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 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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## ~~~ 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: ISOPHORONE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYISOPHORONE 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...........................................................................17

C. Androgen Pathway.........................................................................17

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 Isophorone.......................................................................7
Table 2. Estrogenic/Anti-Estrogenic Pathway for Isophorone ........................................9
Table 3. Androgenic/Anti-Androgenic Pathway for Isophorone .....................................12
Table 4. Thyroid Pathway for Isophorone ..............................................................15
APPENDIX 1. EDSP Tier 1 Screening Assays.................................................................21

APPENDIX 2. Other Scientifically Relevant Information (OSRI)...........................................32ISOPHORONE FINAL

iii Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration
F2 Second filial g eneration
Fcd Fecundit yISOPHORONE FINAL

iv Abbreviation Terminology

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

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated DoseISOPHORONE FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionateISOPHORONE FINAL

vi Abbreviation Terminology

T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 33 Executive Summary

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

In determining whether isophorone interacts with E, A or T hormone pathways, the number and type of effects induced, th e magnitude and pattern of re sponses observed across studies, taxa, and sexes were considere d. Additionally, the conditions under which effects occur were considered, in particular, whet her or not endocrine-related resp onses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On November 20, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WoE) an alysis of the potential interaction of isophorone with the E, A or T hormone pathwa ys. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Isophorone is a cyclic unsaturate d ketone whose acute mode of action is nonpolar narcosis (toxicologically induced and reversib le states of neural disruption, i.e . general anesthesia). There is no known pesticidal mode of action for isophorone, given that it is an inert ingredient and therefore requires testing at or near a limit dose/concentra tion in order to sufficiently challenge the animal.

For the estrogen pathway, the only observed es trogen-related effect, in the absence of overt toxicity, was limited to a decrease in female plasma vitellogenin in the fish short-term reproduction assay (FSTRA) which did not translat e into observed effect s in gonadal tissue or reproductive output. This effect on vi tellogenin in females by itself is not a clear i ndicator of an endocrine effect without other complementary re sponses since a decrease in female vitellogenin
could be due to other non-endoc rine modes of action. Other es trogen-related effects in the FSTRA were only seen in the presence of overt toxicity. Th ere were no complementary or redundant responses seen in other Tier 1 assay or other scientifically relevant information (OSRI).ISOPHORONE FINAL

Page 2 of 33 For the androgen pathway, effects in the Tier 1 male pubertal assay in cluded a decrease in serum testosterone level, reducti on in seminal vesicle weight and a slight delay in unadjusted (for body weight) age (+1.8 days) at preputial se paration (PPS). However, these effects were only seen at a very high dos e (800 mg/kg/day), which appr oached the limit dose of 1000 mg/kg/day at which narcosis and lethargy was se en in the range-finding study. Additionally, in the Tier 1 pubertal assay, increases in absolute (32%) and relative (33%) liver weight were observed. There were no androgen-related eff ects observed in the Tier 1 FSTRA or Part 158
mammalian studies.

There were no thyroid-related effects observed in the amphibian metamorphosis assay (AMA) and the male and female pubertal assays.

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are not recommended for isophorone since there was no convincing evidence of potential interaction with E, A or T pathways.ISOPHORONE FINAL

Page 3 of 33 I. Introduction

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

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

On November 6, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP)conducted a weight-of evidence (WoE) analys is of the potential in teraction of isophorone with the estrogen (E), andr ogen (A) or thyroid (T) signa ling pathways. The T1WoERC
conclusions from the WoE evaluation in this re port are presented by pathway (E, A and then T) beginning with the resu lts 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).

Isophorone is a cyclic unsaturated ketone used as a solvent for pe sticide applications. It is soluble in water (>10,000 mg/L) wi th a partition coefficient (l og octanol/water) of 1.67, and a vapor pressure of 0.3 mm Hg at 20°C (ATSDR , 1989). After release to water or soil, environmental concentrations is anticipated to decrease as a result of volatilization and biodegradation. Isophorone in the atmosphere is removed by photochemical processes with an estimated half-life of about 30 min (based on a mathematical model). In a die-away test, isophorone was biodegraded by approximately 70% within 14 days, and 95% within 28 days. Water solubility, soil adsorption coefficients, and polarity indicate that significant adsorption by suspended solids and sediments is unlikely to occur (IPCS, 1996).

The available information considered in determ ining the potential intera ction of isophorone with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to theISOPHORONE FINAL

Page 4 of 33 Agency, using the WoE approach outlined by th e Agency (USEPA, 2011), is presented in
Section IV. The EDSP Tier 2 studies recommendations are presented in Section V.

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 believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on isophorone (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of eviden ce process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

Page 5 of 33 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

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

Page 6 of 33 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

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

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

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing th e three components descri bed 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 ke y studies; 3) description of inc onsistent or conflicting data; 4) overall strength of evidence supporting a conclusi on; and, 5) what, if a ny, additional data are needed and why. Assessing the need for additiona l data is b ased on a case-by-case analysis
which took all available toxi city data into account.ISOPHORONE FINAL

Page 7 of 33 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

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

.

Table 1: Tier 1 Screening Assays for Isophorone

Tier 1 Assays Test Guideline Test Order Status

Estrogen Receptor (ER) Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48617306)

ER α Transcriptional Activation Assay (Human cell line HeLa-9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48617305)

Androgen Receptor (AR) Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48617303)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48617311)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48617304)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48617309)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48617310)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48617307)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No.48617308)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48617301)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48617302)ISOPHORONE FINAL

Page 8 of 33 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for isophorone 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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WoE assessment.

