UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 41
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 41
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: ISOPHORONE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 41
ISOPHORONE 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)...........................................32 

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ISOPHORONE 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

Page 5 of 41
ISOPHORONE FINAL 

iv 

Abbreviation Terminology 

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

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ISOPHORONE FINAL 

v 

Abbreviation Terminology 

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

Page 7 of 41
ISOPHORONE FINAL 

vi 

Abbreviation Terminology 

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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ISOPHORONE FINAL 

Page 1 of 33

Executive Summary 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether isophorone interacts with E, A or T hormone pathways, the number 

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

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

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

also resulted in general systemic toxicity or overt toxicity. 

On 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) analysis of the potential interaction of 

isophorone with the E, A or T hormone 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).

Isophorone is a cyclic unsaturated ketone whose acute mode of action is nonpolar narcosis 

(toxicologically induced and reversible 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/concentration in order to sufficiently 

challenge the animal. 

For the estrogen pathway, the only observed estrogen-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 translate into observed effects in gonadal tissue or 

reproductive output. This effect on vitellogenin in females by itself is not a clear indicator of an 

endocrine effect without other complementary 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 seen in the presence of overt toxicity. There were no complementary or 

redundant responses seen in other Tier 1 assay or other scientifically relevant information 

(OSRI). 

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ISOPHORONE FINAL 

Page 2 of 33

For the androgen pathway, effects in the Tier 1 male pubertal assay included a decrease in 

serum testosterone level, reduction in seminal vesicle weight and a slight delay in unadjusted 

(for body weight) age (+1.8 days) at preputial separation (PPS). However, these effects were 

only seen at a very high dose (800 mg/kg/day), which approached the limit dose of 1000 

mg/kg/day at which narcosis and lethargy was seen 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 effects 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, mammalian 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. 

 

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ISOPHORONE FINAL 

Page 3 of 33

I. Introduction 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether isophorone interacts with E, A or T hormone pathways, the number and 

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

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

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

also resulted in general systemic toxicity or overt toxicity. 

On November 6, 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) analysis of the potential interaction of isophorone 

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

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

beginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and 

wildlife results, then the results of the cited OSRI for mammalian and wildlife studies (40 CFR 

Part 158 and literature).

Isophorone is a cyclic unsaturated ketone used as a solvent for pesticide applications. It is 

soluble in water (>10,000 mg/L) with a partition coefficient (log 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 determining the potential interaction 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 toxicity studies and other published articles. These 

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

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ISOPHORONE FINAL 

Page 4 of 33

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

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

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

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

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

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

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

Report of the Endocrine Disruptor Review Team on 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 presented at the 2013 FIFRA 

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

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

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

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

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

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

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

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

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

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

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

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

above. 

 

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ISOPHORONE FINAL 

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

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

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

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

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher-levels of biological organization? Finally, an evaluation of 

conditions where responses occur including consideration of whether the responses were 

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

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ISOPHORONE FINAL 

Page 6 of 33

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

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

This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the 2013 FIFRA SAP meeting that evaluated scientific issues associated with the WoE 

evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In 

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

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

evaluated in terms of primary interactions with the endocrine system vs. secondary effects 

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Section IV.A. 

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

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

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

the E, A or T pathway. Adequately addressing the three components described is fundamental to 

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

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

conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) 

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

needed and why. Assessing the need for additional data is based on a case-by-case analysis 

which took all available toxicity data into account. 

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ISOPHORONE FINAL 

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

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

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

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

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

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

A. EDSP Tier 1 Screening Assays 

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

ERα 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) 

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ISOPHORONE FINAL 

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B. Effects on Hypothalamic-Pituitary-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-relevant findings from data evaluated as OSRI. Effects that occurred in 

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

considered further in the WoE assessment. 

Isophorone was negative in the Tier 1 in vitro assays and in the Tier 1 uterotrophic assay. 

Additionally, there were no estrogen-related effects observed in the female pubertal rat assay. 

However, in the Tier 1 FSTRA, plasma vitellogenin (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 almost completely suppressed and multiple other 

endpoints were affected: fecundity (↓94%), fertilization success (↓70%), female plasma VTG 

(↓77%) and a slight increase in oocyte atresia. However, at this concentration, signs of overt 

toxicity included reduced feeding and reduced male and female body weights. No estrogenrelated effects were observed in the Part 158 mammalian studies or OSRI evaluating ER binding 

or transactivation (Nashihara et al., 2000; Massaad and Barouki, 1999). 

