*>EPA

EPA/690/R-23/006F | September 2023 | FINAL

United States
Environmental Protection
Agency

Provisional Peer-Reviewed Toxicity Values for

Fluorene
(CASRN 86-73-7)

PRO1*

SUPERFUND

U.S. EPA Office of Research and Development
Center for Public Health and Environmental Assessment


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A	United $ta»s

Environmental Protection
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EPA/690/R-23/006F
September 2023

https://www.epa.gov/pprtv

Provisional Peer-Reviewed Toxicity Values for

Fluorene
(CASRN 86-73-7)

Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268


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AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGER

Shana White, PhD

Center for Public Health and Environmental Assessment, Cincinnati, OH

CONTRIBUTORS

Jeffry Dean II, PhD

Center for Public Health and Environmental Assessment, Cincinnati, OH
Rachel Schaffer, PhD

Center for Public Health and Environmental Assessment, Washington, DC

SCIENTIFIC TECHNICAL LEAD

Lucina Lizarraga, PhD

Center for Public Health and Environmental Assessment, Cincinnati, OH

DRAFT DOCUMENT PREPARED BY

SRC, Inc.

7502 Round Pond Road
North Syracuse, NY 13212

PRIMARY INTERNAL REVIEWERS

M. Margaret Pratt, PhD

Center for Public Health and Environmental Assessment, Washington, DC
Q. Jay Zhao, PhD, MPH, DABT

Center for Public Health and Environmental Assessment, Cincinnati, OH

PRIMARY EXTERNAL REVIEWERS

Eastern Research Group, Inc.

110 Hartwell Avenue
Lexington, MA 02421-3136

PPRTV PROGRAM MANAGEMENT

Teresa L. Shannon

Center for Public Health and Environmental Assessment, Cincinnati, OH
Allison L. Phillips, PhD

Center for Public Health and Environmental Assessment, Cincinnati, OH
J. Phillip Kaiser, PhD, DABT

Center for Public Health and Environmental Assessment, Cincinnati, OH

Questions regarding the content of this PPRTV assessment should be directed to the U.S. EPA
Office of Research and Development (ORD) Center for Public Health and Environmental
Assessment (CPHEA) website at https://ecomments.epa.gov/pprtv.

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TABLE OF CONTENTS

COMMONLY USED ABBREVIATIONS AND ACRONYMS	iv

BACKGROUND	1

QUALITY ASSURANCE	1

DISCLAIMERS	2

QUESTIONS REGARDING PPRTVs	2

1.	INTRODUCTION	3

2.	REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER ONLY)	8

2.1.	HUMAN STUDIES	11

2.2.	ANIMAL STUDIES	22

2.2.1.	Oral Exposures	22

2.2.2.	Inhalation Exposures	28

2.3.	OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)	30

2.3.1.	Supporting Animal Studies	37

2.3.2.	Mode-of-Action/Mechanistic Studies	37

2.3.3.	Metabolism/Toxicokinetic Studies	37

3.	DERIVATION 01 PROVISIONAL VALUES	39

3.1. DERIVATION OF ORAL REFERENCE DOSES	39

3.1.1.	Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	39

3.1.2.	Derivation of Chronic Provisional RfD (Chronic p-RfD)	41

3 .2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	41

3.3.	SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES	41

3.4.	CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR AND PROVISIONAL
CANCER RISK ESTIMATES	42

APPENDIX A. SCREENING PROVISIONAL VALUES	43

APPENDIX B. DATA TABLES	50

APPENDIX C. REFERENCES	61

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COMMONLY USED ABBREVIATIONS AND ACRONYMS

a2u-g

alpha 2u-globulin

IVF

in vitro fertilization

ACGIH

American Conference of Governmental

LC50

median lethal concentration



Industrial Hygienists

LD50

median lethal dose

AIC

Akaike's information criterion

LOAEL

lowest-observed-adverse-effect level

ALD

approximate lethal dosage

MN

micronuclei

ALT

alanine aminotransferase

MNPCE

micronucleated polychromatic

AR

androgen receptor



erythrocyte

AST

aspartate aminotransferase

MOA

mode of action

atm

atmosphere

MTD

maximum tolerated dose

ATSDR

Agency for Toxic Substances and

NAG

7V-acetyl-P-D-glucosaminidase



Disease Registry

NCI

National Cancer Institute

BMC

benchmark concentration

NOAEL

no-observed-adverse-effect level

BMCL

benchmark concentration lower

NTP

National Toxicology Program



confidence limit

NZW

New Zealand White (rabbit breed)

BMD

benchmark dose

OCT

ornithine carbamoyl transferase

BMDL

benchmark dose lower confidence limit

ORD

Office of Research and Development

BMDS

Benchmark Dose Software

PBPK

physiologically based pharmacokinetic

BMR

benchmark response

PCNA

proliferating cell nuclear antigen

BUN

blood urea nitrogen

PND

postnatal day

BW

body weight

POD

point of departure

CA

chromosomal aberration

PODadj

duration-adjusted POD

CAS

Chemical Abstracts Service

QSAR

quantitative structure-activity

CASRN

Chemical Abstracts Service registry



relationship



number

RBC

red blood cell

CBI

covalent binding index

RDS

replicative DNA synthesis

CHO

Chinese hamster ovary (cell line cells)

RfC

inhalation reference concentration

CL

confidence limit

RfD

oral reference dose

CNS

central nervous system

RGDR

regional gas dose ratio

CPHEA

Center for Public Health and

RNA

ribonucleic acid



Environmental Assessment

SAR

structure-activity relationship

CPN

chronic progressive nephropathy

SCE

sister chromatid exchange

CYP450

cytochrome P450

SD

standard deviation

DAF

dosimetric adjustment factor

SDH

sorbitol dehydrogenase

DEN

diethylnitrosamine

SE

standard error

DMSO

dimethylsulfoxide

SGOT

serum glutamic oxaloacetic

DNA

deoxyribonucleic acid



transaminase, also known as AST

EPA

Environmental Protection Agency

SGPT

serum glutamic pyruvic transaminase,

ER

estrogen receptor



also known as ALT

FDA

Food and Drug Administration

SSD

systemic scleroderma

FEVi

forced expiratory volume of 1 second

TCA

trichloroacetic acid

GD

gestation day

TCE

trichloroethylene

GDH

glutamate dehydrogenase

TWA

time-weighted average

GGT

y-glutamyl transferase

UF

uncertainty factor

GSH

glutathione

UFa

interspecies uncertainty factor

GST

g 1 ut a t h i o nc - V-1 ra n s fc ra sc

UFC

composite uncertainty factor

Hb/g-A

animal blood-gas partition coefficient

UFd

database uncertainty factor

Hb/g-H

human blood-gas partition coefficient

UFh

intraspecies uncertainty factor

HEC

human equivalent concentration

UFl

LOAEL-to-NOAEL uncertainty factor

HED

human equivalent dose

UFS

subchronic-to-chronic uncertainty factor

i.p.

intraperitoneal

U.S.

United States of America

IRIS

Integrated Risk Information System

WBC

white blood cell

Abbreviations and acronyms not listed on this page are defined upon first use in the
PPRTV assessment.

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DRAFT PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
FLUORENE (CASRN 86-73-7) [Noncancer Values]

BACKGROUND

A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund program. PPRTVs are derived after a review of the relevant
scientific literature using established U.S. Environmental Protection Agency (U.S. EPA)
guidance on human health toxicity value derivations.

The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.

Currently available PPRTV assessments can be accessed on the U.S. EPA's PPRTV
website at https://www.epa.gov/pprtv. PPRTV assessments are eligible to be updated on a 5-year
cycle and revised as appropriate to incorporate new data or methodologies that might impact the
toxicity values or affect the characterization of the chemical's potential for causing
toxicologically relevant human-health effects. Questions regarding nomination of chemicals for
update can be sent to the appropriate U.S. EPA eComments Chemical Safety website at
https://ecomments.epa.gov/chemicalsafetv/.

QUALITY ASSURANCE

This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure
data are of known and acceptable quality to support their intended use. Surveillance of the work
by the assessment managers and programmatic scientific leads ensured adherence to QA
processes and criteria, as well as quick and effective resolution of any problems. The QA
manager, assessment managers, and programmatic scientific leads have determined under the
QA program that this work meets all U.S. EPA quality requirements. This PPRTV assessment
was written with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP),
the QAPP titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-
Reviewed Toxicity Values (PPRTVs) and Related Assessments/Documents
(L-CPAD-0032718-QP), and the PPRTV assessment development contractor QAPP titled
Quality Assurance Project Plan—Preparation of Provisional Toxicity Value (PTV) Documents
(L-CPAD-0031971-QP). As part of the QA system, a quality product review is done prior to
management clearance. A Technical Systems Audit may be performed at the discretion of the
QA staff.

All PPRTV assessments receive internal peer review by at least two CPHEA scientists
and an independent external peer review by at least three scientific experts. The reviews focus on
whether all studies have been correctly selected, interpreted, and adequately described for the
purposes of deriving a provisional reference value. The reviews also cover quantitative and
qualitative aspects of the provisional value development and address whether uncertainties
associated with the assessment have been adequately characterized.

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DISCLAIMERS

The PPRTV document provides toxicity values and information about the toxicologically
relevant effects of the chemical and the evidence on which the value is based, including the
strengths and limitations of the data. All users are advised to review the information provided in
this document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.

Other U.S. EPA programs or external parties who may choose to use PPRTVs are
advised that Superfund resources will not generally be used to respond to challenges, if any, of
PPRTVs used in a context outside of the Superfund program.

This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.

QUESTIONS REGARDING PPRTVS

Questions regarding the content of this PPRTV assessment should be directed to the
U.S. EPA ORD CPHEA website at https://ecomments.epa.gov/pprtv.

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1. INTRODUCTION

Fluorene is a discrete organic chemical and a member of the polycyclic aromatic
hydrocarbon (PAH) class of chemicals. Fluorene is a component of petroleum (NCBI, 2021). It
is used as an intermediate for pesticides and dyes (NCBI, 2021). Fluorene is listed as active in
commerce on the Toxic Substances Control Act (TSCA) public inventory (U.S. HP A. 2021c) and
is registered with Europe's Registration, Evaluation, Authorization, and Restriction of Chemicals
(REACH) program (ECHA. 2021).

The 2006 Inventory Updating Reporting (IUR Rule), a precursor to the U.S. EPA's
Chemical Data Reporting (CDR) database, reported that the aggregate volume of fluorene
produced and imported in the United States was <500,000 pounds in 2005 (U.S. HP A. 202 la). Its
use was reported as "other" for commercial and consumer purposes; industrial use is reported as
a fuel (U.S. HP A. 202 la). More recent data were not available. Fluorene can be isolated from
coal tar by distillation and recrystallization of the fluorene fraction, or by continuous
countercurrent crystallization from higher fluorene fractions (Schmidt et al.. 2015). A patented
synthetic route describes passing 2-methylbiphenyl over a palladium-charcoal catalyst at
400-500°C, forming fluorene through cyclodehydrogenation (Orchin. 1947).

The empirical formula for fluorene is C13H10 and its structure is shown in Figure 1.

Table 1 summarizes the physicochemical properties for fluorene. Experimental property data
were selected preferentially over estimated property data. In the absence of experimental data,
estimated values from the U.S. EPA's CompTox Chemicals Dashboard were reported and
indicated with a footnote. Based on the reported vapor pressure, fluorene will exist primarily in
the vapor phase if released to the atmosphere. Fluorene is moderately volatile from water and
moist soil surfaces based on its Henry's law constant; however, the soil adsorption coefficient
indicates that fluorene will strongly sorb to soil and sediment, potentially limiting volatilization
from these surfaces. Due to strong sorption to soil and low water solubility, the potential to leach
to groundwater or undergo runoff after precipitation is low.



Figure 1. Fluorene (CASRN 86-73-7) Structure

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Table 1. Physicochemical Properties of Fluorene (CASRN 86-73-7)

Property (unit)

Value3

Physical state

Solid

Boiling point (°C)

295

Melting point (°C)

115

Density (g/cm3)

1.10 (predicted average)

Vapor pressure (mm Hg at 25°C)

6.00 x 10-4

pH (unitless)

NA

Acid dissociation constant (pKa) (unitless)

NA

Solubility in water (mol/L)

1.15 x 10-5

Octanol-water partition coefficient (log Kow)

4.18

Henry's law constant (atm-m3/mole)

9.62 x 10-5

Soil adsorption coefficient (L/kg)

5.01 x 103

Atmospheric OH rate constant (cm3/molecule-sec)

1.32 x 10-11

Relative vapor density (air =1)

NA (solid)

Molecular weight (g/mol)

166.223

Flash point (°C)

126 (predicted average)

aData were extracted from the U.S. EPA CompTox Chemicals Dashboard (fluorene, CASRN 86-73-3;

https://comptox.epa.gov/dashboard/dsstoxdb/results7searcliFDTXSID8024105; accessed August 4, 2022) (U.S.
EPA. 2021b). All values are experimental averages unless otherwise specified.

NA = not applicable.

A summary of available toxicity values for fluorene from the U.S. EPA and other
agencies/organizations is provided in Table 2. Reference values are based on chronic exposure
unless otherwise indicated.