Isophorone was negative in the Tier 1 in vitro assays and in the Tier 1 uterotrophic assay. Additionally, there were no estroge n-related effects observed in th e female pubertal rat assay. However, in the Tier 1 FSTRA, plasma vite llogenin (VTG) was significantly decreased ( ↓ 44%) in female fish at the mid-concentration (7.62 mg/L) in the absence of overt toxicity. At the high concentration (79.8 mg/L), reproduction was almo st completely suppressed and multiple other endpoints were affected: fecundity ( ↓ 94%), fertilization success ( ↓ 70%), female plasma VTG ( ↓ 77%) and a slight increase in oocy te atresia. However, at this concentration, signs of overt toxicity included reduced feed ing and reduced male and female body weights. No estrogenrelated effects were observed in the Part 158 ma mmalian studies or OSRI evaluating ER binding or transactivation (Nas hihara et al., 2000; Ma ssaad and Barouki, 1999).ISOPHORONEFINALPage9of33Table 2: Estrogenic/Anti-Estrogenic Pathway for IsophoroneLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Isophorone1StudyType/LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Gonadal Staging and Histopatholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48617306)NERTA(MRID 48617305)NAromatase(MRID 48617304)NSteroidogenesis(MRID 48617311)NUterotrophic(MRID 48617309)NNNFemale PubertalRat(MRID 48617307)NNNNNN↑LWNFSTRA(MRID 48617301)NENNNN↓44%(M)X4(H)OSRIERαtranscriptionalactivation(Massaad andBarouki, 1999)NSubchronictoxicity-Rat(MRID 00123977 )NN NISOPHORONEFINALPage10of33Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Isophorone1StudyType/LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Gonadal Staging and Histopatholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronictoxicity- Dog(MRID 00123976)NN NCarcinogenicityRat (MRID00151527)N NN NCarcinogenicityMouse(MRID 00151527)N NN N1 Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated oris not applicable in this assay. LW= Liver weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Reduced feeding and reduced male and female body weights at 79.8 mg/L.P Positive findings (in vitro)N Negative findings (in vitro); No effect (in vivo)NE Not ExaminedISOPHORONE FINAL

Page 11 of 33 2. Effects on Androgen Pathway

The potential for isophorone to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WoE assessment.

Isophorone was negative in the Tier 1 in vitro assays and in the Tier 1 Hershberger assay. However, in the Tier 1 male pubertal rat assay, th ere was a statistically si gnificant decrease in serum testosterone (58% lower than controls) at the highest dose tested (800 mg/kg/day). Other effects found at 800 mg/kg/day included a ~24% reduction in the weight of seminal vesicles and a 1.8 day delay in pr eputial separation (PPS). No e ffects were seen at lower doses (50 and 200 mg/kg/day). In the FSTRA, no andr ogen-related effects in male fish were observed. Additionally, no androgen-related eff ects were observed in the Part 158 mammalian
studies.ISOPHORONEFINALPage12of33Table 3: Androgenic/Anti-Androgenic Pathway for IsophoroneLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Isophorone1Study Type AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights Age and Weight at PPS\2° Sex Characteristics Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48617303)NSteroidogenesis(MRID 48617311)NHershberger(MRID 48617310)NNNNN NMale Pubertal Rat(MRI D 48617308)↓58%(H)NNNNN↓24% SV(H)↑1.8 d (H)↑LW NFSTRA(MRID 48617301)NENNNNX4(H)OSRISubchronic toxicityRat(MRID 00123977 )NNX NSubchronic toxicityDog(MRID 00123976)NNN NCarcinogenicity-Rat(MRID 00151527)NNN NCarcinogenicity-Mouse(MRID 00151527)NNN N1Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. SV=Seminal vesicle; LW=Liver weight2The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. AISOPHORONEFINALPage13of333The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4Reduced feeding and reduced male and female body weights at 79.8 mg/L.N Negative findings (in vitro); No effect (in vivo)ISOPHORONE FINAL

Page 14 of 33 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

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

The potential for isophorone to interact w ith thyroid regulation is summarized in Table 4. The various targets of the thyroid pathway across the rele vant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HP T-relevant findings fr om 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 furthe r in the WoE assessment.

There were no thyroid-related effects observed in the Tier 1 female and male pubertal assays. Additionally, there were no effects on amphibian development or t hyroid histopathology observed in the AMA. In the AMA, on day 21, reductions in body weight (15%) and s nout-vent length (SVL; 5 %) were observed at the high test concentration (59 mg/L). No other thyroid -related effects were observe d in the Part 158 mammalian
studies.ISOPHORONE FINAL

Page 15 of 33 Table 4: Thyroid Pathway for Isophorone Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Isophorone 1

Study TypeThyroid WeightThyroid : Gross andHistopathologySerum T4 LevelsSerum TSH LevelsPituitary WeightGrowth (BW, SVL)Developmental Stage(+ or asynchronous,HLL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal- Rat (MRID 48617308) N N N N N N ↑ LW N

Female Pubertal- Rat (MRID 48617307) N N N N N N ↑ LW N

AMA (Frog) (MRID 48617302) N D21:
BW ↓ 15%; SVL ↓ 5% (H) N N

OSRI

Subchronic toxicity-Rat (MRID 00123977 ) N N X N

Subchronic toxicity- Dog (MRID 00123976) N N N N

Carcinogenicity-Rat (MRID 00151527) N N N N

Carcinogenicity-Mouse (MRID 00151527) N N N N

1. Key to responses L=Low treatment, M=Medium treatme nt, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routin ely evaluated or is not applicable in this assay. LW = Liver weight; BW=Body weight; SVL=Snout ventral length
2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A
3. The overt toxicity in the Tier 1 assa ys 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 findingsISOPHORONE FINAL

Page 16 of 33 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of isophorone to interact with endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence. The WoE evaluation includes considerations of biologi cal 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 availabl e 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 pertur bations of the endocrine system per se or alternatively a sequelae of systemic effects. Endocrine modes of acti on may evoke a number of phenotypic consequences
other than those evaluated in the Tier 1 assays.