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ISOPHORONE FINAL 

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Table 2: Estrogenic/Anti-Estrogenic Pathway for Isophorone 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Isophorone1Study Type/Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Gonadal Staging 

and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

EDSP Tier 1 Assay 

ER Binding (MRID 48617306) N ERTA (MRID 48617305) N Aromatase (MRID 48617304) N Steroidogenesis (MRID 48617311) N Uterotrophic (MRID 48617309) N Female Pubertal Rat (MRID 48617307) 

N N N N N N 

FSTRA (MRID 48617301) NE N N N N ↓

OSRI 

ERαtranscriptional activation (Massaad and Barouki, 1999) 

 N 

Subchronic toxicity-Rat (MRID 00123977 ) 

 N 
ISOPHORONE FINAL 

Page 10 of 33

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Isophorone1Study Type/Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Gonadal Staging 

and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

Subchronic toxicity- Dog (MRID 00123976) 

 N 

CarcinogenicityRat (MRID 00151527) 

 N N 

CarcinogenicityMouse (MRID 00151527) 

 N N 

1 Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the respo

indicates that is parameter is not routinely evaluated or is not applicable in this assay. LW= Liver weight 

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

this column. For details see Section IV. A 

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

details see Section IV. A 

4. 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 Examined
ISOPHORONE FINAL 

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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 Tier 1 assays are delineated so 

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

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

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

table and not considered further in the WoE assessment. 

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, there was a statistically significant 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 preputial separation (PPS). No effects were seen at lower doses 

(50 and 200 mg/kg/day). In the FSTRA, no androgen-related effects in male fish were 

observed. Additionally, no androgen-related effects were observed in the Part 158 mammalian 

studies.

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ISOPHORONE FINAL 

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Table 3: Androgenic/Anti-Androgenic Pathway for Isophorone 

Lines of Evidence Indicating Potential Interaction with the 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 

EDSP Tier 1 Assay 

AR Binding (MRID 48617303) N Steroidogenesis (MRID 48617311) N Hershberger (MRID 48617310) N N N N Male Pubertal Rat (MRI D 48617308) 

↓58% 

(H) N N N N N ↓24% SV (H) ↑1.8 d (H) 

FSTRA (MRID 48617301) NE N N N 

OSRI 

Subchronic toxicityRat (MRID 00123977 ) 

N N 

Subchronic toxicityDog (MRID 00123976) 

N N 

Carcinogenicity-Rat (MRID 00151527) N N Carcinogenicity-Mouse (MRID 00151527) N N 

1 Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. 

that is parameter is not routinely evaluated or is not applicable in this assay. SV=Seminal vesicle; LW=Liver weight 

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 

this column. For details see Section IV. A 
ISOPHORONE FINAL 

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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 th

see Section IV. A 

4 Reduced feeding and reduced male and female body weights at 79.8 mg/L. 

N Negative findings (in vitro); No effect (in vivo)
ISOPHORONE FINAL 

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

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

affect hypothalamic or pituitary regulation of thyroid hormone production, but it does include three in vivo 

assays that 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 with thyroid regulation is summarized in Table 4. The various targets 

of the thyroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of 

potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as 

OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WoE assessment. 

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 thyroid histopathology observed in the AMA. In the 

AMA, on day 21, reductions in body weight (15%) and snout-vent length (SVL; 5%) were observed at the 

high test concentration (59 mg/L). No other thyroid-related effects were observed in the Part 158 mammalian 

studies. 

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Table 4: Thyroid Pathway for Isophorone 

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Isophorone1

Study Type 

Thyroid Weight Thyroid : Gross and Histopathology Serum T4 Levels Serum TSH Levels Pituitary Weight Growth (BW, SVL) Developmental Stage (+ or asynchronous ,HLL) Systemic Toxicity Observed2

Overt Toxicity Observed3

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

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

N Negative findings

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ISOPHORONE FINAL 

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IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight 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 recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

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

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

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

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

includes considerations of biological plausibility of the findings from the different lines of 

evidence by examining the consistency, coherence, and interrelationships among the measured 

endpoints within and across studies. The available findings from standard toxicology studies on 

the substance may contribute to the WoE evaluation in helping elucidate if effects seen in the 

Tier 1 assay are related to perturbations of the endocrine system per se or alternatively a sequelae 

of systemic effects. Endocrine modes of action may evoke a number of phenotypic consequences 

other than those evaluated in the Tier 1 assays. 