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Table 2. Summary of Available Toxicity Values and Qualitative Conclusions
Regarding Carcinogenicity for Fluorene (CASRN 86-73-7)

Source/Parameterab

Value (applicability)

Notes

Reference0

Noncancer

IRIS (RfD)

0.04 mg/kg-d

Based on decreased RBC count,
packed blood cell volume, and
hemoglobin concentration in a
13-wk mouse gavage study

U.S. EPA (1990)

HEAST (sRfD)

0.4 mg/kg-d

Based on decreased RBC count
in a 13-wk mouse gavage study

U.S. EPA (201 lb)

DWSHA (RfD)

0.04 mg/kg-d

Based on a NOAEL for
hematological effects in mice

U.S. EPA (2018): U.S.
EPA (1991)

ATSDR (intermediate
oral MRL)

0.4 mg/kg-d

Based on increased relative liver
weight in a 90-d mouse gavage
study

ATSDR (1995)

IPCS

NV

NA

IPCS (1998)

CalEPA

NV

NA

CalEPA (2021):
CalEPA (2020)

OSHA

NV

NA

OSHA (2021a): OSHA
(2021b): OSHA (2021c)

NIOSH

NV

NA

NIOSH (2018)

ACGIH

NV

NA

ACGIH (2020)

DOE (PAC)

PAC-1: 6.6 mg/m3
PAC-2: 72 mg/m3
PAC-3: 430 mg/m3

PAC-1 and PAC-2 based on
TEELs; PAC-3 based on mouse
i.p. LD50

DOE (2018)

USAPHC (air-MEG)

1-h critical: 500 mg/m3
1-h marginal: 150 mg/m3
1-h negligible: 25 mg/m3

Based on TEELs

U.S. APHC (2013)

USAPHC (water-MEG)

1-yr negligible: 5.6 mg/L

5 L intake rate; based on IRIS
subchronic noncancer study

U.S. APHC (2013)

USAPHC (soil-MEG)

1-yr negligible: 5,200 mg/kg

Developed by USAPHC outside
the standard methodology

U.S. APHC (2013)

Cancer

IRIS (WOE)

Group D, not classifiable as
to human carcinogenicity

Based on no human data and
inadequate data from animal
bioassays

U.S. EPA (1990)

PPRTV (cancer OSF)

NV

NA

U.S. EPA (2002a)

HEAST

NV

NA

U.S. EPA (2011b)

DWSHA

D, not classifiable as to
human carcinogenicity

NA

U.S. EPA (2018)

NTP

NV

NA

NTP (2016)

IARC

Group 3, not classifiable as
to its carcinogenicity to
humans

Available studies in experimental
animals were considered
inadequate to permit evaluation

IARC (2010)

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Table 2. Summary of Available Toxicity Values and Qualitative Conclusions
Regarding Carcinogenicity for Fluorene (CASRN 86-73-7)

Source/Parameterab

Value (applicability)

Notes

Reference0

CalFPA

NV

NA

CalEPA (20211
CalEPA (2020)

ACGIH

NV

NA

ACGIH (2020)

aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DOE = U.S. Department
of Energy; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health Effects Assessment
Summary Tables; IARC = International Agency for Research on Cancer; IPCS = International Programme on
Chemical Safety; IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety
and Health; NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration;
PPRTV = Provisional Peer-Reviewed Toxicity Value; USAPHC = U.S Army Public Health Command.

Parameters: MEG = military exposure guideline; MRL = minimum risk level; OSF = oral slope factor;
PAC = protective action criteria; RfD = reference dose; sRfD = subchronic reference dose; WOE = weight of
evidence.

°Reference date is the publication date for the database and not the date the source was accessed.

i.p. = intraperitoneal; LD5o = median lethal dose; NA = not applicable; NOAEL = no-observed-adverse-effect level;
NV = not available; RBC = red blood cell; TEEL = Temporary Emergency Exposure Limit.

Literature searches were conducted in June 2019 and updated most recently in July 2023
for studies relevant to the derivation of provisional toxicity values for fluorene. Searches were
conducted using the U.S. EPA's Health and Environmental Research Online (HERO) database of
scientific literature. HERO searches the following databases: PubMed, TOXLINE1 (including
TSCATS1), Scopus, and Web of Science. The National Technical Reports Library (NTRL) was
searched for government reports from 2018 through September 20202. The following resources
were searched outside of HERO for health-related values: American Conference of
Governmental Industrial Hygienists (ACGIH), Agency for Toxic Substances and Disease
Registry (ATSDR), California Environmental Protection Agency (CalEPA), Defense Technical
Information Center (DTIC), European Centre for Ecotoxicology and Toxicology of Chemicals
(ECETOC), European Chemicals Agency (ECHA), the U.S. EPA Chemical Data Access Tool
(CDAT), the U.S. EPA ChemView, the U.S. EPA Integrated Risk Information System (IRIS),
the U.S. EPA Health Effects Assessment Summary Tables (HEAST), the U.S. EPA Office of
Water (OW), International Agency for Research on Cancer (IARC), the U.S. EPA
TSCATS2/TSCATS8e, the U.S. EPA High Production Volume (HPV), Chemicals via
International Programme on Chemical Safety (IPCS) INCHEM, Japan Existing Chemical Data
Base (JECDB), Organisation for Economic Co-operation and Development (OECD) Screening
Information Data Sets (SIDS), OECD International Uniform Chemical Information Database
(IUCLID), OECD HPV, National Institute for Occupational Safety and Health (NIOSH),

'Note that this version of TOXLINE is no longer updated

(https://www.nlm.nih.gov/databases/download/toxlinesubset.html'): therefore, it was not included in the literature
search update from July 2023.

2NTRL was a subset of TOXLINE until December 2019 when TOXLINE was discontinued. Searches of NTRL
were conducted starting in 2018 to ensure that references were not missed due to delays in importing items into the
database.

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National Toxicology Program (NTP), Occupational Safety and Health Administration (OSHA),
and World Health Organization (WHO).

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2. REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER ONLY)

Table 3 provides an overview of the relevant noncancer evidence base for fluorene and
includes all potentially relevant repeated-dose, short-term-, subchronic-, and chronic studies, as
well as reproductive and developmental toxicity studies. Principal studies used in the PPRTV
assessment are identified in bold. The phrase "statistical significance" and term "significant,"
used throughout the document, indicates ap-value of < 0.05 unless otherwise specified.

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Table 3. Summary of Potentially Relevant Noncancer Data for Fluorene (CASRN 86-73-7)

Category"

Number of Male/Female, Strain
Species, Study Type, Reported
Doses, Study Duration

Dosimetryb

Critical Effects

NOAELb

LOAELb

Reference
(comments)

Notes0

Human

1. Oral (mg/kg-d)

ND

2. Inhalation (mg/m3)

ND

Animal

1. Oral (mg/kg-d)

Short-term

ND

Subchronic

8 M/0 F, Wistar rat, gavage,
60 d

0,1,10,100

Increased relative liver weight.

1

10

Peiffer et al. (2016)

PS,
PR



Subchronic

20 M/20 F, Crl:CD-l mouse,
gavage, 13 wk

0, 125, 250,
500

Decreased RBC count, packed cell volume,
and hemoglobin; increased serum total
bilirubin and cholesterol and decreased BUN;
increased absolute and relative liver and
spleen weights; increased incidences of
centrilobular cytomegaly, cytoplasmic
alteration, and pigmentation of Kupffer cells
in the liver (males only), and hemosiderosis
and hematopoietic cell proliferation in the
spleen (both sexes).

125

250

TRL (1989)

NPR,
IRIS

Chronic

ND

Reproductive/
Developmental

ND

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Table 3. Summary of Potentially Relevant Noncancer Data for Fluorene (CASRN 86-73-7)

Category"

Number of Male/Female, Strain
Species, Study Type, Reported
Doses, Study Duration

Dosimetryb

Critical Effects

NOAELb

LOAELb

Reference
(comments)

Notes0

2. Inhalation (mg/m3)

Short-term

18 M/0 F, Wistar Han rat,
nose-only vapor inhalation, 6 h/d,
7 d/wk, 2 wk

0, 0.003, 0.3

No toxicologically relevant effects on
behavior were observed.

0.3

NDr

Peiffer et al. (2013)
(Other than
behavior, no
toxicological
endpoints were
evaluated in
fluorene-exposed
rats.)

PR

Subchronic

ND

Chronic

ND

Reproductive/
Developmental

ND

aDuration categories are defined as follows: Acute = exposure for <24 hours; short-term = repeated exposure for 24 hours to <30 days; long-term (subchronic) = repeated
exposure for >30 days or <10% life span for humans (>30 days up to approximately 90 days in typically used laboratory animal species); and chronic = repeated
exposure for >10% life span for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002b).

bDosimetry: Doses are presented as ADDs (mg/kg-day) for oral noncancer effects and as HECs (in mg/m3) for inhalation noncancer effects. The HEC from animal
studies was calculated using the equation for extrarespiratory effects from a Category 3 Gas (U.S. EPA. 1994): HECer = continuous concentration in mg/m3 x ratio of
animal:human blood gas partition coefficients (default value of 1 applied).

°Notes: Used by the IRIS program to derive a chronic oral RfD (U.S. EPA. 1990): NPR = not peer reviewed; PR = peer reviewed; PS = principal study.

ADD = adjusted daily dose; BUN = blood urea nitrogen; ER= extrarespiratory; F = female(s); HEC = human equivalent concentration; IRIS = Integrated Risk
Information System; LOAEL = lowest-observed-adverse-effect level; M = male(s); ND = no data; NDr = not determined; NOAEL = no-observed-adverse-effect level;
RBC = red blood cell; RfD = reference dose.

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2.1. HUMAN STUDIES

Select human studies that reported associations between biomarkers of exposure to
fluorene and health effects are presented in Table 4 and briefly summarized below. In general,
studies that reported human data were confounded by co-exposures to other PAHs and did not
specify the route of exposure (i.e., oral, inhalation, and other routes were likely in all cases.)
Although quantitative biomonitoring data were reported, information does not exist to support
the calculation of direct fluorene exposure (i.e., external dose) from reported exposure biomarker
concentrations (i.e., physiologically based pharmacokinetic [PBPK] models were not identified
for fluorene). For these reasons, no human studies were considered suitable for quantitative
dose-response analysis, and no-observed-adverse-effect level (NOAEL) or
lowest-observed-adverse-effect level (LOAEL) values were not identified.

Monitoring levels of the mono-hydroxylated fluorene metabolites, 2-OH fluorene, 3-OH
fluorene, and 9-OH fluorene, is a common proxy used for estimating fluorene exposure in
humans (Gmeiner et al.. 2002; Grantham. 1963; Dewhurst 1962; Neish. 1948). Nearly all
available studies evaluated associations between estimated fluorene exposure (based on urinary
or blood plasma levels of fluorene or the mono-hydroxylated metabolites) and health outcomes
(or biomarkers of health outcomes). Numerous studies reported associations between exposure to
fluorene from PAH mixtures and a variety of negative health outcomes for males and females,
including reduced lung function (Peng et al.. 2023; Alhamdow et al.. 2021; Cakmak et al.. 2017).
reduced liver function in adults (Mallah et al.. 2023). increased risk for cardiovascular disease in
a population of adult petrochemical workers (SUNY. 2023). greater likelihood of risk factors for
metabolic syndrome and dyslipidemia (Shahsavani et al.. 2022; Guo et al.. 2018b; Rani bar et al..
2015). associations with markers of age-related diseases (Yang et al.. 2023; Chen and Chen.
2022). increased inflammation in adolescents (Verheven et al.. 2021). increased biomarkers of
oxidative stress in adults (Verheven et al.. 2021; Zhu et al.. 2021). and obesity during childhood
and/or adolescence (Liu et al.. 2023; Dobraca et al.. 2020; Uche et al.. 2020) or adulthood (Wang
et al.. 2022; Ranibar et al.. 2015). There are also many studies that found sex-specific effects of
fluorene due to the nature of the endpoint evaluated (e.g., sperm effects observed in males) or
that reported results stratified by sex in which only one sex was found to be negatively affected
by exposure. In males, fluorene exposure has been linked to deleterious sperm effects (Chen et
al.. 2021; Yang et al.. 2017; Han et al.. 2011). Exposure in females has been associated with
oxidative stress in pregnant mothers working outside the home (Lou et al.. 2019). negative
effects on parameters of liver function (Xu et al.. 2021). delays in breast developments (without
effect on the age at pubertal transition) (Dobraca et al.. 2020). and increases in all-cause
(noncancer) mortality (Chen et al.. 2020). A few studies found evidence for a relationship
between fluorene exposure and negative health outcomes but the results were not statistically
significant; these include increases in serum biomarkers of cardiovascular disease (Clark et al ..
2012). presence of esophageal squamous dysplasia (Mwachiro et al .. 2021). risk for preterm
labor (Agarwat et al .. 2017). risk of osteoporosis and decreased bone density (Guo et al .. 2018a).
and risk of low-birth-weight offspring (Kumar et al .. 2020).

11

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Mortality

Chen et al. (2020) (United States)
Cross-sectional study of 1,409 subjects
(692 males and 717 females aged >20 yr)
fromNHANES (2001-2006).

Based on urinary levels
of 2-OH fluorene and
3-OH fluorene.

Mortality outcomes were
evaluated from the National
Death Index (linked to death
certificate data).

No significant association was found
between levels of 2-OH fluorene or
3-OH fluorene and cardiovascular or
cancer mortality. In females only, levels
of 3-OH fluorene were significantly
associated with an increased risk of
all-cause mortality (HR = 2.2, 95%
CI = [1.2, 3.8]).

Environmental exposure
to fluorene in adults was
associated with
noncarcinogenic mortality,
as also found for some of
the other PAHs evaluated.

Cardiovascular effects

Clark et al. (2012) (United States)

Cross-sectional study of 3,219 subjects
(1,547 males and 1,672 females aged
>20 yr) from NHANES (2001-2004).

Based on urinary levels
of 2-OH fluorene,
3-OH fluorene, and
9-OH fluorene.

Serum biomarkers of
cardiovascular disease
(fibrinogen, homocysteine, and
WBC counts) were measured.

Though not significant, an interquartile
increase in level (75th vs. 25th percentile)
for each fluorene metabolite was
positively associated with measurements
of cardiovascular disease in nonsmoking
subjects.

There was not strong
evidence for a relationship
between PAH exposure
(including fluorene) and
markers of cardiovascular
disease after controlling
for tobacco use.

Guo et al. (2018b) (China)
Cross-sectional study of 2,476 subjects,
n = 1,884 (675 males and 1,209 females,
mean age = 51.6 yr) without metabolic
syndrome (MetS) and n = 592
(193 males and 399 females, mean
age = 57.7 yr) with MetS from the
Wuhan-Zhuhai cohort.

Based on the summed
urinary levels of 2-OH
fluorene and 9-OH
fluorene.

Heart rate variability indices
(including very low frequency
[VLF], low frequency [LF],
high frequency [HF], and total
power [TP]) were measured.

High levels of fluorene metabolites were
significantly associated with decreased
VLF, LF, and TP in subjects with MetS
(graphs representing 95% CI of OR for
highest tertile vs. reference do not
include zero for these indices). There
were no significant associations in
subjects without MetS.