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

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects 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, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ w eights or blood chemistry).ISOPHORONE FINAL

Page 17 of 33 1. Tier 1 in vivo Assays

Systemic toxicity in the mammalian in vivo assays at the high dose (800 mg/kg/day) was limited to increased absolute (32% in males and 18% in females) and relative (33% in males and 17% in females) liver weights in the pubertal assays. In the range-finding study for the male pubertal assay, rats received isophorone at the Limit Dose (1000 mg/kg/day). By test day 3 (after 2 doses of isophorone), both male and female rats had lost body weight (17 and 20 g, respectively) and feed consumption was markedly decreased, ranging from 0.7-6.8 g/day in these animals. Clinical signs included decreased activity, slow respiration, uncoordinated gait and perineal/perioral soiling. These animals were euthanized for humane reasons on Day 3. In the FSTRA, overt toxicity at the high dose (80 mg/ kg/day) manifested as significant reduction in male and female body weights, reduced feeding, and an observable decrease in body size in males compared to
the controls.

2. OSRI

In the subchronic toxicity study with rats dietary administrati on of isophorone at 312 mg/kg/day resulted in greater than 10% decrease in ab solute body weight in male rats; no body weight effects were seen in females. No other syst emic toxicity was seen in the OSRI studies.

B. Estrogen pathway

Isophorone is a cyclic unsaturate d ketone whose acute mode of action is nonpolar narcosis (toxicologically induced and reversib le states of neural disruption, i.e . general anesthesia). There is no known pesticidal mode of action for isophorone, given that it is an inert ingredient, and therefore requires testing at or near a limit dose/concentra tion in order to sufficiently challenge the animal.

The only observed estrogen-related effect in th e absence of overt toxicity, was limited to a decrease in female plasma vitellogenin in th e FSTRA which did not translate into observed effects in gonadal tissue or repr oductive output. This effect on v itellogenin in females by itself is
not a clear indicator of and e ndocrine effect without other co mplementary responses since a decrease in female vitellogenin could be due to other non-endocrine modes of action. Other estrogen-related effects in the FSTRA were only s een in the presence of overt toxicity. There were no complementary or redundant responses seen in other Tier 1 assay or OSRI.

C. Androgen pathway

Effects in the Tier 1 male pubertal assay incl uded a decrease in serum testosterone level, reduction in seminal vesicle weight and a slig ht delay in unadjusted (for body weight) age (+1.8 days) at PPS. However, these effects were only seen at a very hi gh dose (800 mg/kg/day), which approached the limit dos e of 1000 mg/kg/day for which na rcosis and lethargy was seenISOPHORONE FINAL

Page 18 of 33 in the range-finding study. Additiona lly, in the pubertal assay, in creases in absolute (32%) and relative (33%) liver weight were observed. There were no androge n-related effects observed in
the Tier 1 FSTRA or Part 158 mammalian studies.

D. Thyroid pathway

There were no thyroid-related e ffects observed in the AMA and the male and female pubertal

assays.

E. Conclusions

Based on a WoE of the available data, there is no convincing evidence fo r potential interaction with the estrogen, androgen or t hyroid pathways for isophorone.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are
not recommended for isophorone since there was no convincing evidence of poten tial interaction with E, A or T pathways.ISOPHORONE FINAL

Page 19 of 33 VI. References

ATSDR (1989). Agency for Toxic Substances and Di sease Registry (1989) Toxicological Profile for isophorone.

Coady, K. et al. (2011) Isophorone: The Am phibian Metamorphosis Assay with ( Xenopus laevis ). Dow Chemical Company, Midland, Mi chigan. Laboratory Report Number 101063. Unpublished. (MRID 48617302).

Coady, K.K. and Sosinski, L.K. (2011) Evalua tion of Isophorone in the Human Recombinant Aromatase Assay. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Labor atory Project Study ID: 101140. Unpublished. (MRID 48617304).

Geter, D., Coady, K., LeBaron, M., and Marty, M. (2012) Isophorone: Evaluation of potential estrogen, androgen, or thyroid effects. . Toxi cology & Environmental Re search and Consulting, Dow Chemical Company, Midland, Michigan. Laboratory Study ID NS000077. Unpublished.

International Programme on Chemical Safe ty (1995) IPCS re port on isophorone.

LeBaron, M.J., Kan, H.L., (2011) Evaluation of isophorone in an in vitro estrogen receptor transcriptional activation assay in Human cell Line hER α -HELA-9903. Unpublished (MRID 4861705).

LeBaron, M., Kan, H., Perala, A. (20 11) Evaluation of isophorone in the in vitro steroidogenesis assay. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101185, November 14, 2011. Unpublished (MRID 48617311).

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011). Evaluation of isophorone in an in vitro estrogen receptor binding assa y. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Labor atory Project ID: 111119, Unpublished (MRID 48617306).

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011) Evaluation of isophorone in an in vitr o androgen receptor binding assay. Toxicology & E nvironmental Research and Consulting, The Dow Chemical Company, Midland, MI. Labor atory Project ID: 111112. Unpublished. (MRID 48617303).

Marino, T.A., K.K. Coady, L.K., Sosinski, and Thomas, J. (2012). Isophorone: The Fish Shortterm Reproduction Assay Using the Fathead Minnow, Pimephales promelas . Toxicology and Environmental Research and Consulting, Mi dland, Michigan. Lab Study No.: 101057 and K001389-014. Unpublished. (MRID 48617301).

Marty, M.S., Zablotny, C. L., and Hukkanen, R. R. (2011). Isophorone: pubertal development and thyroid function in intact juvenile/peripubertal female Crl:CD(SD) rats. Toxicology &ISOPHORONE FINAL

Page 20 of 33 Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111100, D ecember 22, 2011. Unpublished. (MRID 48617307).

Marty, M.S., Andrus, A. K., and Hukkanen, R. R. (2011). Isophorone: pubertal development and thyroid function in intact j uvenile/peripubertal male Cr l:CD(SD) rats. Toxicology & Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111101, Unpublished. (MRID 48617308).

Marty, M.S. and Brooks, K.J. (2011). Isophorone: Ut erotrophic Assay in Ovariectomized Adult Female CrL:CD(SD) Rats. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 101109. Unpublished. (MRID 48617309).

