high-dose males and females, respectively; liver weights were 110 and 112% of cont rol 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. Li ver weight increases were not as sociated with histopathological
lesions and were consider ed to be the result of enzyme induc tion. 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 co ntrol animals; therefore, the incidence of nephropathy rated as mild was considered an indi cator of toxicity. Males given the two highest concentrations of acetone had increases in the in cidence 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/dos e) 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, re spectively, and for females 0, 900, 2000, 4200, 5900, and 11,000 mg/kg/day, respectively. No d eaths 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 cytolo gy, were not affected in either sex. Organ weights in treatedACETONE FINAL

Page 32 of 32 males were similar to cont rol; 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 hypertro phy, observed in tw o high-dose females, considered to be due to enzyme induction. Mild hepatic change s (observed in males exposed to

≥ 20,000 ppm for 14 days) did not persis t after 13 weeks of exposure, suggesting the development of tolerance toward acetone. In summary, the liver wa s 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, we re not evident in mice. The LOAELs for male and female mice were 4900 and 11,000 mg/kg-day, resp ectively (high dose), and the NOAELs were 2300 and 5900 mg/kg-day, respectively (high- mid dos e). The LOAEL for male mice was selected by the study authors on th e basis of the transient fi ndings in the 14-day study.