The association between
PAH exposure (including
fluorene) and heart rate
variability differed by
MetS status.

SUNY (2023) (China)

Cross-sectional study of 746 (601 males
and 145 females, median age = 49 yr)
petrochemical workers from two
different plants in the largest industrial
petroleum and petrochemical production
region in China.

Based on the summed
urinary levels of 2-OH
fluorene and 3-OH
fluorene.

Cardiovascular measurements
of hypertension (including
systolic blood pressure,
diastolic blood pressure,
cardiac frequency, and pulse
rate) were obtained.

The summed fluorine metabolites were
significantly associated with increases in
systolic blood pressure (r = 0,151),
diastolic blood pressure (r = 0.139), and
cardiac frequency (r = 0.121).

PAH exposure (including
fluorene) was associated
with increased risk of
hypertension in a
population of
petrochemical workers.

12

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Liver effects

Mallah et al. (2023) (United States)
Cross-sectional study of 2,515 subjects
aged >18 yr (1,211 males, 1,304 females,
mean age = 45.71 yr) from NHANES
(2003-2016).

Based on urinary levels
of 2-OH fluorene,
3-OH fluorene, and
9-OH fluorene.

Liver function indices (ALT,
AST, GGT, LDH, and total
bilirubin) and blood lipid
levels (TG, LDL-C, HDL-C,
and TC) were measured.

There were significant positive
relationships between each metabolite
and GGT: 2-OH fluorene (OR = 1.61,
95% CI = [1.23, 2.11]), 3-OH fluorene
(OR = 1.54, 95% CI = [1.21, 1.95]), and
9-OH fluorene (OR = 2.11, 95%
CI = [1.52, 2.95]).

PAH exposure (including
fluorene) was negatively
associated with liver
function in adults.

Xu et al. (2021) (United States)

Cross-sectional analysis of
3,194 adolescents (1,648 males, mean
age = 15.5 yr; 1,546 females, mean
age = 15.4 yr) from NHANES
(2003-2016).

Based on levels of

2-OH	fluorene and

3-OH	fluorene.

Liver function indices (ALT,
AST, GGT), inflammation
markers (C-reactive protein
and WBC count), and
indicators of blood lipid levels
(TG, LDL-C, HDL-C, and TC;
log 10-transformed for
analysis) were measured.

In females, 2-OH fluorene was
significantly associated with interquartile
increases in % change (A«/t) for ALT
(A% = 5.07, 95% CI = [1.83, 8.29]),
WBC count (A% = 3.56, 95% CI = [1.21,
5.96]), TG levels (A% = 6.99, 95%
CI = [0.73, 13.64]), and TC levels
(A% = 1.70, 95% CI = [0.12, 3.31 [).

There were no significant associations
among males for either fluorene
metabolite.

PAH exposure (including
fluorene) was negatively
associated with liver
function in female
adolescents.

Respiratory effects

Alhamdow et al. (2021) (Sweden)
Cross-sectional study using data from a
subset (n = 1,000, median age = 22.6 yr)
of 2,223 subjects from the Barn/Child,
Allergy, Milieu, Stockholm,
Epidemiology cohort.

Based on the summed
urinary levels of 2-OH
fluorene and 3-OH
fluorene.

Measurements of respiratory
function (FEVi, FVC) were
obtained via standard practice
and fractional exhaled nitric
oxide concentration (FeNo)
was measured as a biomarker
for eosinophilic
pulmonary inflammation.

There were significant inverse
relationships between summed fluorene
metabolites and FEVi (P = -73, 95%
CI = [-115, -30]) as well as FVC
((3 = -59, 95% CI = [-111, -6.5]).

Low-level exposure to
PAHs (including fluorene)
was associated with
reduced lung function in
young adults.

13

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Cakmak et al. (2017) (Canada)
Cross-sectional study of 3,531 subjects
from the Canadian Health Measures
Survey cycles 2 (2009-2011) and
3 (2012-2013), aged 6-79 yr.

Based on individual
and summed urinary
levels of 2-OH
fluorene,

3-OH fluorene, and
9-OH fluorene.

Measurements of respiratory
function (FEVi, FVC) were
obtained via standard practice.

All fluorene metabolites were
significantly associated with interquartile
increases in % change for FEVi and FVC
with combined levels reaching the
highest level of magnitude (FEVi:
A% = -1.41, 95% CI = [-2.68, -0.14];
FVC: A% = -1.28, 95% CI = [-2.46,
-0.10]).

Exposure to PAHs
(including fluorene) may
negatively impact lung
function.

Peng et al. (2023) (United States)
Cross-sectional study of 3,015 subjects
(500 individuals with incidence of COPD
and 2,015 without incidence of COPD)
aged 20-79 yr from NHANES
(2007-2016).

Based on individual
and summed urinary
levels of 2-OH
fluorene,

3-OH fluorene, and
9-OH fluorene.

Diagnostic criteria for COPD
were based on cutoffs for
measurements of respiratory
function (FEVi/FVC <70%)
obtained following inhaled
beta2-adrenergic
bronchodilator medication for
individuals in study yr
2007-2012 and self-reports of
an affirmative response to the
question "Have you ever been
told that you have COPD" for
individuals in study yr
2013-2017.

Increasing levels of urinary biomarkers
for fluorene exposure were significantly
and positively associated with risk for
diagnosis of COPD in 2-OH fluorene
(OR = 2.29. 95% CI = [1.42, 3.68] for
tertile 3) and 9-OH (OR = 1.72, 95%
CI = [1.04, 2.84] for tertile 2). Summed
levels of fluorene metabolites were
significantly associated with COPD for
the highest tertile of exposure
(OR = 2.74, 95% CI = [1.77, 4.23]).

Exposure to PAHs
(including fluorene) was
associated with risk for a
positive diagnosis of
COPD.

Pregnancy outcomes

Agarwal et al. (2017) (India)
Case-control study of 84 healthy,
pregnant women recruited from a
medical college; controls (n = 55)
included gestational age >36 wk
(full-term delivery) undergoing
spontaneous labor at term; cases (n = 29)
included gestational age <36 wk
(preterm delivery) undergoing preterm
labor.

Based on fluorene
levels in placental
tissue samples.

Levels of MDA and GSH were
measured in placental tissue
samples as biomarkers of
redox status.

Though not significant, higher levels of
fluorene (placental levels; |ig/L) were
observed in women with full-term
deliveries (controls; mean = 0.012,
SD = 0.06) compared to women with
preterm deliveries (cases;
mean = 0.0007, SD = 003).

Observations of increased
MDA and decreased GSH
in cases relative to
controls suggest a possible
role for PAHs (other than
fluorene) in early delivery.

14

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Kumar et al. (2020) (India)

Case-control study of 175 pregnant
women recruited from a medical college;
controls (n = 120, mean age = 23.7 yr)
with normal-birth-weight offspring;
cases (n = 55, mean age = 22.3 yr) with
low-birth-weight offspring.

Based on fluorene
levels in maternal
blood (placental and
cord blood levels of
fluorene were not used
due to low detection
rates).

Birth weights were measured
and evaluated
(low vs. normal).

Fluorene exposure above the median
level as measured in maternal blood was
associated with an increased likelihood
of low-birth-weight offspring
(OR = 9.36, 95% CI = [0.50, 175.04],
p = 0.135).

The blood concentrations
of some PAHs (including
fluorene) were associated
with low-birth-weight
offspring.

Lou et al. (2019) (China)
Cross-sectional study of 188 pregnant
women (mean age = 29.2 yr) randomly
recruited during regular pregnancy
checks; cases (n = 138) with pregnant
women working outside the home and
controls (n = 24) with pregnant women
not working outside the home.

Based on urinary levels
of 2-OH fluorene.

Urinary levels of 8-OHdG, as a
biomarker of DNA oxidative
damage, were measured.

2-OH fluorene was significantly
(r = 0.496; p < 0.01) associated with
urinary 8-OHdG in pregnant women
working outside the home. No significant
associations were found in pregnant
women not working outside the home.

Exposure to some PAHs
(including fluorene) was
associated with oxidative
stress in pregnant women
who worked in jobs
outside the home.

Sperm parameters

Chen et al. (2021) (China)
Cross-sectional analysis (n = 656) based
on subjects from the Male Reproductive
Health in Chongqing College Students
cohort study with data available at
baseline (2013) and 1-yr follow-up
(2014); average age at baseline = 20 yr.

Based on individual
urinary levels of 2-OH
fluorene as well as
fluorene levels
measured via
PM2.5 sampling.

Sperm parameters (sperm
concentration, progressive
motility [%], normal
morphology [%], and sperm
DNA integrity, fragmentation,
and stainability via sperm
chromatin structural assay) and
serum biomarkers for
reproductive health (estradiol,
FSH, LH, prolactin,
progesterone, and testosterone)
were measured.

2-OH fluorene was significantly
negatively associated with sperm
progressive motility ((3 = -4.347, 95%
CI = [-7.628, -0.949]) and serum
progesterone levels ((3 = -7.877, 95%
CI = [-14.137, -1.162]). Fluorene
exposure as measured via
PM2.5 sampling was positively
associated with serum estradiol
(estimates not reported) and inversely
associated with LH ((3 = -13.9, 95%
CI = [-18.5, -8.9]), prolactin
((3 = -100.0, 95% CI = [-100.0,
-100.0]), and testosterone ([3 = -15.6,
95% CI = [-21.9, -8.9]).

Environmental exposure
to PAHs (including
fluorene) was negatively
associated with male
reproductive function.

15

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Han et al. (2011) (China)
Cross-sectional study of 232 subjects
from the general male population (mean
age = 31.89 yr) assessed in December
2007.

Based on urinary levels
of 2-OH fluorene.

Semen quality, sperm
apoptotic markers (Annexin V
assay), and sperm DNA
damage (comet assay) were
evaluated.

2-OH fluorene was significantly
negatively associated with Annexin
V /PI spermatozoa (living cells without
PS translocation) ((3 = -11.10, 95%
CI = [-17.31, -4.88]) and positively
associated with PI+ (necrotic) cells
((3 = 8.91, 95% CI = [2.99, 14.84]); it
was also weakly associated with tail %
((3 = 5.04, 95% CI = [-0.99, 11.07];
p = 0.07).

Environmental exposure
to PAHs (including
fluorene) was associated
with sperm DNA damage.

Yang et al. (2017) (China)
Cross-sectional study of 793 male
partners in subfertile couples (mean
age = 32 yr) with sufficient unprocessed
urine for analysis of PAH metabolites.

Based on urinary levels
of 2-OH fluorene and
9-OH fluorene.

Sperm indices (sperm DNA
damage via the comet assay
and apoptosis via the
Annexin V/PI assay) were
measured.

9-OH fluorene was significantly
positively associated with tail length and
comet length (p values for trend = 0.05
and 0.01, respectively) and inversely
associated with percentage of
Annexin V /PI spermatozoa (i.e., living
cells without PS translocation; p < 0.10).

Environmental exposure
to PAHs (including
fluorene) was associated
with increased sperm
DNA damage and
apoptosis.

16

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Obesity in children and adults (also timing of puberty)

Dobraca et al. (2020) (United States)
Cohort study of 404 girls (aged 6-8 yr at
baseline, mean = 7.4 yr) enrolled in the
Northern California site of the Breast
Cancer and the Environment Research
Program cohort.

Based on summed
urinary levels of

2-OH	fluorene,

3-OH	fluorene, and
9-OH fluorene.

Adiposity (BMI and
waist-to-height ratio) and
pubertal onset according to the
Tanner stages of breast and
pubic hair development were
evaluated from age 7 through
16 yr.

At baseline (approximately 7 yr):
The highest tertiles of fluorene
metabolites were associated with higher
adiposity.

Over the follow-up period (7-16 yr):
High tertiles of fluorene metabolites
were significantly associated with
increased BMI (7.0 kg/m2 increase at the
high tertile compared to 6.0 kg/m2
increase at the lowest tertile). The
highest tertiles of fluorene metabolites
were significantly associated with
increased waist-to-height ratio. Breast
development occurred significantly later
at the highest tertiles of fluorene
metabolites (10.3 yr) compared to the
lowest tertile (9.9 yr) in normal-weight
girls. A nonsignificant delay in pubic
hair development was also observed in
normal-weight girls with higher tertiles
of fluorene metabolites.

Exposure to PAHs
(including fluorene)
during childhood may
influence adiposity during
adolescence and affect
pubertal timing.

Liu et al. (2023) (Canada. Iran. Korea,
and United States)

Meta-Analysis of eight cross-sectional
studies with a pooled sample size of
68,454 individuals (aged >3 yr in all
studies).

Based on pooled effect
estimates of urinary
and blood levels of

2-OH	fluorene,

3-OH	fluorene, and
9-OH fluorene.

Obesity criteria were defined
by the individual studies
included in the meta-analysis.

9-OH fluorene was significantly

positively associated with obesity after

adjusting for physical activity

(OR = 1.37, 95% CI = [1.11, 1.69]) and

in subgroup analyses for individuals aged

3-19 yr (OR = 1.53, 95% CI = [1.20,

1.96]).

The association between
PAH exposure (including
fluorene) was positively
associated with increased
risk for obesity in children
and adolescents.

17

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Ranibaret al. (2015) (United States)

Cross-sectional study of 4,765 subjects
(aged >20 yr) from NHANES
(2001-2008). The mean ages of
non-obese (n = 3,085) and obese
(,n = 1,680) subjects were 45.1 and
47.2 yr, respectively.

Based on urinary levels
of 2-OH fluorene and
3-OH fluorene.

Obesity (based on BMI
>30 kg/m2), having three or
more risk factors for metabolic
syndrome (3RFMetS), type 2
diabetes, hypertension, and
dyslipidemia were evaluated.

For both fluorene metabolites, the
highest quintiles of exposure were
significantly associated with a greater
likelihood of 3RFMetS (2-OH fluorene:
OR = 1.66, 95% CI = [1.05, 2.62]; 3-OH
fluorene: OR = 1.80, 95% CI = [1.25,
2.59]) and dyslipidemia (2-OH fluorene:
OR = 1.54, 95% CI = [1.20, 1.97]; 3-OH
fluorene: OR = 1.57, 95% CI = [1.21,
2.05]).