Marty, M.S., Marshall, V.A., and Jeong, YC. (2011). Isophorone: Hershberger Assay in Castrated Male Crl:CD(SD) Rats. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Labor atory Study No.: 101077. Unpublished. (MRID 48617310).

National Toxicology Program (1986) NTP techni cal report on the toxico logy and carcinogenesis studies of isophorone (CAS # 78-59-1) in F344/N rats and B6C3F 1 mice (gavage studies). (MRID 00151527)

Rohm & Hass Co. (1972a) 90-day subchronic toxi city of isophorone in the dog. Unpublished. (MRID 00123976)

Rohm & Hass Co. (1972b) 90-day subchronic toxici ty of isophorone in the rat. Unpublished. (MRID 00123977)

K.A. Traul et al., (1984) Inhalation Teratol ogy Study in Rats and Mice. Unpublished. (MRID 00151531).

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coordina tion and Policy. U.S. Environmental Protection Agency, Washington, DC.

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

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

Page 21 of 33 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (Frog); OCSPP 890.1100

The 21-day assay (MRID 48617302) of isophor one on amphibian metamorphosis of Xenopus
laevis was conducted under flow-through conditions. Amphibian larvae (80 tadpoles/treatment group) at Nieuwkoop-Faber (NF) Stage 51 were exposed to isophorone (98.7% purity) at 0 (negative control), 0.600, 6.00, a nd 60.0 mg a.i./L. Mean-measured concentrations were <0.199 (<LOQ; control), 0.584, 5.63, and 59.0 mg a.i./L. The test system was maintained at 21.7 to 22.8°C and a pH of 7.0 to 7.6.

One mortality was observed in the mid treatment group during the study, and this mortality was
not considered treatment-related. At test terminat ion, tadpoles with bent tails were observed in the control and all treatment groups ( 17-29%); these observations were notconsidered to be treatmentrelated.

The only treatment-related effects observed in th is study were significan t (p<0.05) reductions in Day 21 wet weight ( ↓ 15%) and Day 21 snout-vent length (SVL; ↓ 5%) in the high treatment group. Isophorone had no significant effect (p>0.05) on nor malized (for SVL) hind-limb length (HLL) at any treatment level on Day 7 or 21. There was no significant increase or delay of median NF developmental stage on Day 7 or 21. No asynchronous development was observed in any treatment group. Mild follicular cell hypertrophy was observed in the control and all treatment groups and did not appear to be treatment-related.

All effects are reported based on comparison to the negative (clean water) c ontrol and analysis of Day 21 wet weight, snout-vent length (SVL), HLL, and normalized HLL endpoints excluded performance of late-stage tadpoles (i.e., Day 21 NF >60). There were several individuals from the control and isophorone-treated groups to mature beyond NF stage 60 by Day 21 (range of NF=61- 62, with no clear patte rn in occurrence). Late stage individua ls did not comprise more than 20% of any treatment level and there was no evidence to suggest a dose-dependent relationship for late stage individuals (i.e., 13% of i ndividuals were NF>60 in the nega tive control group, compared to 8, 10, and 18% in the low, mid, and high treatment groups, respectively).

Androgen Receptor (AR) Bi nding Assay; OCSPP 890.1150

In an androgen receptor (AR) binding assay (MRID 48617303), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment,
which was conducted to measure the bi nding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations of isophorone (logarithmic increase from 10

− 10 to 10

− 3 M). Ethanol was used as a vehi cle at a final assay concentrati on of <3%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.ISOPHORONE FINAL

Page 22 of 33 The saturation binding experiment was conducted in duplicate and summarized in a submitted study profile (MRID 48614902). In the saturation binding experiment, the maximum binding capacity (B max ) was 3.245 fmol/100 μg protein and the dissociation constant (K d ) was 0.4641

nM. These values were below the range of values from th e validation studies (0.685-1.57 nM). The Scatchard plot indicated a linear response across the concentrations of ligand added. Nonspecific binding as a percent of total binding was less than 20% across the entire concentration range in the saturation binding assa ys (range 6.2-19.8%), with the exception of the high concentration (10 nM) in one assay, which was 24.6%.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 8.978 M) and the weak positive control, dexamethasone (

− 4.412 M), were within the expected ranges. The mean RBA for the weak positive control was 0.0027% . Confidence in these numbers is high due
to the small variation. The solv ent control responses indicated no drift in the study assay. All performance criteria were met.

Isophorone had no significant effect on specific binding of the [ 3 H]-ligand; therefore, a log IC 50 and RBA could not be calculated for isophorone as it did not disp lace 50% of the radioligand at any test concentration.

Based on the results from the three runs, isophor one 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 48617304), isophorone (98.7% a.i., lot#: 1136041301) in ethanol (1%) was incubated with hu man recombinant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) for 15 minutes at 37 °C to assess the potential of isophorone to inhibit aromatase activit y. Isophorone was tested at logarithmic concentrations from 10 –10 M to 10

− 3 M in three independent runs.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Thr ee independent runs were conducted and each run included a full activity c ontrol, a background activity control, a positive control series (10

− 10 to
10

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

− 10 to 10

− 3 M) with 3 repetitions per concentration.

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, Hill Slope, log IC 50 , and coefficient of variation (CV) for replicates of eachISOPHORONE FINAL

Page 23 of 33 concentration within runs. Fo r 4-OH ASDN, the estimated log IC 50 averaged

− 7.38 M and the slope was

− 0.912.

For isophorone, aromatase activity av eraged 0.224±0.018 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.187±0.027 nmol·mg-protein

− 1 ·min

− 1 at the highest tested concentration (10

− 3 M). The lowest portion of the respons e curve across runs was greater than 75% activity at all concentrations of isophorone. The average dose-response curve indicated that aromatase activity at concentra tions of isophorone ranging from 10

− 10 M to 10

− 3 M was essentially equivalent to the activity obs erved in the full activity controls.