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

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

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

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

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

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

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

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

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

A. Systemic/Overt toxicity in the in vivo Tier 1 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 below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects observed which may be considered 

overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% 

decrease in mammalian body weight) was sufficient for a dose/concentration to be considered 

overtly toxic. However, in other instances, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

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

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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 administration of isophorone at 312 mg/kg/day 

resulted in greater than 10% decrease in absolute body weight in male rats; no body weight 

effects were seen in females. No other systemic toxicity was seen in the OSRI studies. 

B. Estrogen pathway 

Isophorone is a cyclic unsaturated ketone whose acute mode of action is nonpolar narcosis 

(toxicologically induced and reversible 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/concentration in order to sufficiently 

challenge the animal. 

The only observed estrogen-related effect in the absence of overt toxicity, was limited to a 

decrease in female plasma vitellogenin in the FSTRA which did not translate into observed 

effects in gonadal tissue or reproductive output. This effect on vitellogenin in females by itself is 

not a clear indicator of and endocrine effect without other complementary 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 seen 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 included a decrease in serum testosterone level, 

reduction in seminal vesicle weight and a slight delay in unadjusted (for body weight) age 

(+1.8 days) at PPS. However, these effects were only seen at a very high dose (800 mg/kg/day), 

which approached the limit dose of 1000 mg/kg/day for which narcosis and lethargy was seen 

Page 25 of 41
ISOPHORONE FINAL 

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in the range-finding study. Additionally, in the pubertal assay, increases in absolute (32%) and 

relative (33%) liver weight were observed. There were no androgen-related effects observed in 

the Tier 1 FSTRA or Part 158 mammalian studies. 

D. Thyroid pathway 

There were no thyroid-related effects 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 for potential interaction 

with the estrogen, androgen or thyroid pathways for isophorone. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, mammalian 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. 

 

Page 26 of 41
ISOPHORONE FINAL 

Page 19 of 33

VI. References 

ATSDR (1989). Agency for Toxic Substances and Disease Registry (1989) Toxicological Profile 

for isophorone. 

Coady, K. et al. (2011) Isophorone: The Amphibian Metamorphosis Assay with (Xenopus 

laevis). Dow Chemical Company, Midland, Michigan. Laboratory Report Number 101063. 

Unpublished. (MRID 48617302). 

Coady, K.K. and Sosinski, L.K. (2011) Evaluation of Isophorone in the Human Recombinant 

Aromatase Assay. Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company, Midland, MI. Laboratory 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. . Toxicology & Environmental Research and Consulting, 

Dow Chemical Company, Midland, Michigan. Laboratory Study ID NS000077. Unpublished. 

International Programme on Chemical Safety (1995) IPCS report 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 hERα-HELA-9903. Unpublished (MRID 

4861705). 

LeBaron, M., Kan, H., Perala, A. (2011) 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 assay. Toxicology & Environmental Research and Consulting, The 

Dow Chemical Company, Midland, MI. Laboratory Project ID: 111119, Unpublished (MRID 

48617306). 

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011) Evaluation of isophorone in an in vitro 

androgen receptor binding assay. Toxicology & Environmental Research and Consulting, The 

Dow Chemical Company, Midland, MI. Laboratory 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, Midland, 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 & 

Page 27 of 41
ISOPHORONE FINAL 

Page 20 of 33

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project Study ID: 111100, December 22, 2011. Unpublished. (MRID 48617307). 

Marty, M.S., Andrus, A. K., and Hukkanen, R. R. (2011). Isophorone: pubertal development and 

thyroid function in intact juvenile/peripubertal male Crl: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: Uterotrophic 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. Laboratory Study No.: 101077. Unpublished. (MRID 

48617310). 

National Toxicology Program (1986) NTP technical report on the toxicology and carcinogenesis 

studies of isophorone (CAS # 78-59-1) in F344/N rats and B6C3F1 mice (gavage studies). 

(MRID 00151527) 

Rohm & Hass Co. (1972a) 90-day subchronic toxicity of isophorone in the dog. Unpublished. 

(MRID 00123976) 

Rohm & Hass Co. (1972b) 90-day subchronic toxicity of isophorone in the rat. Unpublished. 

(MRID 00123977) 

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

00151531). 

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

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

Safety and Pollution Prevention Science Coordination and Policy. U.S. Environmental Protection 

Agency, Washington, DC. 