Exposure to PAHs
(including fluorene) was
associated with obesity
and obesity-related
cardiometabolic health
risk factors.

Siiahsavani et al. (2022) (Iran)

Cross-sectional analysis of

200 individuals (mean age = 40.2 yr).

Based on urinary levels
of 2-OH fluorene.

Levels of biomarkers for lipid
peroxidation (urinary MDA)
and metabolic factors (blood
serum FBS, LDL-C, HDL-C,
TC, TG, and other blood
biochemical parameters) were
measured. Body measurements
(weight, height, and waist
circumference), blood pressure
parameters, and MetS status
were measured and/or
evaluated by trained health
professionals using
standardized protocols.

2-OH fluorene was significantly
associated with increased systolic blood
pressure (r = 0.11), diastolic blood
pressure (r = 0.92), TG level (r = 0.03),
waist circumference (r = 0.16), and
hemoglobin (r = 0.44).

Environmental exposure
to PAHs (including
fluorene) was related to an
increase in risk for
metabolism- and
obesity-related health
outcomes.

Uche et al. (2020) (United States)

Cross-sectional study of 50,048 children
and adolescents (aged 6-17 yr; mean
age =11.5 yr) from NHANES
(1999-2016).

Based on various
environmental factors,
including urinary
levels of 9-OH
fluorene (not further
specified).

Obesity was measured (BMI
and waist-to-height ratio).

9-OH fluorene was significantly
positively associated with obesity based
on BMI (OR = 1.509, 95% CI = [1.230,
1.851]) and abdominal obesity
(OR = 1.478, 95% CI = [1.182, 1.847])
in children/adolescents. Though not
significant, females with higher levels of
9-OH fluorene were more likely than
males to be obese.

Environmental factors
(including exposure to
fluorene and other PAHs)
were associated with
childhood obesity.

18

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Wane et al. (2022) (United States)

Cross-sectional study of

2,691 nonsmoking subjects

(959 non-Hispanic white,

585 non-Hispanic black, and

767 Hispanic subjects aged >20 yr) from

NHANES (2001-2016).

Based on urinary levels
(individual and
summed) of 2-OH
fluorene,

3-OH fluorene, and
9-OH fluorene.

FM% of the trunk and legs was
determined via dual-energy
x-ray absorptiometry results,
and body measurements
(weight, height, and waist
circumference) were obtained
by trained health professionals
using standardized protocols.

In the total population, 3-OH fluorene
was significantly inversely correlated
with multiple outcomes including total
FM% (r = -0.07) and trunk FM%
(r = -0.08), while 9-OH was
significantly positively correlated with
trunk FM% (r = 0.09), trunk/leg ratio
(r = 0.10), and waist circumference
(r = 0.08). In analyses stratified by
race/ethnicity, 9-OH fluorene was
significantly positively correlated with
waist circumference (r = 0.18), trunk
FM% (r = 0.22), trunk/leg ratio
(r = 0.21), and total FM% (r = 0.20) in
the non-Hispanic black population.

PAH exposure (including
fluorene) was associated
with increased risk for
obesity-related health
outcomes and the
associations varied based
on race/ethnicity.

Musculoskeletal effects

Guo et al. (2018a) (United States)

Cross-sectional study of 1,768 women
(aged >20 yr) from NHANES
(2005-2010).

Based on urinary levels
of 2-OH fluorene,
3-OH fluorene, and
9-OH fluorene.

Bone mass density and
osteoporosis were evaluated.

Compared with the first tertile, the third
tertile of 2-OH fluorene in women was
associated with significantly decreased
bone mass density (femur: OR = -0.014,
95% CI = [-0.028, -0.001]; trochanter:
OR = -0.016, 95% CI = [-0.028,
-0.004]). The third tertile of 9-OH
fluorene was associated with an
increased likelihood of osteoporosis in
women relative to the first tertile
(overall: OR = 1.97, 95% CI = [1.07,
3.63]). Though the differences did not
reach statistical significance, associations
were strengthened in postmenopausal
women relative to premenopausal
women.

Associations between
PAH exposure (including
fluorene) and bone mass
density or osteoporosis
varied by bone site and
menopausal status.

19

Fluorene


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EPA/690/R-23/006F

Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Chronic endocrine stress, inflammation, oxidative stress

Verheven et al. (2021) (Belgium)
Cross-sectional study of 393 adolescents
(183 males and 210 females with mean
age = 14.8 yr) from the fourth Flemish
Environment and Health Study
(2016-2017).

Based on summed
urinary levels of 2-OH
fluorene and 3-OH
fluorene.

Biomarkers of chronic
endocrine stress (HCC),
inflammation (NLR), and
oxidative stress (8-oxodG in
urine) were measured.

Combined fluorene metabolites were
significantly positively associated with
NLR ((3= 1.06, 95% CI = [1.01, 1.13]).
In sex-stratified analyses for NLR,
associations in females were similar to
the primary analysis, while associations
in males were slightly attenuated
(females: (3 = 1.10, 95% CI = [1.02,
1.18]; males: (3 = 1.02, 95% CI = [0.94,
1.10]).

Environmental exposure
to PAHs (including
fluorene) was associated
with inflammation in
adolescents.

Zhu et al. (2021) (United States)

Longitudinal analysis of 19 healthy
volunteers (11 males, 8 females, with
overall mean age = 34 yr) over a 44-d
study period.

Based on summed
urinary levels of 2-OH
fluorene,

3-OH fluorene, and
9-OH fluorene.

Biomarkers of oxidative stress
(diY, 8-OHdG, MDA, and four
F2-isoprostane isomers
[8-isoprostaglandinF2a,
1 ip-prostaglandinF2a,
15(R)-prostaglandinF2a, and
8-iso, 15(R)-prostaglandinF2a])
were measured in urine.

Over the course of the study period, there
were significant increases in levels of
8-OHdG (9.8%), MDA (12%), and diY
(14%) attributed to every 1 unit increase
in the log-transformed level of combined
fluorene metabolites.

Continuous exposure to
environmental PAHs
(including fluorene) was
associated with increased
levels of oxidative stress.

Markers of Aged-Related Diseases

Chen and Chen (2022) (United States)
Cross-sectional study of 2,597 subjects
(1,318 men and 1,279 women aged
>20 yr) from NHANES (2015-2016).

Based on urinary levels
of 2-OH fluorene and
3-OH fluorene.

Serum klotho levels were
measured as a biomarker of
premature aging.

In the total population, 3-OH fluorene
was inversely associated with klotho
((3 = -0.026, 95% CI = [-0.046,
-0.005]). In sex-stratified analyses, both
metabolites were significantly associated
with decreased serum klotho levels in
men but not women (2-OH fluorene
[men]: (3 = -0.014, 95% CI = [-0.027,
0.000]; [women]: |3 = -0.005, 95%
CI = [-0.021, 0.011], 3-OH fluorene
[men]: p = -0.034, 95% CI = [-0.059,
-0.008]; [women]: p = -0.015, 95%
CI = [-0.049, 0.018]).

Exposure to PAHs
(including fluorene) was
associated with decreased
serum klotho levels.

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Table 4. Selected Studies Evaluating Associations between Fluorene Exposure and Health Outcomes

Citation (Location); Study Type, Size
and Description of Population

Methods for Fluorene
Exposure

Methods for Outcome
Assessment

Summary of Results"

Conclusions

Yang et al. (2023) (United States)

Cross-sectional study of 1,460 subjects
(716 men and 1,279 women aged >20 yr)
from NHANES (2015-2016).

Based on urinary levels
of 2-OH fluorene and
3-OH fluorene.

Telomere length of DNA
obtained from blood samples
was measured via quantitative
polymerase chain reaction
methods to calculate telomere
length ratios relative to a
standard reference.

Levels of 2-OH fluorene were
significantly and inversely associated
with telomere length ((3 = -0.01, 95%
CI = [-0.1,-0.004]).

Exposure to PAHs
(including fluorene) was
associated with decreased
telomere length.

Cancer

Mwachiro et al. (2021) (Kenva)
Cross-sectional analysis of 289 adults
(158 males, 138 females; 157 aged
<50 yr, 132 aged >50 yr) from the Study
of Tenwek Esophageal Squamous
Dysplasia Prevalence.

Based on levels of
urinary 2-OH fluorene
and 3-OH fluorene.

Esophageal squamous
dysplasia was evaluated based
on results from Lugol's iodine
chromoendoscopy.

Positive, but nonsignificant, associations
were detected between urinary fluorene
metabolites and moderate or severe
esophageal squamous dysplasia.

There were no significant
associations between
urinary fluorene
metabolites and risk of
moderate or severe
esophageal squamous
dysplasia.

Significant results imply statistical significance at the level p < 0.05 as reported by the study authors. ORs and HRs are considered significant if the 95% CI does not
include one; (3 (regression) coefficients and average interquartile differences of percent change (A«/t) are considered significant if the 95% CI does not include zero.
Differences in subpopulations are considered significant if the 95% CIs for the corresponding measurements do not overlap.

2-OH fluorene = 2-hydroxyfluorene; 3-OH fluorene = 3-hydroxyfluorene; 8-OHdG = 8-hydroxy-2'-deoxyguanosine; 8-oxodG = 8-oxo-7,8-dihydro-2'-deoxyguanosine;
9-OH fluorene = 9-hydroxyfluorene; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BMI = body mass index; CI = confidence interval;

COPD = chronic obstructive pulmonary disease; diY = o,o'-dityrosine; DNA = deoxyribonucleic acid; FBS = fasting blood sugar; FEVi = forced expiratory volume of
1 second; FM% = fat mass percentage; FSH = follicle-stimulating hormone; FVC = forced vital capacity; GGT = gamma-glutamyl transpeptidase; GSH = glutathione;
HCC = hair Cortisol concentration; HDL-C = high-density lipoprotein cholesterol; HR = hazard ratio; LDH = lactate dehydrogenase; LDL-C = low-density lipoprotein
cholesterol; LH = luteinizing hormone; MDA = malondialdehyde; MetS = metabolic syndrome; NHANES = National Health and Nutrition Examination Survey;
NLR = neutrophil-lymphocyte ratio; OR = odds ratio; PAH = polycyclic aromatic hydrocarbon; PI = propidium iodide; PM2.5 = particulate matter 2.5;
PS = phosphatidylserine; SD = standard deviation; TC = total cholesterol; TG = triglycerides; WBC = white blood cell.

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2.2. ANIMAL STUDIES
2.2.1. Oral Exposures

The oral noncancer database for fluorene is limited to two subchronic gavage studies:
(1) a 60-day study in male rats that evaluated behavioral endpoints (Peiffer et al.. 2016) and (2) a
13-week study in male and female mice that evaluated a comprehensive set of toxicological
endpoints CTRL. 1989). and two subchronic to chronic dietary studies: (1) 104- and 453-day
studies in rats (sex unspecified) with limited data reported (Wilson et al.. 1947) and (2) 6- and
18-month studies in female rats that evaluated a limited number of non-neoplastic endpoints
(Morris et al.. 1960). The TRL (1989) study was used by the IRIS program to derive a chronic
oral reference dose (RfD) for fluorene (U.S. EPA. 1990).

Short-Term Studies

No studies were identified.

Subchronic Studies

Peiffer et al (2016)

In a published, peer-reviewed study, Wistar rats (eight males/group, aged 8-9 weeks)
were administered fluorene (98% pure) in vegetable oil (a mixture of sunflower, rapeseed, and
grape seed oils with no PAH contamination) via gavage at 0 (vehicle control), 1, 10, or
100 mg/kg-day for 60 days. Body weights were recorded on Study Days 2, 7, 14, 21, and 28.
Four behavioral tests, initiated on Study Day 28 and performed through Study Day 60, were
conducted 30 minutes after daily gavage administration (during the dark phase of the circadian
cycle). Tests included an elevated-plus maze test on Study Day 28 to evaluate anxiety, an open-
field test on Study Day 29 to evaluate motor and exploratory activity, an eight-arm maze on
Study Days 30-44 (7 days of food restriction, 3 days familiarization, and 5 days of testing) to
evaluate spatial learning and memory, and an aversive light stimulus avoidance test (Test
d'Evitement d'un Stimulus Lumieux Aversif, or TESLA) on Study Days 45-53 (7 days
acclimatization, 1 day habituation, and 1 day of recall) to evaluate learning and memory. At
sacrifice on Study Day 60, blood samples were collected, and brain and liver weights were
recorded.

The outcomes measured in the elevated-plus maze (a raised maze consisting of three
main areas: two open arms [ledges without enclosure] and two closed arms [ledges enclosed by
vertical surrounding walls] intersecting at a central area to form a "plus" shape) included number
of arm entries (total, open, and closed), time spent in each area (open arms, closed arms, and
central area), total head dipping, percent head dipping in open arms, total rearing, and percent
rearing in closed arms during a 5-minute period. Decreased open arm entries, decreased time
spent in open arms, and increased occurrences of head dipping and rearing were considered
indicative of anxiety.

In the open-field test, levels of activity on a platform containing 32 equivalent sections
and three concentric zones (central, intermediate, and peripheral) were observed for 5 minutes
and quantified by recording the numbers of squares crossed, number of rears, and amount of time
spent in each zone.

The eight-arm maze, an enclosed maze consisting of eight arms (containing food pellets
during the 5-day testing phase) joined in a central circular area, tested learning and spatial
memory by measuring the ability of rats to position themselves within the maze using external

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visual cues located within the testing room. Parameters evaluated included total time to complete
the maze (time taken to visit each arm with a cutoff value at 15 minutes), total arm entries, arm
entries before the first error, and number of arms visited per minute.

The TESLA evaluated reference memory. Rats were placed in a box with high-intensity
lighting and two pedals: an active lever that turned off the light for a 30-second period and an
inactive lever that did not turn the light off. The active lever is deactivated while the light is
turned off and regains its active status after the light has been off for 30 seconds; cumulative
lever presses of the deactivated active lever do not result in longer periods of light reduction.
After 1 day of habituation, rats were observed for 20 minutes and reference memory was
evaluated based on discrimination of the active lever, discrimination of the active (light) period,
discrimination of the lever and the active period, and total number of lever presses.