Based on the data from the average response curve, isophorone 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 48617306) for isophorone, uterine cytosol from Sprague Dawley rats was used as the source of ER for a competitive binding experiment,
which was conducted to measure the bi nding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 10 to 10

− 3 M) of isophorone. Ethanol was used as the solvent vehicle at a final concentration of <3%. A total of three valid 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 ligand
reference material.

Summary data pertaining to the saturation binding experiment we re reported separately in the study profiles submitted by the test order recipient. The K d for [ 3 H]-17 β -estradiol was 0.1032
nM and the B max (nM) was 0.07097 for the prepared ra t uterine cytosol used in these experiments. The K d for the run was within the expect ed range of 0.03 to 1.5 nM, and the B max was within the expected range of 10-150 fmol/100 μ g protein. The data produced a linear Scatchard plot.

In the competitive binding experiment, the estimated mean log IC 50 was

− 8.917 M for 17 β

estradiol and

− 5.436 M for the weak positive control. The mean RBA was 0.033% for the weak positive control. Confidence in these numbers is high due to the small variation. All performance criteria were met.

Isophorone had no effect on sp ecific binding of the [ 3 H]-ligand; therefore, a log IC 50 and RBA could not be calculated for isophorone.ISOPHORONE FINAL

Page 24 of 33 Based on the results of the three runs, isophorone is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activatio n (ERTA) Assay; OCSPP 890.1300

In an estrogen receptor transcript ional activation assay (MRID 48617305), hER α -HeLa-9903
cells cultured in vitro were exposed to isophorone (98.7% a.i., Lot # 1136041301) at logarithmically increasing concentrations from 10

− 9 M to 10

− 3 M in DMSO (0.1%) in three independent runs. Experiments were performe d in 96-well plates and each concentration was tested in triplicate (3 wells/plate) in each run. Cells were exposed to the test agent for approximately 24 hrs to induce reporter (luciferas e) gene products. Luciferase expression in response to activation of the estrogen receptor was measured upon addition of a luciferase
substrate and detection with a luminometer with acceptable sensitivity. Isophorone was tested up to the guideline limit of 10

− 3 M. Performance criteria were ac ceptable for all of the reference chemicals. The response of 17 α -methyltestosterone indicates incr eased sensitivity to very weak agonists. The RPC Max was 5.9% for the first run, 7.8% for the second run and 9.9% for the third
run. Because the RPC Max < PC 10 in all three assay runs, isophorone was considered negative for estrogen receptor transcriptional activation in this test system.ISOPHORONE FINAL

Page 25 of 33 Fish Short-term Reproduction Assay (FSTRA); OCSPP 890.1350

The 21-day short-term reproduction assay (MRID 48617301) of isophorone with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 6 months old), were exposed to isophorone (98.7% purity) at nominal concentrati ons of 0 (control), 0.8, 8.0, and 80 mg a.i./L; mean-measured concentrations were <LOQ (<0.199), 0.748, 7.62, and 79.8 mg a.i./L. The test system was maintained at 24.6 to 25.4oC and a pH of 7.0 to 7.5.

Adult survival in both sexes at all treatment levels was 100% . Male and female body weight were both significantly (p<0.05) reduced by 24% re lative to the control at the high treatment
level. There were no notable observations on seconda ry sex characteristics or clinical signs of toxicity for any treatment group compared to th e controls. However, th e study author reported reduced feeding in both sexes with an observa ble decrease in body size for males in the high treatment group compared to the controls.

Spawning occurred at least every 4 days and fecundity in controls was 31.6 eggs/female/day/replicat e (range 19.1-36.5); fertiliz ation success in the control group averaged 98.4%. Fecundity was significantly (p<0.05; Jonc kheere-Terpstra) reduced 94% at the high
treatment level. There was also a significant reduction in fert ility of 70%, (p<0.05; JonckheereTerpstra) at the high treatment le vel compared to the control.

Female vitellogenin (VTG) was significantly (p< 0.05; Jonckheere-Terpstra) reduced 44 and 77% of control at the mid and high treatment le vels, respectively. Ther e were no significant
differences in male VTG between the treatment groups and the control.

There were no significant diffe rences in gonado-somatic index (GSI) for males or females compared to the control. Tubercle scores in ma les in the treatment groups were not significantly different from the control; tubercles were not observed in any female. Although not analyzed statistically, there were no repor ted treatment-related histopathologica l observations in males, but there was a reported increase in inci dence of mild to moderate oocyte atresia in female fish at the high treatment level compared to the control. Sex-steroids were not measured. Observation of dorsal fat (nape) pads was reported but furt her analysis of the data was not reported

.

Hershberger Assay; OCSPP 890.1400

In a Hershberger assay (MRID 48617310) te sting for androgenic activity, isophorone ( ≥ 98.7±0.1%, lot nos. 1137080201 and 1136041301) was admini stered daily in corn oil via oral gavage to groups of seven 55-day old castrated ma le Sprague Dawley rats/g roup at dose levels of 0 (vehicle), 100, 400, or 800 mg/kg/day for 10 c onsecutive days. The study included an androgenic positive control group of seven castrated male rats dosed daily with testosterone propionate (TP) in corn oil at 0.4 mg/kg/da y via subcutaneous (s.c.) injection.ISOPHORONE FINAL

Page 26 of 33 To screen for possible anti-androgenic activity, additional groups of seven 55-day old castrated male Sprague Dawley rats/group were dosed daily via oral gavage with isophorone in corn oil at 100, 400, or 800 mg/kg/day for 10 consecutive days in conjunction with a daily s.c. injection of TP in corn oil at 0.4 mg/kg/day. The negative control group consisted of seven castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the positive control group consisted of seven castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day for 10 days in conjugation with a da ily s.c. dose of TP (0.4 mg/kg/day).