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

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

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (Frog); OCSPP 890.1100 

The 21-day assay (MRID 48617302) of isophorone 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, and 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 termination, tadpoles with bent tails were observed in the 

control and all treatment groups (17-29%); these observations were not considered to be treatmentrelated. 

The only treatment-related effects observed in this study were significant (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 normalized (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) control 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 pattern in occurrence). Late stage individuals 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 individuals were NF>60 in the negative control group, compared to 

8, 10, and 18% in the low, mid, and high treatment groups, respectively). 

Androgen Receptor (AR) Binding 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 binding 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 vehicle at a final assay concentration of <3%. The assay included 

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

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ISOPHORONE FINAL 

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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 (Bmax) was 3.245 fmol/100 μg protein and the dissociation constant (Kd) was 0.4641 

nM. These values were below the range of values from the 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 assays (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 IC50s 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 solvent 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 IC50

and RBA could not be calculated for isophorone as it did not displace 50% of the radioligand at 

any test concentration. 

Based on the results from the three runs, isophorone 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 human recombinant aromatase and tritiated 

androstenedione (1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) for 15 minutes at 37 °C to 

assess the potential of isophorone to inhibit aromatase activity. 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). Three independent runs were conducted and each run 

included a full activity control, a background activity control, a positive control series (10−10 to 

10−5 M) 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 ranges for the top of the 

curve, bottom curve, Hill Slope, log IC50, and coefficient of variation (CV) for replicates of each 

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ISOPHORONE FINAL 

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concentration within runs. For 4-OH ASDN, the estimated log IC50 averaged −7.38 M and the 

slope was −0.912. 

For isophorone, aromatase activity averaged 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 response curve across runs was greater than 

75% activity at all concentrations of isophorone. The average dose-response curve indicated that 

aromatase activity at concentrations of isophorone ranging from 10−10 M to 10−3

 M was 

essentially equivalent to the activity observed 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) Binding Assay; OCSPP 890.1250 

In an estrogen receptor (ER) binding assay (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 binding of a single concentration of [3H]-17β-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 17β-estradiol as the natural ligand 

reference material. 

Summary data pertaining to the saturation binding experiment were reported separately in the 

study profiles submitted by the test order recipient. The Kd for [3H]-17β-estradiol was 0.1032 

nM and the Bmax (nM) was 0.07097 for the prepared rat uterine cytosol used in these 

experiments. The Kd for the run was within the expected range of 0.03 to 1.5 nM, and the Bmax

was within the expected range of 10-150 fmol/100 μg protein. The data produced a linear 

Scatchard plot. 

In the competitive binding experiment, the estimated mean log IC50 was −8.917 M for 17βestradiol 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 specific binding of the [3

H]-ligand; therefore, a log IC50 and RBA 

could not be calculated for isophorone. 

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ISOPHORONE FINAL 

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

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay; OCSPP 890.1300 

In an estrogen receptor transcriptional activation assay (MRID 48617305), hERα-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 10−3M in DMSO (0.1%) in three 

independent runs. Experiments were performed 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 (luciferase) gene products. Luciferase expression in 

response to activation of the estrogen receptor was measured upon addition of a luciferase 

substrate and detection with a luminometer with acceptable sensitivity. Isophorone was tested up 

to the guideline limit of 10−3

 M. Performance criteria were acceptable for all of the reference 

chemicals. The response of 17α-methyltestosterone indicates increased sensitivity to very weak 

agonists. The RPCMax was 5.9% for the first run, 7.8% for the second run and 9.9% for the third 

run. Because the RPCMax < PC10 in all three assay runs, isophorone was considered negative for 

estrogen receptor transcriptional activation in this test system. 

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ISOPHORONE FINAL 

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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 concentrations 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% relative to the control at the high treatment 

level. There were no notable observations on secondary sex characteristics or clinical signs of 

toxicity for any treatment group compared to the controls. However, the study author reported 

reduced feeding in both sexes with an observable 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/replicate (range 19.1-36.5); fertilization success in the control group averaged 

98.4%. Fecundity was significantly (p<0.05; Jonckheere-Terpstra) reduced 94% at the high 

treatment level. There was also a significant reduction in fertility of 70%, (p<0.05; JonckheereTerpstra) at the high treatment level 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 levels, respectively. There were no significant 

differences in male VTG between the treatment groups and the control. 

There were no significant differences in gonado-somatic index (GSI) for males or females 

compared to the control. Tubercle scores in males 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 reported treatment-related histopathological observations in males, but 

there was a reported increase in incidence 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 further analysis of the data was not reported. 