Based on data presented for eight animals/group, it was presumed that no mortality
occurred at any of the dosing levels (Peiffer et al.. 2016). Body-weight data were not provided;
however, percent body-weight gain (compared to the first day of treatment) was presented
graphically with indicators of statistical significance. No statistically significant changes in body-
weight gain measured on Study Days 2, 7, 14, 21, and 28 were observed for rats in the first two
dose groups (1 and 10 mg/kg-day) relative to controls. Rats treated at the high dose of
100 mg/kg-day lost approximately 3 and 6% of their body weight by Study Days 2 and 7,
respectively (based on analysis of the graphical data using the MATLAB tool, GRAB IT3; see
Table B-l), but then gained weight thereafter. Overall, rats in this group showed decreased
percent weight gain relative to controls through Study Day 28 (p < 0.01 at each time point). The
study authors reported that rats treated at the high dose exhibited lower average body weights
throughout the 60-day study (data not shown). No treatment-related, toxicologically relevant
effects on anxiety were observed based on the results of the elevated-plus maze; rats treated at
the low dose (but not other doses) showed reduced anxiety (as evidenced by significantly
decreased time spent in the closed arms and significantly increased time spent in the central area
relative to controls) (Peiffer et al.. 2016). Motor activity was unaffected by treatment (open-field
test data not shown). In the eight-arm maze to evaluate learning and memory, a trend for
decreased time to visit all arms (p = 0.074) as well as a statistically significant trend for the
increase in the number of arms visited per minute (p < 0.01) were observed in all groups
(including controls) based on the time of testing (i.e., all rats became more efficient at the maze
as testing progressed). Rats treated at the high dose had fewer arm entries before the first error
(i.e., performed worse in this behavioral test) compared to the other groups regardless of time of
testing (p = 0.098). In addition to this overall effect, the only statistically significant interaction
between time of testing and treatment group was for reduction in the number of arm entries
before the first error (p < 0.05), which was particularly evident at the high dose (reduced by 24%
on Study Day 5 relative to Study Day 1 compared to an 8% reduction in the control group and
increases [i.e., improved performance] in the other dose groups). In the TESLA, another test that
evaluated learning and memory, ability to discriminate between active (light) and inactive (dark)
periods was significantly improved based on the time of testing for rats from all dosing groups
(i.e., during testing relative to habituation; p < 0.05). The total number of lever presses increased
based on treatment group (i.e., higher-dose groups typically had a higher number of lever

'GRABIT (https://www.mathwoiks.com/matlabcentral/fileexchange/7173-grabit') is an application of MATLAB and
extracts data points from an image file using a graphical user interface.

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presses; p = 0.073) and significantly decreased based on time of testing (i.e., there were fewer
lever presses during testing than habituation; p < 0.01). There were no statistically significant
interactions between treatment group and time of testing for this effect or any of the other
TESLA endpoints. Based on overall results from the behavioral battery, the study authors
concluded that learning and memory were not affected by treatment.

Absolute brain and liver weights were not reported (Peiffer et al.. 2016). Relative brain
and liver weight data were presented graphically by dose group, with indicators of statistical
significance for treatment groups relative to the control group. Nonsignificant increases in
relative brain weight were observed at the mid and high dose (4 and 8% higher than controls,
respectively). Relative liver weight showed statistically significant increases (p < 0.05) at all
doses (by approximately 6, 17, and 37% relative to controls at 1, 10, and 100 mg/kg-day,
respectively, based on analyses using the MATLAB tool, GRAB IT; see Table B-l). Increases in
relative liver weight at the mid and high doses (10 and 100 mg/kg-day) were considered to be
biologically significant.

Limitations of the study include the lack of data reported for biological endpoints of
interest, including absolute brain and liver weights at study termination. Data were presented
graphically, but not reported numerically, for relative brain and liver weights as well as body
weights measured on Study Days 2, 7, 14, 21, and 28. No clinical chemistry evaluations or
histological examinations were performed. Despite these limitations, aNOAEL of 1 mg/kg-day
is identified from these data based on biologically significant (>10%) increases in relative liver
weight in male rats at 10 and 100 mg/kg-day, which were also reported to be statistically
significant (p < 0.01). The administered doses of 0, 1, 10, and 100 mg/kg-day correspond to
human equivalent doses (HEDs) of 0, 0.2, 2.4, and 23.6 mg/kg-day, respectively4.

TRL (1989) [citedas U.S. EPA, 1989 by U.S. EPA (1990)]

In an unpublished, non-peer reviewed study, Crl:CD-l mice (20/sex/group, aged 35 days)
were administered fluorene (>98% pure) in corn oil via daily gavage at 0 (vehicle control), 125,
250, or 500 mg/kg-day for 13 weeks. Additional groups of five animals/sex were designated as
satellite animals; these animals were dosed in parallel to the main group and, if necessary, used
as substitutes for animals lost by technical error. Endpoints evaluated included mortality, clinical
signs of toxicity, ophthalmologic examinations, food consumption, body weight, hematology
(red blood cell [RBC] count, total and differential white blood cell [WBC] count, reticulocyte
counts if animals exhibited signs of anemia, hemoglobin, RBC packed cell volume [PCV], mean
cell volume [MCV], mean cell hemoglobin [MCH], and mean cell hemoglobin concentration
[MCHC]) and clinical chemistry (glucose, blood urea nitrogen [BUN], cholesterol, total
bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride,
total carbon dioxide, and the activities of alkaline phosphatase [ALP], alanine aminotransferase
[ALT], aspartate aminotransferase [AST], and lactate dehydrogenase [LDH]) in
10 mice/sex/group. Organ weights (brain, heart, liver, spleen, kidneys, and testes; as paired
organs when applicable) and gross and microscopic pathology (-40 tissues in control animals,
high-dose animals, and animals that died or were sacrificed moribund; liver, lungs, spleen,

4Administered doses were converted to HEDs by multiplying by a dosimetric adjustment factor (DAF) of
0.236 calculated as follows: DAF = (BWa1/4 ^ BWh14), where BWa = animal body weight, and BWh = human body
weight. In the absence of data for study-specific time-weighted average (TWA) animal body weights, the reference
value for the body weight of male Wistar rats in a subchronic study of 0.217 kg was used (U.S. EPA. 1988). For
humans, the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988).

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kidneys, testes, and gross lesions of low- and mid-dose animals) were also evaluated. Only one
member of an organ pair (with the exception of the thyroid gland) was examined unless a
potentially treatment-related lesion was detected.

Six animals died during the study (TRL. 1989). Two high-dose females that died during
Week 1 were replaced with animals from the satellite group; the others (one control male during
Week 3 and one control, one low-dose, and one-high dose female during Weeks 5, 7, and 9,
respectively) were not replaced; upon histological examination, all deaths were attributed to
gavage error. Overall survival was not significantly different among groups, including controls.
Clinical signs of toxicity with significantly increased incidence compared to controls in both
males and females included salivation (all treated groups) and hypoactivity (high-dose group
only) (see Table B-2). Other clinical signs noted (but not significantly increased) were labored
respiration in high-dose animals of both sexes and ptosis, urine wet abdomen, and unkempt
appearance, predominantly in high-dose males but also seen in other dose groups. The study
authors indicated that retinal degeneration observed at Week 13 was spontaneous rather than
treatment-related; however, data (including the numbers of animals and dose groups affected)
were not shown. Significantly increased food consumption was observed in mid- and high-dose
males (8-22% higher than controls) and high-dose females (8-19% higher than controls) for
most weeks of the study (see Table B-3). Significantly increased food consumption was also
observed (albeit less frequently) in low-dose males and mid-dose females. No significant effects
on body weight occurred in males (see Table B-4). In females, small, statistically significant
increases in body weight were observed at the high dose (6—8% higher than controls) for most
weeks of the study (especially starting on Week 5). There were no statistically significant effects
on body weight in females treated at the low or mid dose. During the duration of the study
(Weeks 1-13), high-dose males and females gained 20 and 30% more weight than controls,
respectively (see Table B-4).

Hematological changes indicative of anemia included significant reductions in RBC
count (10-21%) lower than controls in high-dose males and mid- and high-dose females), PCV
(10-22%) lower than controls in mid- and high-dose males and females), and hemoglobin (16 and
13%o lower than controls in high-dose males and females, respectively) (see Table B-5) (TRL.
1989). There were small (<10%>), but statistically significant, increases in some of the calculated
RBC indices (MCV, MCH, and MCHC), primarily in the mid- and/or high-dose groups. WBC
count was significantly increased in high-dose females (46%> higher than controls) but was
within the range of natural variation as measured within the study; differential WBC counts
(i.e., neutrophil, monocyte, eosinophil, and basophil counts) were not significantly impacted by
treatment. Significant clinical chemistry effects observed in mid- and/or high-dose males and
females included increased serum cholesterol (>25% higher than controls), decreased BUN
(20-24%) lower than controls), and increased total bilirubin (61-71%) higher than controls);
statistically significant trends for decreased BUN and increased bilirubin were also reported
(see Table B-6). Other statistically significant changes in clinical chemistry parameters
(i.e., potassium levels and the activities of ALT and ALP) were not considered treatment-related
for one or more of the following reasons: the magnitude of change was small (within range of
natural variation), changes were not dose-related, changes were not consistent across sexes or
endpoints (e.g., changes in serum ALT were not accompanied by changes in other parameters
indicative of liver cell damage, such as serum AST or LDH), and/or changes were of uncertain
toxicological significance.

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Significant effects on organ weights are shown in Table B-7 (TRI.. 1989). In males,
absolute and relative liver weights were biologically and statistically significantly increased at
the mid and high dose (by 17-35% relative to controls). Relative (but not absolute) liver weight
was statistically significantly increased at the low dose; however, the magnitude of change was
small (7% higher than controls). Significant increases in absolute and relative kidney weight
were observed in high-dose males only (12 and 8% higher than controls, respectively). Large and
statistically significant increases in absolute and relative spleen weights were observed at the mid
dose (28-31% higher than controls) and at the high dose (99-106% higher than controls). In
females, absolute and relative liver weights were biologically and statistically significantly
increased at the mid dose (21—25% higher than controls) and the high dose (35—45% higher than
controls); relative liver weight was slightly, but statistically significantly, increased at the low
dose (8%> higher than controls). No treatment-related effects on kidney weights were observed.
Absolute and relative spleen weights were increased by 33—35% relative to controls in mid-dose
females and by 84-99% relative to controls in high-dose females. There were no significant
changes in absolute brain weight; however, a slight, but significant, decrease in relative brain
weight (9% lower than controls) was observed in high-dose females. This effect was attributed
by the study authors to increased body weight of high-dose females at necropsy (8% higher than
controls).

No treatment-related effects were observed at gross necropsy CTRL. 1989). Microscopic
examination revealed significant increases in histopathological effects in male mice, including
increased incidences of brown pigment (with an appearance reminiscent of hemosiderin) in
Kupffer cells, centrilobular cytomegaly (i.e., enlarged hepatocytes), and centrilobular cytological
alterations (more homogeneous and eosinophilic cytoplasm) in the livers of high-dose males;
significant increases in the incidence and severity of hemosiderosis and hematopoietic cell
proliferation in the spleens of mid- and high-dose males; and degenerative lesions (characterized
by the presence of giant spermatid cells) in the testes of high-dose males (see Table B-8). The
latter effect was accompanied by observations of hypospermia in 2/20 high-dose males
(compared to 0/20 controls). In females, a nonsignificant increased incidence of liver
pigmentation was observed in high-dose females (4/20 at the high dose compared to 0/20 for
controls). The incidence and severity of hemosiderosis of the spleen were significantly increased
in all groups of treated females; the incidence and severity of hematopoietic cell proliferation
were significantly increased in mid- and high-dose females only.

The changes in hematology and clinical chemistry parameters were consistent with the
organ weight changes and histopathological effects seen in the liver and spleen. Effects in the
mid- and/or high-dose males and females included decreased RBC count, PCV, and hemoglobin;
increased serum total bilirubin and cholesterol and decreased BUN; increased absolute and
relative liver and spleen weights; and histopathological changes in the liver (pigmentation in
Kupffer cells, centrilobular cytomegaly, and cytoplasmic alterations) and spleen (hemosiderosis
and hematopoietic cell proliferation). Although an increased incidence of minimal hemosiderosis
was also observed in low-dose females, it was not considered to be biologically relevant due to
the absence of hematological changes and hematopoietic cell proliferation suggestive of RBC
destruction and restorative proliferation. The NOAEL and LOAEL values based on biologically
relevant effects are 125 and 250 mg/kg-day, respectively. The administered doses of 0, 125, 250,

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and 500 mg/kg-day correspond to HEDs of 0, 18.3, 36.6, and 73.7 mg/kg-day for males, and 0,
17.4, 34.7, and 70.1 mg/kg-day for females5.

Wilson et al. (1947)

Groups of rats (sex, strain, and number not specified) were administered fluorene (purity
not specified) in the diet for 104 days. Exposure concentrations ranged from 65.74 to
1,060 mg/kg-day (equivalent to approximately 0.062-1.0% [unspecified number of dosage
levels]) in the diet6. There was no indication that a control group was used and the comparison
group for effects reporting in fluorene-treated rats was not specified. Effects reported in fluorene-
treated rats (and the concentrations at which they were observed, when specified) included
yellow staining of the fur near the urethra, significantly decreased growth (at 530 and
1,060 mg/kg-day), increased liver weight (>265.1 mg/kg-day), decreased spleen weights (all
concentrations), and decreased testes weight (at 1,060 mg/kg-day). It was indicated that animals
appeared normal, and that there were no significant changes in gross or microscopic pathology.
The lack of detailed reporting precludes the identification of effect levels for this study;
accordingly, it is not summarized in Table 3.

Chronic Studies

Wilson et al. (1947)

Wilson et al. (1947) reported a second experiment in which groups of rats (sex, strain,
and number not specified) were administered fluorene (purity not specified) at 0.125, 0.25, or
0.5% in the diet for 453 days. These concentrations are equivalent to approximately 104.7, 209.5,
and 419 mg/kg-day, respectively7. There was no indication that a control group was used. No
treatment-related effects on body weight or gross pathology were observed (data not shown).
Fluorene-treated rats showed yellow staining of the fur and histological effects in the lung
(inflammation and/or metaplasia of the bronchial epithelium; not considered by the study authors
to be treatment-related), heart (pericarditis), urinary bladder (worms), and testes (moderate
atrophy). A small benign tubular adenoma of the kidney was also noted in one of the low-dose
animals. The lack of detailed reporting precludes the identification of effect levels for this study;
accordingly, it is not summarized in Table 3.