Body weights, body weight gains, and food c onsumption were measured. Serum hormone
concentrations were not determin ed, and histology was not performed. Animals were euthanized approximately 24 hours after the final dose admini stration. The five androgen-dependent tissues, adrenals, kidneys, and liver were weighed and macroscopically examined. No adverse, isophorone treatment-related effects were observed on mortality, clinical signs of toxicity, body weights, body wei ght gains, food consumption, or gross pathology. No treatmentrelated effects were observed at any dose on the weights of any of the accessory sex tissues [seminal vesicles, ventral prostate, levator ani-bulbocavernosus muscle (LABC), Cowper's glands or glans penis], or on the adrenals or kidneys in the androge nic and anti-androgenic studies. Liver weights were increased (p ≤ 0.05) by isophorone treatment in both the androgenic and anti-androgenic portions of the study by 27-28% at 400 mg/kg/day and by 32–34% at 800 mg/kg/day. Although there was no evidence of t oxicity at the highest dose tested (800 mg/kg/day), the dose range finding assay did pr ovide sufficient evidence that a 1000 mg/kg/day dose is excessive (body weight losses, marked fo od consumption decrease, and clinical signs of distress leading to early animal termination). Th erefore, dosing was consid ered adequate in this study.

For the androgenic portion of the study, the TP positive control group demonstrated treatmentrelated increases (p ≤ 0.05) in the weights of the seminal vesicles w ith coagulating glands ( ↑ 1079%), ventral prostate ( ↑ 995%), LABC ( ↑ 96%), Cowper’s glands ( ↑ 603%), and glans penis ( ↑ 77%), indicating that the test system was sensitive to an andr ogenic response. TP
administration also resulted in increased food consumption and body weight gains, consistent with an androgenic response. For the anti-a ndrogenic portion of the study, the FT positive control group demonstrated treatment-related decreases (p ≤ 0.05) in weights of the seminal vesicles with coagulating glands ( ↓ 78%), ventral prostate ( ↓ 68%), LABC ( ↓ 38%), Cowper’s glands ( ↓ 54%), and glans penis ( ↓ 17%) compared to weights in the TP-treated group. These data indicate that the test system was sensitive to an anti-androgenic response

In the androgen agonist assay, the guideline sp ecified m aximum %CV levels were exceeded in the ventral prostate at 800 mg /kg/day (55.2% vs 45% criterion) and LABC at 400 mg/kg/day (35.5% vs 30% criterion). The anti-androgen assay failed to meet the following guidelineISOPHORONE FINAL

Page 27 of 33 criteria for maximum %CV levels: (i) LA BC at 400 mg/kg/day with TP (23.5% vs 20% criterion); (ii) LABC in the FT positive contro l (26.2% vs 20% criteri on); and (iii) Cowper's gland in the FT positive contro l (35.1% vs 30% criterion).

The report also compared two control groups: one control group was admini stered the vehicle by oral gavage only while the othe r group was administered the ve hicle by oral gavage and s.c. injection. Body weights in animals given corn oil by both the oral and s.c. routes were similar to the body weights in animals given corn oil by or al gavage only. However, a minor decrease (p ≤ 0.05) in overall (Days 1–11) body weight gain of 11% was not ed in the animals given corn oil by both the oral and s.c. rout es compared to animals given co rn oil by gavage only. Minor decreases (p ≤ 0.05) of 6–8% of food consumption duri ng the intervals of Days 1–4 and 7–11 were observed in animals given corn oil by both th e oral and s.c. routes compared to the food consumption in animals given corn oil by oral gavage only. This effect corresponded to the
minor decrease noted in overall (Days 1–11) body weight gain. No differences (p>0.05) were detected in any organ weight between the two control groups.

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

Pubertal Female Assay; OCSPP 890.1450

In a Female Pubertal Assay (MRID 48617307), 16 Sprague Dawley rats/dose group were treated daily via oral gavage (4 mL/kg) with isophorone (98.8  0.1%, batch/lot # 1137080201) in corn oil at doses of 0 (corn oil), 50, 200, or 800 mg/k g/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment was recorded. Following sacrifice on P ND 42, total thyroxine (T 4 ) levels were determined using a chemilumi nescent immunoassay and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay; serum was also analyzed for
levels of selected clinical chem istry parameters. Adrenal, liver, pituitary, thyroid, and urogenital organ weights were recorded and microscopic ex aminations of the ovaries, uterus, thyroid, and kidneys were performed.

One animal each in the control and 200 mg/kg/ day groups died prior to study termination, and one animal in the 50 mg/kg/day group was removed from the study due to accidental injury. All
other rats survived until scheduled sacrifice.

No treatment-related effects were observed on cl inical signs, body weight, body weight gains, or gross or microscopic pathology. There were no effects of treatment on age or body weight atISOPHORONE FINAL

Page 28 of 33 attainment of VO, mean age at first vaginal estrus, mean cycle length, percent cyc ling, or percent regularly cycling. There were no dose-related effects on serum T 4 or TSH.

At 800 mg/kg/day, adjusted, absolute, and relativ e liver weights were increased (p<0.05) by 16- 18% compared to control. There were no effects on liver weights at doses ≤ 200 mg/kg/day. There were no dose-related effects on the weight s of kidneys, pituitary glands, adrenal glands, ovaries, uterus, or thyroid.

The high-dose level of isophorone se lected for this study (800 mg/ kg/day) was considered to be an adequate dose based on data from an earlier study showing reduced body weight, reduced food consumption, and clinical signs after two days of dosing at 1000 mg/kg/day.

Pubertal Male Assay; OCSPP 890.1500

In a Male Pubertal Assay (MRID 48617308), 16 Sp rague Dawley rats/dose group were treated daily via oral gavage (4 mL/kg) with isophorone (98.8  0.1% a.i., Lot # 1137080201) in corn oil at doses of 0 (corn oil), 50, 200, or 800 mg/kg/da y from post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) da ily beginning on PND 30, and age and weight at day of attainment was recorded. Following sacrifice on PND 53, total thyroxine (T 4 ) and
testosterone levels were determined usi ng chemiluminescent immunoassays and thyroid stimulating hormone (TSH) levels were determin ed using a radioimmunoa ssay. Serum was also analyzed for levels of selected clinical chemistr y parameters. Adrenal, li ver, pituitary, thyroid, and urogenital organ weights were recorded, a nd microscopic examinations of the testes, epididymides, thyroid, and kidneys were performed.