Hershberger Assay; OCSPP 890.1400 

In a Hershberger assay (MRID 48617310) testing for androgenic activity, isophorone 

(≥98.7±0.1%, lot nos. 1137080201 and 1136041301) was administered daily in corn oil via oral 

gavage to groups of seven 55-day old castrated male Sprague Dawley rats/group at dose levels of 

0 (vehicle), 100, 400, or 800 mg/kg/day for 10 consecutive 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/day via subcutaneous (s.c.) injection. 

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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 daily s.c. dose of TP (0.4 mg/kg/day). 

Body weights, body weight gains, and food consumption were measured. Serum hormone 

concentrations were not determined, and histology was not performed. Animals were euthanized 

approximately 24 hours after the final dose administration. 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 weight 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 androgenic 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 toxicity at the highest dose tested (800 

mg/kg/day), the dose range finding assay did provide sufficient evidence that a 1000 mg/kg/day 

dose is excessive (body weight losses, marked food consumption decrease, and clinical signs of 

distress leading to early animal termination). Therefore, dosing was considered 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 with 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 androgenic response. TP 

administration also resulted in increased food consumption and body weight gains, consistent 

with an androgenic response. For the anti-androgenic 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 specified maximum %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 guideline 

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criteria for maximum %CV levels: (i) LABC at 400 mg/kg/day with TP (23.5% vs 20% 

criterion); (ii) LABC in the FT positive control (26.2% vs 20% criterion); and (iii) Cowper's 

gland in the FT positive control (35.1% vs 30% criterion). 

The report also compared two control groups: one control group was administered the vehicle by 

oral gavage only while the other group was administered the vehicle 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 oral gavage only. However, a minor decrease 

(p≤0.05) in overall (Days 1–11) body weight gain of 11% was noted in the animals given corn 

oil by both the oral and s.c. routes compared to animals given corn oil by gavage only. Minor 

decreases (p≤0.05) of 6–8% of food consumption during the intervals of Days 1–4 and 7–11 

were observed in animals given corn oil by both the 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/kg/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 PND 42, total thyroxine (T4) 

levels were determined using a chemiluminescent immunoassay and thyroid stimulating 

hormone (TSH) levels were determined using a radioimmunoassay; serum was also analyzed for 

levels of selected clinical chemistry parameters. Adrenal, liver, pituitary, thyroid, and urogenital 

organ weights were recorded and microscopic examinations 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 clinical signs, body weight, body weight gains, or 

gross or microscopic pathology. There were no effects of treatment on age or body weight at 

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attainment of VO, mean age at first vaginal estrus, mean cycle length, percent cycling, or percent 

regularly cycling. There were no dose-related effects on serum T4 or TSH. 

At 800 mg/kg/day, adjusted, absolute, and relative 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 weights of kidneys, pituitary glands, adrenal glands, 

ovaries, uterus, or thyroid. 

The high-dose level of isophorone selected 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 Sprague 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/day from post-natal day (PND) 23 to 53. Animals 

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

day of attainment was recorded. Following sacrifice on PND 53, total thyroxine (T4) and 

testosterone levels were determined using chemiluminescent immunoassays and thyroid 

stimulating hormone (TSH) levels were determined using a radioimmunoassay. Serum was also 

analyzed for levels of selected clinical chemistry parameters. Adrenal, liver, pituitary, thyroid, 

and urogenital organ weights were recorded, and 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 the 800 mg/kg/day group were excluded from the study 

due to a vascular abnormality which was considered to be unassociated with treatment. 

No treatment-related effects were observed on mortality, clinical signs, body weights, body 

weight gains, or gross or microscopic pathology. At 800 mg/kg/day, the age of attainment of 

PPS, adjusted for body weight on PND 23, was delayed (p<0.05) compared to the control, from 

an adjusted mean of 44.7 days in the control group 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 significant. There were no effects on age at attainment of PPS for 

the 50 and 200 mg/kg/day dose groups. Body weight at attainment of PPS was similar across all 

dose groups. 

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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 + coagulating glands (with and without fluid) was decreased 

(p<0.05) by 24-26%. Seminal vesicles weights in the 50 and 200 mg/kg/day groups were similar 

to controls. There were no dose-related effects on the weights of kidneys, pituitary glands, 

adrenal glands, ventral prostate, dorsolateral prostate, levator ani-bulbocavernosus muscle 

(LABC), epididymides, testes, or thyroid. 