Morris et al. (1960)

Groups of Buffalo strain rats (18-20 females/group) were administered fluorene (stated
to be "highly purified") at 0.05% in the diet for 6 months (diet low in protein and fat and
containing 3% propylene glycol) or 18 months (diet containing natural foodstuffs and 3% corn
oil) (Morris et al.. 1960). The study authors reported that the daily average intakes and the total
intakes of fluorene per rat were 4.3 and 796 mg, respectively, for the 6-month study and 4.6 and

5Administered doses were converted to HEDs by multiplying by DAFs of 0.146, 0.146, and 0.147 for low-, mid-,
and high-dose males and 0.139, 0.139, and 0.140 for low-, mid-, and high-dose females calculated as follows:
DAF = (BWa1/4 ^ BWh1/4), where BWa = animal body weight, and BWh = human body weight. Study-specific TWA
animal body weights of 0.032, 0.032, and 0.033 kg for low-, mid-, and high-dose males, respectively, and 0.026,
0.026, and 0.027 kg for low-, mid-, and high-dose females, respectively, were used (U.S. EPA. 19881. For humans,
the reference value of 70 kg was used for body weight, as recommended by U.S. EPA (1988).

' Dose estimates were calculated using reference values for food consumption and body weight (U.S. EPA. 19881.
The reference body weight and food consumption values for male and female rats (unspecified strain, averaged
across sexes) in a subchronic study are 0.152 kg and 0.0161 kg/day, respectively.

Dose estimates were calculated using reference values for food consumption and body weight (U.S. EPA. 1988).
The reference body weight and food consumption values for male and female rats (unspecified strain, averaged
across sexes) in a chronic study are 0.305 kg and 0.0255 kg/day, respectively.

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2,553 mg, respectively, for the 18-month study. These daily intakes are equivalent to
approximately 18.8 mg/kg-day for the 6-month study and 20.1 mg/kg-day for the 18-month
study8. Two groups of control animals were fed diets containing 3% propylene glycol or 3% corn
oil for about 12 months. The age of animals at study initiation was variable (0.9 and 3.0 months
in the fluorene-treated groups; 1.8 and 3.5 months in the control groups). Animals were
monitored daily for mortality and clinical signs of toxicity. Body weights and food consumption
were measured weekly. At study termination, all animals were subjected to necropsy; tissues
(number not specified) were examined microscopically in 11/20 animals from the 6-month study
and 18/18 animals from the 18-month study (as the tissues of these animals were deemed
"satisfactory for microscopic examinations").

No data for mortality, clinical signs of toxicity, body weights, food consumption, or gross
pathology were reported (Morris et al.. 1960). Non-neoplastic lesions in rats treated with
fluorene for 6 months included acanthosis and hyperkeratosis of the forestomach (incidence:
5/11), squamous metaplasia of the kidney (incidence: 7/11) and uterus (incidence: 1/11),
epithelial ulcer of the small intestine (incidence: 1/11), and cirrhosis of the liver (incidence:
3/11). Neoplastic lesions in rats treated for 6 months were squamous cell carcinomas of the
kidney (incidence: 1/11) and ureter (incidence: 1/11). Non-neoplastic lesions in rats treated for
18 months were limited to hyperplasia of the pituitary (incidence: 2/18). Neoplastic lesions in
rats treated for 18 months were fibrosarcoma of the uterus (incidence: 1/18), carcinosarcoma of
the uterus (incidence: 1/18), granulocytic leukemia of the reticuloendothelial system (incidence:
1/18), and adenoma of the pituitary (incidence: 4/18). None of these lesions were reported in
controls. Lesions seen in the 6-month study were not observed in the 18-month study; it is
possible that these effects might be attributed to the composition of the diet and/or the vehicle
used (propylene glycol or corn oil). Limitations associated with the study (including incomplete
data reporting, lack of concurrent control groups, and confounding factors such as the
vehicle/diet and the age of the animals at study initiation) precludes the identification of effect
levels for this study; accordingly, it is not summarized in Table 3.

Reproductive and Developmental Studies

No studies were identified.

2.2.2. Inhalation Exposures

The noncancer inhalation toxicity database of fluorene is limited to a short-term study
that evaluated effects on behavior in fluorene-exposed rats (Peiffer et al.. 2013).

Short-Term Studies

Peiffer et al. (2013)

In a published, peer-reviewed study that adhered to OECD guidelines (OECD Test
Guideline [TG] 403), male Wistar Han rats (18/group, aged 8-9 weeks) were exposed nose-only
to fluorene (purity >99%) as a vapor at target concentrations of 0 (air control), 1.5, or 150 ppb (0,
0.010, or 1.02 mg/m3)9 6 hours/day, 7 days/week, for 2 weeks. To discriminate between
stress- and treatment-related effects, two control groups were employed. One group of controls
was exposed via inhalation tubes (restrained controls); the other was exposed in chambers that

8Dose estimates were calculated using a reference value for body weight (U.S. EPA. 19881. The reference body
weight value for female rats (unspecified strain) in a chronic study is 0.229 kg.

9Values in the study report were given in ppb. Values in mg/m3 = exposure in ppm x molecular weight (MW) of
fluorene 24.45. The MW of fluorene is 166.22 g/mol (U.S. EPA. 2021b).

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permitted movement (freely moving rats). Rats designated to the restrained control and fluorene-
exposed groups were habituated to exposure conditions for 8 days prior to initiation of the study.
Parameters measured to evaluate the stress response in control rats included body weights, blood
corticosterone levels (at four time points: prior to habituation, on the day prior to initiation of
exposure, after 7 days of exposure, and after 14 days of exposure), and brain, liver, and adrenal
gland weights (after 14 days of exposure). During each exposure period, fluorene concentrations,
temperature, and relative humidity were monitored. Following the last exposure, 12 rats/group
were used for neurobehavioral analyses (conducted during the dark phase of the circadian cycle);
the remaining 6 rats/group were used for measurements of blood and brain levels of fluorene and
mono-hydroxylated metabolites. In addition to the open-field test to evaluate locomotor activity,
the elevated-plus maze to evaluate anxiety, and the eight-arm maze to evaluate learning and
spatial memory (3 days of acclimatization and 12 days of testing), performed as described by
(Peiffer et al.. 2016) in Section 2.2.1, short-term memory was evaluated in the Y-maze. The
parameters measured in the Y-maze (a maze formed by three arms) included the percent
spontaneous alternation (as a measurement of working memory) and numbers of total arm
entries, arms visited per minute, and arm entries in the first minute (as measurements of activity).

Based on data presented for 6 or 12 animals/group, it was presumed that no mortality
occurred at any of the dosing levels (Peiffer et al.. 2013). No fluorene was detectable in the
control chambers (both control groups). Measured fluorene concentrations were 1.30-1.60 ppb
(0.00884-0.0109 mg/m3) in the low exposure group and 144.3-157.2 ppb (0.9810-1.069 mg/m3)
in the high-exposure group. Measured fluorene concentrations in the low-exposure group did not
vary significantly from the target concentration; however, measured fluorene concentrations at
the high concentration were slightly higher than the target concentration on Day 1 and slightly
lower than the target concentration on Day 4 (see Table B-9). During exposure, temperature and
relative humidity measurements did not differ significantly from target values (22 ± 1°C and
55 ± 10%, respectively).

Most physiological parameters used to evaluate restraint stress did not vary significantly
among freely moving rats and restrained controls (i.e., blood corticosterone levels and relative
brain, liver, and adrenal gland weights) (Peiffer et al.. 2013). The body weights of restrained
controls were slightly, but statistically significantly, decreased at three of the four measured time
points (4-6% lower than freely moving controls) (see Table B-10). Data for body and organ
weights in fluorene-exposed rats were not reported. Anxiety and memory-related behaviors were
reportedly unaffected by restraint stress (presumably based on results from the elevated-plus and
eight-arm mazes; data not shown). Restrained controls showed a significant increase in the total
number of crossed squares in the open-field test (27% higher than freely moving controls) and
the total arms visited in the Y-maze (29% higher than freely moving controls). The study authors
reported that these results indicated that restrained controls displayed a nonspecific increase in
activity compared to freely moving controls in the absence of detrimental effects on
physiological parameters, anxiety, or learning.

The results of neurobehavioral tests in fluorene-exposed rats compared to freely moving
controls were not provided (Peiffer et al.. 2013). Compared to restrained controls, rats exposed to
fluorene (both exposure groups) showed significantly increased numbers of crossed squares in
the central (unprotected) area of the open-field test (78—87% higher than controls); rats exposed
at the low concentration also spent significantly more time in the central area (69% higher than
controls) (based on analyses using the MATLAB tool, GRAB IT; see Table B-l 1). The study

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authors reported that these results were indicative of decreased anxiety in fluorene-exposed rats.
However, these effects were not dose-related, and there were no statistically significant effects
on other open-field test parameters (including the total number of crossed squares). In the
elevated-plus maze, another test that evaluated anxiety, rats exposed to fluorene spent
significantly more time in the central (decision-making) area compared to restrained controls
(20-25% higher than controls); a decreased percentage of head dipping in the open area was also
reported (p = 0.08). These effects occurred in the absence of significant differences in open and
closed arm times, total head dipping, or other parameters (i.e., providing no evidence of
decreased anxiety in fluorene-exposed rats). In the eight-arm maze (data not shown), reductions
in the total time required to visit all arms and total numbers of arm entries and significant
increases in arm entries before first error and numbers of arms visited per minute during the
exposure period (i.e., based on time of testing from Day 1 to 12) were observed in both
restrained controls and fluorene-exposed rats; there were no significant differences among
exposed rats and controls. Results from the Y-maze test showed that the percentage of
spontaneous alteration was not significantly impacted by exposure; activity parameters were
likewise unaffected. Although standard toxicological endpoints (e.g., body weights, clinical
chemistry parameters, organ weights, and gross or microscopic pathology) were not measured in
fluorene-exposed rats, behavioral endpoints were evaluated. A free-standing NOAEL of 1 mg/m3
(the highest tested concentration) was identified based on no effects on behavioral endpoints in
male rats. The concentrations of 0, 0.010, and 1.02 mg/m3 correspond to human equivalent
concentrations (HECs) of 0, 0.003, and 0.3 mg/m3, respectively10.

Subchronic Studies

No studies were identified.

Chronic Studies

No studies were identified.

Reproductive and Developmental Studies

No studies were identified.

2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)

Tables 5 A and 5B provide overviews of other supporting studies of fluorene and
metabolism/toxicokinetic studies of fluorene, respectively.

"The HEC was calculated using the equation for extrarespiratory effects from a Category 3 Gas (U.S. EPA. 19941:
HECer = continuous concentration in mg/m3 x ratio of animal:human blood gas partition coefficients. A default
value of 1 applied was applied due to the lack of data for partition coefficients for fluorene in humans and rats.

Fluorene

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Table 5A. Other Studies

Test

Materials and Methods

Results

Conclusions

References

Supporting animal studies

Acute i.p. exposure

Not reported.

Mouse LD5o >2,000 mg/kg (no further details
were available).

The acute toxicity of fluorene is low
via the i.p. route of exposure.

NLM (2021)

Subchronic i.p.
exposure

Male Wistar rats (eight/group)
were exposed to fluorene via
gavage at 0, 1, 10, or 100 mg/kg-d
for 60 d. Body weights were
measured regularly (reported
through Study Day 28). On Study
Days 28-60, rats were subjected
to behavioral tests (open field test
to evaluate motor activity,
elevated-plus maze to evaluate
anxiety, and an eight-arm maze
and TESLA to evaluate learning
and memory). Brain and liver
weights were recorded.

Rats treated at the high dose lost weight
during the first week of treatment and showed
decreased body-weight gain (approximately
5%) relative to controls through Day 28.

Rats treated at the low and mid dose showed
decreased anxiety (based on significantly
more time spent in the central unprotected
area of the elevated-plus maze). Locomotor
activity and learning ability were unaffected
by treatment.

Relative liver weight was biologically and
statistically significantly increased (by
approximately 30% as estimated from
graphical data; p < 0.01) at the high dose
only. Relative brain weight was not
significantly impacted by treatment.

Low doses of fluorene decreased
anxiety, with no effect on motor
activity or learning; the highest dose
of fluorene caused decreased body
weight gain and increased relative
liver weight.

Peiffer et al. (2016)

Mode of action/mechanistic

Inovo

White leghorn chicken eggs
(n >10/group) were exposed to
fluorene (total dose: 1.36 mg/egg
or 300 mg/kg) via three daily
injections into the air sac on
Days 9-11 of incubation.
Viability, fetal weights, liver
weights, and liver histopathology
were evaluated at study
termination (Day 12 or 18).

Viability was 93% in the fluorene-treated
group. No significant effects on fetal weights,
absolute or relative fetal liver weights, or
liver histology were observed on Days 12 or
18 relative to control groups.

No developmental anomalies were
observed in chickens treated in ovo
with fluorene.

latroooulos et al.
(2017)

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Table 5A. Other Studies

Test

Materials and Methods

Results

Conclusions

References

Cytotoxicity

HepG2 cells were exposed to
fluorene at 0, 25, or 50 ng/mL for
3 d with or without metabolic
activation. Cytotoxicity was
measured via neutral red assay.

Cytotoxicity was 115 and 108% of controls at
25 and 50 |ig/mL. respectively, in the absence
of activation and 105 and 101% of controls at
25 and 50 ng/mL, respectively, in the
presence of activation.

Fluorene was not cytotoxic in a
human liver tumor cell line.

Babichetal. (1988)



Cytotoxicity

Rat hepatocytes were exposed to
fluorene at 10 4 M. Cell damage
was measured as a function of
LDH levels.

LDH activity was increased by about 20%
relative to controls; variation of up to 15%
was observed in duplicate cultures.

Fluorene induced little cytotoxicity in
rat hepatocytes.

Mcciueen and

Williams (19821



CYP induction

CYP1A1 induction (as measured
by EROD) was evaluated in rat
hepatocytes.