One animal each in the 200 and 800 mg/kg/day groups died prior to study termination. All other rats survived until scheduled sacrifice. No clinical signs of toxicity were observed in any of the surviving rats. The results for one rat in th e 800 mg/kg/day group were excluded from the study due to a vascular abnormality which was consid ered to be unassociated with treatment.

No treatment-related effects were observed on mortality, clinical signs, body weights, body weight gains, or gross or mi croscopic pathology. At 800 mg/kg/day, the age of attainment of PPS, adjusted for body weight on PND 23, was delaye d (p<0.05) compared to the control, from an adjusted mean of 44.7 days in the control gr oup to an adjusted mean of 46.5 days in the 800 mg/kg/day group. Although the unadjusted age at attainment of PPS was delayed in the 800 mg/kg/day group (+1.7 days compared to the unadjusted mean in the control group), the difference was not statistically si gnificant. There were no effects on age at attainment of PPS for the 50 and 200 mg/kg/day dose grou ps. Body weight at attainment of PPS was similar across all dose groups.ISOPHORONE FINAL

Page 29 of 33 Relative liver weight was increased (p<0.05) at 50 mg/kg/day by 7%. Adjusted, absolute, and relative liver weights were increased (p<0.05) at 200 mg /kg/day by 16-20%, and at 800 mg/kg/day by 32%-33% compared to controls. At 800 mg/kg/day, the adjusted, absolute, and relative weight of seminal vesicles + coagulatin g glands (with and without fluid) was decreased (p<0.05) by 24-26%. Seminal vesi cles weights in the 50 and 200 mg/kg/day groups were similar
to controls. There were no dos e-related effects on the weight s of kidneys, pituitary glands, adrenal glands, ventral prostate , dorsolateral prostate, levato r ani-bulbocavernosus muscle (LABC), epididymides, testes, or thyroid.

At 800 mg/kg/day, the level of se rum testosterone was decreased (p<0.05) by 58% compared to
the control. Levels were also decreased at 50 and 200 mg/kg/day, however the decreases were not statistically si gnificant. Serum urea nitrogen levels were decreased (p< 0.05) in the 200 and 800 mg/kg/day dose groups ( ↓ 25%, and ↓ 42%, respectively). Leve ls of creatinine were increased (p<0.05) in rats dosed at 800 mg/kg/day ( ↑ 400%). Levels of alkaline phosphatase were decreased (p<0.05) by 15% at 50 mg/kg/ day, by 18% at 200 mg/kg/day, and by 25% at 800 mg/kg/day. Levels of chloride were decreased (p<0.05) by 2% at 50 and 200 mg/kg/day and by 4% at 800 mg/kg/day. There were no dose-related effects on serum T 4 , serum TSH, or the other evaluated clinical chemistry parameters.

The high-dose level of isophorone se lected for this study (800 mg/ kg/day) was considered to be an adequate dose based on data from an earlier study showing reduced body weight, reduced food consumption, and clinical signs after two days of dosing at 1,000 mg/kg/day.

Steroidogenesis Assay; OCSPP 890.1550

In a steroidogenesis assay (M RID 48617311), H295R cells cultured in vitro in 24-well plates were incubated with isophorone (98.7±1.3% purity, Lot #1136041301) at concentrations of

0.0001 to 100 μ M for 48 hours in triplicate for three independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicl e, at a final con centration of 0.1%.

Testosterone and estradiol levels were measured by using LC-APPI-MS/MS. A Quality Control (QC) plate was run concurrently with each i ndependent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol a nd testosterone production.

The Guideline minimum basal hormone production levels for testosterone (500 pg/mL) were generally met in both blank and solvent control (SC) wells for te stosterone. Although the basal estradiol production was generally less than th e recommended lower limit, values were more than 2.5-fold greater than the MDL. Testoste rone and estradiol production in the presence ofISOPHORONE FINAL

Page 30 of 33 10 μM forskolin met induction requirements. A lthough testosterone inhibition was greater than the required 50% in the presence of 1 μM prochl oraz, inhibition of estradiol only reached 40% in Runs #2 and #3. Because all other guideline requirements were met, these results were deemed acceptable.

Precipitation was not reported to occur, and cytotoxicity was not obs erved at any tested isophorone concentration. The within-plate variabilities of the ve hicle controls in the test assays were 3.3–13.9% CV, and the between-plate variabili ty was 30.2% for testosterone and 22.1% for estradiol. Although the % CV estim ate for testosterone is slightly higher than the Guidelinerecommended limit of ≤ 30%, the reviewers consider these data acceptable. The % CVs within
and between each run on the testosterone and estr adiol QC plates were reported to be 0-19.6% and 1.9-19.0%, but these data could not be verified by the reviewers.

No effect on testosterone producti on was observed at any test conc entration. Slightly decreased
estradiol levels were observed at two mid-level isophorone concentr ations in a single run, and a slight increase was noted at the highest concentration in a separa te run. These effects were not reproducible or concen tration-dependent.

Based on hormone responses in at least three independent runs, isophorone treatment did not result in statistically signifi cant and reproducible alterations in testosterone or estradiol production.

Uterotrophic Assay; OCSPP 890.1600

In an uterotrophic assay (MRID 48617309) conducted to screen for potential estrogenic activity, isophorone (98% a.i., lot # 1137080201) in corn oil was administered daily via subcutaneous (s.c.) injection to groups of 6 ovariectomized female, Sprague-Dawl ey rats at dose levels of 0 (untreated and vehicle contro ls), 5, 15, or 50 mg/kg/day on postnatal days (PND) 56-58. A positive control group was treated with 17 α -ethynyl estradiol (EE) in co rn oil by s.c. injection at a dose level of 0.27 μg/kg/day. All animals were terminated and necropsied approximately 24
hours after the final dose on PND 59 to determ ine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any isophorone treated groups. No effects on body weights were observed for the isophorone treated groups; however , overall body weight gains fo r the 50 mg/kg/day group were decreased (p<0.05) by 32% compared to the vehicl e control. In the positive control group (EE), overall body weight gains were decreased (p<0.05) by 28% on Days 1-4. Uterine weights in the isophorone treated groups were comparable to th e controls. As expected, absolute wet and blotted uterus weights for the positive control (E E) group were increased (p<0.05) by 86% each.ISOPHORONE FINAL

Page 31 of 33 The 32% decrease in overall body weight gains in the high-dos e group indicated that these animals were dosed at marginally adequate levels.