At 800 mg/kg/day, the level of serum 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 significant. Serum urea nitrogen levels were decreased (p<0.05) in the 200 and 

800 mg/kg/day dose groups (↓25%, and ↓42%, respectively). Levels 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 T4, serum TSH, or the other 

evaluated clinical chemistry parameters. 

The high-dose level of isophorone selected 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 (MRID 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 μM for 48 hours in triplicate for three independent experiments. Dimethyl 

sulfoxide (DMSO) was used as the vehicle, at a final concentration 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 independent 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 and 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 testosterone. Although the basal 

estradiol production was generally less than the recommended lower limit, values were more 

than 2.5-fold greater than the MDL. Testosterone and estradiol production in the presence of 

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10 μM forskolin met induction requirements. Although testosterone inhibition was greater than 

the required 50% in the presence of 1 μM prochloraz, 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 observed at any tested 

isophorone concentration. The within-plate variabilities of the vehicle controls in the test assays 

were 3.3–13.9% CV, and the between-plate variability was 30.2% for testosterone and 22.1% for 

estradiol. Although the % CV estimate 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 estradiol 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 production was observed at any test concentration. Slightly decreased 

estradiol levels were observed at two mid-level isophorone concentrations in a single run, and a 

slight increase was noted at the highest concentration in a separate run. These effects were not 

reproducible or concentration-dependent. 

Based on hormone responses in at least three independent runs, isophorone treatment did not 

result in statistically significant 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-Dawley rats at dose levels of 0 

(untreated and vehicle controls), 5, 15, or 50 mg/kg/day on postnatal days (PND) 56-58. A 

positive control group was treated with 17α-ethynyl estradiol (EE) in corn 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 determine 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 for the 50 mg/kg/day group were 

decreased (p<0.05) by 32% compared to the vehicle 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 the controls. As expected, absolute wet and 

blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 86% each. 

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The 32% decrease in overall body weight gains in the high-dose group indicated that these 

animals were dosed at marginally adequate levels. 

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

negative in the uterotrophic assay.

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

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

In a 90-day subchronic oral toxicity study (MRID 00123977), groups of Sprague-Dawley rats 

(10 rats/sex/dose) were administered isophorone (unspecified purity) 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 chemistry and urinalysis measurements at four weeks and at 

termination. Decreased body weights were observed only in high-dose male rats. No effects 

were observed on testes or thyroid weights. No histopathological findings were noted in the 

testes, prostate, ovaries, uterus, pituitary, or thyroid. For 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 Toxicity in Non-Rodents (Dog) 

In a 90-day subchronic oral toxicity study (MRID 00123976), groups of beagle dogs (4 

dogs/sex/dose) were administered isophorone (unspecified purity) in gelatin 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, pituitary, or thyroid. 

The NOAEL was 150 mg/kg/day (highest dose tested); a LOAEL was not established. Note: 

this study was classified as unacceptable; the highest dose tested (150 mg/kg/day) was 

considered insufficient, as no effects of treatment were observed. 

Prenatal Developmental in Rodents (Rat) 

In a developmental toxicity study (MRID 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, there were no treatment-related effects on mortality, 

clinical signs, food consumption, or cesarean parameters. Mean GD 18 body weights (corrected 

for uterine weight) were decreased (p<0.05) by 6% at 115 ppm. The maternal LOAEL for mice 

was 115 ppm, based on decreased GD 18 body weights; the maternal NOAEL for mice was 50 

ppm. The developmental LOAEL was not established; the developmental NOAEL was 115 

ppm. In rats, there were no treatment-related 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 decreased (p<0.01) at 115 ppm 

during GD 6-20 (↓10%) and GD 0-20 (↓8%). Body weight was decreased (p<0.05) at 115 ppm 

during GD 12 (↓6%) and 15 (↓7%). The maternal LOAEL for rats was 50 ppm, based on an 

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increased incidence of clinical 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 developmental 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 preputial gland were increased in high-dose male 

rats. Under the conditions of this study, there was some evidence of carcinogenicity in male 

F344/N rats as shown by the occurrence of renal tubular cell adenomas and adenocarcinomas, 

and carcinomas of the preputial gland. There were no effects on the weights of the testes, 

ovaries, or thyroid. No histopathological 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 albino 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-related 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 effects were seen in the testes, 

prostate, ovaries, uterus mammary, or thyroid. There was no evidence of carcinogenicity of 

isophorone in female B6C3Fl mice. 

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