No CYPlAl-catalzyed EROD activity was
observed in fluorene-treated hepatocytes.

Fluorene was not an inducer of
CYP1A1 in rat hepatocytes.

Till et al. (1999)



CYP = cytochrome; EROD = 7-ethoxyresorufin-O-deethylase; i.p. = intraperitoneal; LD5o = median lethal dose; LDH = lactate dehydrogenase; TESLA = Test
d'Evitement d'un Stimulus Aversif (avoidance test of an adverse light stimulus).

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Table 5B. Metabolism and Toxicokinetic Studies

Test

Materials and Methods

Results

Conclusions

References

Human studies

Human (dietary)

The excretion half-times of
mono-hydroxylated metabolites were
determined in nine nonsmoking
volunteers, aged 23-61 yr (no
occupational exposure to PAHs),
administered a PAH-containing
lunch. Urine samples were collected
15 h before exposure to 60 h after
exposure.

Urinary 9-OH fluorene, 3-OH fluorene, and

2-OH	fluorene were increased by a median of 27-,
36-, and 28-fold, respectively, from pre-exposure
to post-exposure. The observed time to reach
maximum urinary fluorene metabolite
concentrations (Wax) was 3.8-3.9 h after exposure.
Modeled median elimination half-time values were
3.1, 6.1, and 2.9 hfor 9-OH fluorene,

3-OH	fluorene, and 2-OH fluorene, respectively.

Urinary levels of OH metabolites
increased rapidly after exposure and
returned to background levels 24-48 h
after exposure.

Li et al. (2012)

Human (dietary,
inhalation, dermal)

Twenty male subjects were exposed
to PAHs via outdoor barbeque for
2.5 h. Dietary, inhalation, and dermal
exposures occurred via ingestion of
grilled food, intake of atmospheric
PAHs, and absorption of PAH fumes
from exposed skin and clothing,
respectively. Subjects were divided
into three groups: Group A = dietary,
dermal, and inhalation exposure
(cooked and ate barbeque; n = 7),
Group B = dermal and inhalation
exposure (cooked but did not eat
barbeque; n = 7), and
Group C = dermal absorption only
(cooked barbeque while wearing
protective hood; n = 6). Food,
clothing, and air samples were
collected and analyzed for PAH.
Urine samples, collected prior to
exposure and for 35 h following
exposure, were evaluated for
hydroxylated metabolites (including
2-OH fluorene).

Peak levels of OH metabolites in the urine were
seen within 10 h of exposure, declining to initial
levels within 24 h. Urinary levels of OH
metabolites were increased the most by dietary
exposure (Group A-Group B), but also increased
by dermal and/or inhalation exposure (Groups B
and C). Ratios of excretion to intake via the
dietary, dermal, dermal + inhalation, and inhalation
routes were 0.38, 0.11, 0.11, and 0.097,
respectively. Dermal absorption was estimated by
the study authors to account for a higher proportion
of dermal + inhalation intake than inhalation (61%,
compared to 39% via inhalation).

Urinary levels of PAH metabolites
(including 2-OH fluorene) increased
rapidly in the urine and returned to
initial levels within 24 h after
exposure. Ratios of excretion to intake
provided evidence for the highest
availability of PAHs (including
fluorene) from dietary exposure;
availability was higher via dermal
absorption than inhalation exposure for
low molecular weight PAHs (including
fluorene).

Lao et al.
(2018)

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Table 5B. Metabolism and Toxicokinetic Studies

Test

Materials and Methods

Results

Conclusions

References

Human (inhalation
and/or dermal)

Firefighters (37 males and 4 females)
responding to controlled residential
fires were monitored for urinary
levels of PAH metabolites (including
hydroxylated fluorenes) at pre- and
post-firefighting time points.

Median urinary concentrations of summed
hydroxylated fluorenes increased significantly at
the post-firefighting time point (relative to the
pre-firefighting time point); peak excretion
occurred 3 h after exposure.

Urinary PAH metabolite
concentrations (including fluorene
metabolites) increased rapidly after
exposure.

Dermal absorption likely contributed
to fluorene exposure, as workers
protected their airways using SCBA.

Fent et al.
(2019)

Human (inhalation
and/or dermal)

Levels of mono-hydroxylated
metabolites of fluorene
(2-OH fluorene, 3-OH fluorene, and
9-OH fluorene) were measured in the
urine of six firefighting instructors
who completed five 2-h training
sessions.

Concentrations of summed fluorene metabolites in
the urine increased after training sessions, peaking
at approximately 1 h (9-OH fluorene) or 3 h (2-OH
fluorene and 3-OH fluorene) after the end of
training. Estimated elimination half-lives for
2-OH fluorene, 3-OH fluorene, and 9-OH fluorene
were 4.8, 9.3, and 3.5 h, respectively.

Fire training is associated with rapid
uptake of PAHs, including fluorene.
Based on SCBA use, dermal
absorption is presumed to be a
significant route of exposure.

Rossbach et al.

(2020)

Human
(unspecified)

Human tissue samples (brain, liver
kidney, lung, heart, spleen, and
abdominal fat), obtained from eight
cadavers at autopsy, were evaluated
for levels of PAHs, including
fluorene.

Fluorene was detected in 88% of liver, lung, and
abdominal fat samples and 100% of brain, kidney,
heart, and spleen samples. Concentrations of
fluorene were highest in abdominal fat > heart >
brain > kidney > liver > lung > spleen.

PAHs, including fluorene, are
distributed throughout the body and
accumulate preferentially in fatty
tissues.

Pastor-Belda et

al. (2019)

Human
(unspecified)

Urinary levels of mono-hydroxylated
PAH metabolites (including 2-OH
fluorene) were measured in
218 children (aged 3 yr) in Krakow,
Poland.

2-OH fluorene was present in nearly all samples.
Higher 2-OH fluorene concentrations were
associated with exposure to environmental tobacco
smoke and gas-based appliances.

Monitoring urinary 2-OH fluorene can
be used to evaluate fluorene exposure.

Sochacka-
Tatara et al.
(2018)

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Table 5B. Metabolism and Toxicokinetic Studies

Test

Materials and Methods

Results

Conclusions

References

Animal studies

Short-term
inhalation exposure

Male Wistar Han rats (18/group)
exposed to fluorene nose-only as a
vapor at 0, 1.5, or 150 ppb (0, 0.010,
or 1.02 mg/m3) 6 h/d, 7 d/wk for
2 wk. At the end of the 2-wk
exposure period, blood and brain
levels of fluorene, 9-OH fluorene,
3-OH fluorene, and 2-OH fluorene
were measured in six rats/group.

Blood (plasma):

•	Mean levels of fluorene were similar to controls
(background) in all treated groups.

•	2-OH fluorene was significantly increased at
both exposure concentrations.

•	9-OH fluorene and 3 -OH fluorene were
significantly increased at the high concentration.

Brain:

•	Mean fluorene levels were significantly
decreased at both exposure concentrations.

•	9-OH fluorene and 2-OH fluorene were
significantly increased at the high concentration.

•	3-OH fluorene was not detected (in exposed rats
or controls).

Hydroxylated metabolites of fluorene
were detected in a dose-related manner
in the blood and brain of rats following
inhalation exposure.

Peiffer et al.
(2013)

Subchronic oral
(gavage) or i.p.
exposure

Male Wistar rats (eight per group)
were exposed to fluorene via gavage
or i.p. injection at 0, 1, 10, or
100 mg/kg-d for 60 d. At the end of
the 60-d exposure period, plasma and
brain tissue were evaluated for levels
of fluorene, 9-OH fluorene,
2-OH fluorene, and 3-OH fluorene.

Gavage-

Blood (plasma):

•	Plasma fluorene levels were similar to controls
(background) in all treated groups.

•	Levels of all three metabolites were significantly
increased in all treatment groups in a dose-
related manner.

Brain:

•	Fluorene and 2-OH fluorene were significantly
increased in all treated groups (and

9-OH fluorene in the mid- and high-dose
groups) in a dose-related manner.

•	Levels of 3-OH fluorene were below the limit of
detection in all groups (including controls).

Fluorene and/or its hydroxy lated
metabolites were detected in a dose-
related manner in the blood and/or
brain of rats following i.p. or gavage
exposure.

Peiffer et al.
(2016)

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Table 5B. Metabolism and Toxicokinetic Studies

Test

Materials and Methods

Results

Conclusions

References





Injection-

Blood (plasma):

•	Plasma fluorene levels were significantly
increased at the high dose.

•	2-OH fluorene and 3 -OH fluorene were
significantly increased at all doses (and

9-OH fluorene was significantly increased at the
mid and high dose) in a dose-related manner.

Brain:

•	Fluorene levels were significantly increased at
the mid and high dose (dose-related).

•	2-OH fluorene and 9-OH fluorene levels were
significantly increased at all doses (dose-
related).

•	3-OH fluorene was not detected (in treated rats
or controls).





In vitro

Rat liver
microsomes

Rat liver preparations (S9) were
incubated with fluorene at 37°C for
20 min; metabolites were identified
by UV spectra, mass spectrometry,
and comparison to reference
standards.

Metabolites of fluorene included 9-fluorenol
(9-OH fluorene), 1-OH fluorene, and 9-fluorenone.
Fluorene also reacts with oxygen to form a
hydroperoxide.

Metabolites of fluorene include
1-OH fluorene, 9-OH fluorene,
9-fluorenone, and hydroperoxides. It is
unknown if these hydroperoxides are a
direct or indirect product of
metabolism.

I ARC (1983):
LaVoie et al.
(1981)

1-OH fluorene = 1-hydroxyfluorene; 2-OH fluorene = 2-hydroxyfluorene; 3-OH fluorene = 3-hydroxyfluorene; 9-OH fluorene = 9-hydroxyfluorene;
i.p. = intraperitoneal; PAH = polycyclic aromatic hydrocarbon; SCB A = self-contained breathing apparatus; UV = ultraviolet.

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2.3.1.	Supporting Animal Studies

The database of supporting animal studies for fluorene comprises two intraperitoneal
(i.p.) exposures: one acute (Nl.M. 2021) and one subchronic (Peiffer et al.. 2016) (see Table 5A).
The acute toxicity of fluorene is low via the i.p. route of exposure (based on a mouse median
lethal dose [LD50] >2,000 mg/kg) (Nl .M. 2021). No further information on this study or other
acute toxicity studies was located.

A subchronic toxicity study conducted via the i.p. route of exposure showed effects
consistent with those observed in the subchronic gavage toxicity study that was run in parallel
(Peiffer et al. 2016). As in the gavage study, male rats treated via i.p. injection showed evidence
of decreased anxiety at low doses (at 1 mg/kg-day and at 1 and 10 mg/kg-day, respectively)
without significant effects on learning or memory. However, there were significant
toxicologically relevant systemic effects at the highest dose only (100 mg/kg-day); relative to the
controls, statistically significant reduced weight gain was observed across time points and
relative liver weight was reported to be significantly increased, both statistically and biologically
(14% increase).

2.3.2.	Mode-of-Action/Mechanistic Studies

Table 5A provides an overview of mode of action/mechanistic studies. Few noncancer
mechanistic studies were identified. In studies conducted in human HepG2 cells and rat
hepatocytes, fluorene exhibited little to no cytotoxicity (Babich et al.. 1988; Mcqueen and
Williams. 1982); fluorene also did not significantly induce CYP1A1 in rat hepatocytes (Till et
al .. 1999). Treatment of chicken eggs with fluorene in ovo had no significant effect on viability
and did not induce developmental anomalies (i.e., no effects on fetal weight, absolute or relative
fetal liver weights, or liver pathology were observed) (latropoulos et al.. 2017).

2.3.3.	Metabolism/Toxicokinetic Studies

Table 5B provides an overview of metabolism/toxicokinetic studies of fluorene. PAHs
are absorbed via oral, inhalation, and dermal exposure. Absorption is influenced by the vehicle
of administration and/or the lipophilicity of the compound (ATSDR. 1995). Data for fluorene
(log Kow of 4.18) suggest that it is absorbed rapidly via all routes. Fluorene metabolites have
been detected in the tissues and urine of humans exposed to PAHs via the oral, inhalation, and/or
dermal routes of exposure, with urinary levels peaking within 1-10 hours of exposure, providing
evidence of rapid absorption (Rossbach et al .. 2020; Fent et al .. 2019; Pastor-Belda et al .. 2019;
Lao et al.. 2018; Sochacka-Tatara et al.. 2018; Li et al.. 2012). In most tissues, but especially in
urine, hydroxylated metabolites are present as glucuronide and sulfate conjugates. Based on
ratios of excretion of fluorene metabolites to fluorene intake, absorption and bioavailability of
fluorene are highest via oral (dietary) exposure, followed by dermal exposure, and then
inhalation exposure (l.ao et al.. 2018). Animal studies conducted via oral and inhalation exposure
also showed that absorption occurs via these routes, as fluorene and/or its hydroxylated
metabolites were detected in a dose-related manner in the blood and brain of exposed rats at
study termination (Peiffer et al.. 2016; Peiffer et al.. 2013).

Once absorbed, PAHs are widely distributed to the tissues (ATSDR. 1995). Fluorene was
detected in tissue samples obtained from human cadavers at autopsy. Levels of fluorene were
highest in abdominal fat > heart > brain > kidney > liver > lung > spleen, suggesting that
fluorene, like other PAHs, is widely distributed throughout the body and accumulates
preferentially in fatty tissues (Pastor-Belda et al.. 2019). Animal studies (short-term inhalation

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and subchronic oral and i.p. exposures) in rats detected metabolites of fluorene in the brain,
demonstrating that fluorene and fluorene metabolites distribute to the brain (Peiffer et al.. 2016;

Peiffer et at.. 2013).

Across a wide number of studies, PAH metabolites have been detected in all tissue types
(ATSDR. 1995). Studies in humans and animals show that fluorene is metabolized to mono-
hydroxylated metabolites, including 2-OH fluorene, 3-OH fluorene, and 9-OH fluorene
(Gmcincr et al.. 2002; Grantham. 1963; Dewhurst. 1962; Neish. 1948). These mono-
hydroxylated metabolites have been detected in the urine of humans after oral, inhalation, and/or
dermal exposure (Rossbach et al.. 2020; Lao et al.. 2018; Sochacka-Tatara et al.. 2018; Li et al..