No statistically sign ificant changes were seen in uterine weight in this assay. Isophorone is negative in the uterotrophic assay.ISOPHORONE FINAL

Page 32 of 33 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

90-Day (Subchronic) Oral T oxicity in Rodents (Rat)

In a 90-day subchronic oral toxicity study (MRID 00123977), groups of Sprague-Dawley rats (10 rats/sex/dose) were admini stered isophorone (unspecified pur ity) in the diet at 0, 750, 1500 or 3000 ppm (equivalent to 0/0, 57/ 78.9, 102.5/163.8, and 233.8/311.8 mg/kg/day in males/females). Additional groups of ten males and ten females were incorporated in the study for hematological, clinical chem istry and urinalysis measurements at four weeks and at termination. Decreased body weights were observe d only in high-dose male rats. No effects
were observed on testes or thyroid weights. No histopatholog ical findings were noted in the testes, prostate, ovaries, uter us, pituitary, or thyroid. Fo r females, the NOAEL was 311.8 mg/kg/day; a LOAEL was not established. For males, the NOAEL was 233.8 mg/kg/day and LOAEL was 102.5 mg/kg/day, based on the decrease in body weight.

90-Day (Subchronic) Oral Toxi city in Non-Rodents (Dog)

In a 90-day subchronic oral toxicity st udy (MRID 00123976), groups of beagle dogs (4 dogs/sex/dose) were administered isophorone (uns pecified purity) in ge latin capsules at 0, 35, 75, or 150 mg/kg/day. No effects were observed on thyroid weights. No histopathological findings were noted in the testes, prostate, seminal vesicle, ovaries, uterus, pituitar y, or thyroid. The NOAEL was 150 mg/kg/day (highest dose tested ); a LOAEL was not established. Note: this study was classified as unacceptable; th e highest dose tested (150 mg/kg/day) was considered insufficient, as no e ffects of treatment were observed.

Prenatal Developmental in Rodents (Rat)

In a developmental toxicity study (MRI D 00151531), isophorone vapor (96.8% purity) was administered by whole-body inhalation to groups of 22 mated female CD-1 mice and 22 mated
female Fischer 344 rats at dose levels of 0, 25, 50, or 115 ppm [0,0.14, 0.28, or 0.64 mg/L] from gestation days (GD) 6 through 15. In mice, ther e were no treatment-relate d effects on mortality, clinical signs, food consumpti on, or cesarean parameters. M ean GD 18 body weights (corrected for uterine weight) were decrea sed (p<0.05) by 6% at 115 ppm. The maternal LOAEL for mice was 115 ppm, based on decreased GD 18 body wei ghts; the maternal NOAEL for mice was 50 ppm. The developmental LOAEL was not es tablished; the developmental NOAEL was 115 ppm. In rats, there were no treatment-relate d effects in mortality or cesarean parameters compared to control. Clinical signs of toxicity were seen at 50 and 115 ppm, and consisted of alopecia (14/22 at 50 ppm and 21/22 at 115 ppm, both compared to one control) and brown staining in the ano-genital or cervical region (11/22 at 50 ppm and 22/22 at 115 ppm, both compared to one control). Food consumption was significantly decreas ed (p<0.01) at 115 ppm during GD 6-20 ( ↓ 10%) and GD 0-20 ( ↓ 8%). Body weight was d ecreased (p<0.05) at 115 ppm during GD 12 ( ↓ 6%) and 15 ( ↓ 7%). The maternal LOAEL for rats was 50 ppm , based on anISOPHORONE FINAL

Page 33 of 33 increased incidence of clinic al signs; the maternal NOAEL was 25 ppm. The developmental LOAEL was 115 ppm, based on decreased (p <0.05) fetal crown-rump distance ( ↓ 3%) in female fetuses; the developm ental NOAEL was 50 ppm.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a carcinogenicity study (MRID 00151527), isophorone (94% or 97% purity) was administered via gavage to 50 F344/N rats/sex/dose in corn oil at dose levels of 0, 250 or 500 mg/kg/day, 5 days/week for 103 weeks. Carcinomas of the pre putial gland were increas ed in high-dose male
rats. Under the conditions of this study, there was some evid ence of carcinogenicity in male F344/N rats as shown by the o ccurrence of renal t ubular cell adenomas and adenocarcinomas, and carcinomas of the preputial gland. There were no effects on the weights of the testes, ovaries, or thyroid. No histopathol ogical effects were seen in the testes, prostate, ovaries, uterus, mammary, or thyroid. There was no evidence of carcinogenicity in female F344/N rats.

Carcinogenicity in Rodents (Mouse)

In a carcinogenicity study (MRID 00151527), isophorone (94% or 97% purity) in corn oil was administered via gavage to 50 B6C3Fl albi no mice/sex/dose at dose levels of 0, 250 or 500 mg/kg/day, 5 days per week for 103 weeks. In the high dose male mice, isophorone exposure
was associated with increased incidences of hepatocellular adenomas and carcinomas, and of mesenchymal tumors of the integumentary system (fibroma, fibro sarcoma, neuro fibrosarcoma, or sarcoma). No compound-re lated neoplastic or non-neoplastic lesions associated with isophorone exposure were seen in female mice. No effects on the weights of the testes, ovaries, mammary, or thyroid were observed. No histopathological effect s were seen in the testes, prostate, ovaries, uterus mamma ry, or thyroid. There was no evidence of carcinogenicity of isophorone in female B6C3Fl mice.