2012).	Rats exposed via oral, inhalation, or i.p. routes of exposure also produced these
metabolites, which were detected in the blood and the brain (Peiffer et al.. 2016; Peiffer et al..

2013).	A study using rat liver preparations showed that, in addition to 2-OH fluorene,
3-OH fluorene, and 9-OH fluorene, 1-OH fluorene, and 9-fluorenone are generated as
metabolites of fluorene after 20 minutes of incubation in vitro; fluorene also reacts with oxygen
to form hydroperoxides (IARC. 1983; I.aVoie et al.. 1981).

PAHs are eliminated via the urine and feces; excretion varies by compound and the route
of exposure (Choi et al .. 2023; Lao et al .. 2018; van Schooten et al .. 1997). The amounts of
mono-hydroxylated PAH metabolites excreted in the urine decrease as molecular weight
increases (ATSDR. 1995). Fluorene is considered a low molecular weight PAH and is excreted
primarily in the urine (Lao et al .. 2018). A study of human subjects exposed via a
PAH-containing lunch showed increased concentrations of mono-hydroxylated fluorene
metabolites in the urine soon after exposure (maximum concentrations were reached within
3.8-3.9 hours after exposure); levels approached background within 48 hours (Li et al.. 2012).
Other human studies showed similar results, with peak urinary levels of metabolites within
1 — 10 hours of exposure, declining to background levels after 24-48 hours (Rossbach et al ..
2020; l ent et al.. 2019; Lao et al.. 2018). Elimination half-lives for 9-OH fluorene, 3-OH
fluorene, and 2-OH fluorene were estimated as 3.1, 6.1, and 2.9 hours, respectively, in one study
in which subjects were exposed by diet (Li et al.. 2012) and 3.5, 9.3, and 4.8 hours, respectively,
in another study in which subjects were exposed primarily by skin contact to fumes (Rossbach et
al.. 2020).n

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3. DERIVATION OF PROVISIONAL VALUES

3.1. DERIVATION OF ORAL REFERENCE DOSES

3.1.1. Derivation of Subchronic Provisional RfD (Subchronic p-RfD)

The database of potentially relevant studies for derivation of an oral subchronic
provisional reference value for fluorene is limited to a published, peer-reviewed, 60-day gavage
study that evaluated behavioral endpoints in male rats (Peiffer et al.. 2016). an unpublished
13-week study that evaluated a comprehensive set of toxicological endpoints in male and female
mice CTRL. 1989). and two subchronic to chronic studies in rats with substantial study
limitations (Morris et al.. 1960; Wilson et al.. 1947).

The Peiffer et al. (2016) and TRL (1989) studies identify the liver as a target of toxicity
following subchronic oral exposure in rodents. Peiffer et al. (2016) observed biologically
significant (>10%), dose-related increases in relative liver weight at >10 mg/kg-day in male rats
that were also statistically significant (p < 0.01). The study did not include investigation of other
liver endpoints. Body-weight gain was significantly reduced at 100 mg/kg-day. The focus of the
study was a neurobehavioral test battery, which evaluated anxiety, motor activity, and learning
and memory; no neurobehavioral or neurotoxic effects relevant to treatment were observed at the
tested doses. TRL (1989) observed statistically and biologically (>10%) significant dose-related
increases in absolute and relative liver weights in male and female mice at >250 mg/kg-day
(p < 0.01). Relative liver weights in both sexes were found to be statistically significant at
125 mg/kg-day (p < 0.05), but the increases at this dose were small (<10%) and not considered
biologically significant. Other changes observed at 250 and/or 500 mg/kg-day in this study
included enlarged and eosinophilic centrilobular hepatocytes and pigmentation reminiscent of
hemosiderin deposition in Kupffer cells (predominantly in males); decreases in RBC count,
PCV, and hemoglobin; increased serum total bilirubin and cholesterol and decreased BUN;
hemosiderosis and hematopoietic cell proliferation in the spleen; and increased absolute and
relative spleen weights. Liver weight was also reportedly increased in rats (sex not specified)
exposed to fluorene in the diet at 265.1 mg/kg-day for 104 days (Wilson et al.. 1947). A study
run in parallel to the 60-day gavage study showed that male rats administered fluorene via
i.p. injection at 100 mg/kg-day for 60 days also had biologically and statistically significantly
increased relative liver weights (Peiffer et al.. 2016).

The Peiffer et al. (2016) and TRL (1989) studies provide dose-response data and, as such,
are considered adequate for quantitative toxicity value derivation. The unpublished study by
TRL (1989) evaluated a comprehensive set of toxicological endpoints in male and female mice
and was used by IRIS to derive a chronic RfD value (U.S. EPA. 1990). However, the NOAEL of
125 mg/kg-day (HEDs = 18.3 and 17.4 mg/kg-day in males and females, respectively) and
LOAEL of 250 mg/kg-day (HEDs = 36.6 and 34.7 mg/kg-day in males and females,
respectively) identified from this study based on liver, hematological, and splenic effects are
1-2 orders of magnitude higher than the NOAEL of 1 mg/kg-day (HED = 0.2 mg/kg-day) and
LOAEL of 10 mg/kg-day (HED = 2.4 mg/kg-day) identified from Peiffer et al. (2016) based on
biologically significant increased relative liver weight in male rats. The relative liver weight data
from Peiffer et al. (2016) are not suitable for benchmark dose (BMD) modeling because variance
data were not presented for the control group. Therefore, a NOAEL (HED) of 0.24 mg/kg-day
for increased relative liver weight in the 60-day gavage study by Peiffer et al. (2016) is selected
as the point of departure (POD) for derivation of the subchronic p-RfD.

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The subchronic p-RfD of 8 x 10 4 mg/kg-day for fluorene is derived by applying a
composite uncertainty factor (UFc) of 300 (reflecting an interspecies uncertainty factor [UFa] of
3, a database uncertainty factor [UFd] of 10, and an intraspecies uncertainty factor [UFh] of 10)
to the selected POD of 0.24 mg/kg-day.

Subchronic p-RfD = POD (HED) UFc

= 0.24 mg/kg-day -^300
= 8 x 10"4 mg/kg-day

Table 6 summarizes the uncertainty factors for the subchronic p-RfD for fluorene.

Table 6. Uncertainty Factors for the Subchronic p-RfD for Fluorene

UF

Value

Justification

UFa

3

A UFa of 3 (10°5) is applied to account for uncertainty associated with extrapolating from animals
to humans when cross-species dosimetric adjustment (HED calculation) is performed.

UFd

10

A UFd of 10 is applied to account for deficiencies in the database. The oral database for fluorene
includes an unpublished 13-wk gavage study in male and female mice evaluating a comprehensive
set of toxicological endpoints; a 60-d gavage study in male rats that evaluated limited standard
toxicological endpoints (body, brain, and liver weights) and performed an extensive
neurobehavioral test battery evaluating anxiety, motor activity, and learning and memory; and two
subchronic to chronic studies in rats with reporting deficiencies (Morris et al. 1960; Wilson et al.
1947). No studies of reproductive or developmental toxicity were located: however, for PAHs with
la rue r databases, reproductive and/or developmental effects have been reported (U.S. EPA. 2017;
EC. 2002; ATSDR. 1995).

UFh

10

A UFh of 10 is applied to account for interindividual variability, in the absence of information to
assess toxicokinetic and toxicodynamic variability of fluorene in humans. As the critical effect in
the principal study was only examined in male rats, it is unclear how these effects may inform
sex-specific human variability and susceptibility.

UFl

1

A UFl of 1 is applied because the POD is a NOAEL.

UFS

1

A UFS of 1 is applied because the subchronic POD was derived from subchronic data.

UFC

300

Composite UF = UFA x UFD x UFH x UFL x UFS.

HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;

NOAEL = no-observed-adverse-effect level; p-RfD = provisional reference dose; PAH = polycyclic aromatic
hydrocarbons; POD = point of departure; UF = uncertainty factor; UFA = interspecies uncertainty factor;
UFc = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor;
UFl = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.

Confidence in the subchronic p-RfD for fluorene is low, as described in Table 7.

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Table 7. Confidence Descriptors for the Subchronic p-RfD for Fluorene

Confidence Categories

Designation

Discussion

Confidence in study

M

Confidence in the orincioal studv (Peiffer et al.. 2016) is medium. The
study examined a limited number of standard toxicological endpoints
(i.e., body, brain, and liver weights) in male rats after 60 d of gavage
exposure. However, an extensive neurobehavioral test battery was
performed and three exposure levels were evaluated, permitting the
identification of both a NOAEL (at the lowest dose) and a LOAEL (at
the mid dose).

Confidence in database

L

Confidence in the database is low. The oral database for fluorene
includes an unpublished 13-wk gavage study in male and female mice
evaluating a comprehensive set of toxicological endpoints; a 60-d
gavage study in male rats that evaluated limited standard toxicological
endpoints (body, brain, and liver weights) and performed an extensive
neurobehavioral test battery evaluating anxiety, motor activity, and
learning and memory; and two subchronic to chronic studies in rats
with rcDortinu deficiencies (Morris et al.. 1960; Wilson et al.. 1947).
No studies of reproductive or developmental toxicity were located;
however, for PAHs with larger databases, reproductive and/or
developmental effects have been reDorted (U.S. EPA. 2017; EC. 2002;
ATSDR. 1995).

Confidence in subchronic
p-RfDa

L

Overall confidence in the subchronic p-RfD is low.

aThe overall confidence cannot be greater than the lowest entry in the table.

L = low; LOAEL = lowest-observed-adverse-effect level; M = medium; NOAEL = no-observed-adverse-effect
level; PAH = polycyclic aromatic hydrocarbon; p-RfD = provisional reference concentration.

3.1.2. Derivation of Chronic Provisional RfD (Chronic p-RfD)

A chronic p-RfD value was not derived because an oral RfD value is available on the
U.S. EPA's IRIS database (U.S. EPA. 19901

3.2.	DERIVATION OF INHALATION REFERENCE CONCENTRATIONS

No subchronic or chronic provisional reference concentration (p-RfC) values can be
derived because the database of fluorene inhalation studies is limited to a 14-day study that
evaluated only behavioral effects in fluorene-exposed rats (Peiffer et al.. 2013). As detailed in
Appendix A, the application of an alternate analogue approach was attempted but screening
p-RfCs could not be derived for fluorene because no candidate analogues with inhalation toxicity
values were identified.

3.3.	SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES

Table 8 presents a summary of noncancer references values.

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Table 8. Summary of Noncancer Reference Values for Fluorene

(CASRN 86-73-7)

Toxicity Type
(Units)

Species/Sex

Critical Effect

p-Reference
Value

POD

Method

POD

(HED/HEC)

UFc

Principal
Study

Subchronic p-RfD
(mg/kg-d)

Rat/M

Increased relative
liver weight

8 x 10-4

NOAEL

0.24

300

Peiffer et al.
(2016)

Chronic p-RfD
(mg/kg-d)

An oral RfD of 0.04 me/ke-d is available on IRIS (U.S. EPA. 1990s).

Subchronic p-RfC
(mg/m3)

NDr

Chronic p-RfC
(mg/m3)

NDr

HEC = human equivalent concentration; HED = human equivalent dose; IRIS = Integrated Risk Information
System; M = male(s); NDr = not determined; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose; RfD = reference dose;
UFC = composite uncertainty factor.

3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR AND PROVISIONAL
CANCER RISK ESTIMATES

A cancer assessment was not performed because a cancer weight-of-evidence (WOE) is
available on the U.S. EPA's IRIS database (U.S. EPA. 1990). which characterized fluorene as
Group D, not classifiable as to human carcinogenicity (based on no human data and inadequate
data from animal bioassays). No newer cancer data were identified.

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APPENDIX A. SCREENING PROVISIONAL VALUES

Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity
Value (PPRTV) assessment, it is inappropriate to derive provisional inhalation toxicity values for
fluorene. However, some information is available for this chemical, which although insufficient
to support derivation of a provisional toxicity value under current guidelines, may be of limited
use to risk assessors. In such cases, the Center for Public Health and Environmental Assessment
(CPHEA) summarizes available information in an appendix and develops a "screening value."
Appendices receive the same level of internal and external scientific peer review as the
provisional reference values to ensure their appropriateness within the limitations detailed in the
document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there could be more uncertainty associated with deriving an appendix screening
toxicity value than for a value presented in the body of the assessment. Questions or concerns
about the appropriate use of screening values should be directed to the CPHEA.

APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH (METHODS)

The analogue approach allows for the use of data from related compounds to calculate
screening values when data for the compound of interest are limited or unavailable. Details
regarding searches and methods for analogue analysis are presented in Wang et al. (2012) and
Lizarraga et al. (2023). Three types of potential analogues (structural, metabolic, and toxicity-
like) are identified to facilitate the final analogue chemical selection. The analogue approach
may or may not be route specific or applicable to multiple routes of exposure. All information is
considered together as part of the final weight-of-evidence (WOE) approach to select the most
suitable analogue both toxicologically and chemically.

An expanded analogue identification approach was developed to collect a more
comprehensive set of candidate analogues for the compounds undergoing U.S. Environmental
Protection Agency (U.S. EPA) PPRTV screening-level assessment. As described below, this
method includes application of a variety of tools and methods for identifying candidate
analogues that are similar to the target chemical based on chemical structure and key features;
metabolic relationships; or related toxic effects and mechanisms of action.

To identify structurally-related compounds, an initial pool of analogues is identified using
automated tools, including ChemlDplus" (Nl.M, 2021). CompTox Chemicals Dashboard (U.S.
EPA. 202 lb), and Organisation for Economic Co-operation and Development (OECD)
Quantitative Structure-Activity Relationship (QSAR) Toolbox (OECD. 2020). to conduct
structural similarity searches. Additional analogues identified as ChemlDplus-related substances,
parent, salts and mixtures, and CompTox-related substances are considered. CompTox
Generalized Read-Across (GenRA) analogues are collected using the methods available on the
publicly available GenRA Beta version, which may include Morgan fingerprints, Torsion
fingerprints, ToxPrints and ToxCast, Tox21, and ToxRef data. For compounds that have very
few analogues identified by structure similarity using a similarity threshold of 0.8 or 80%,
substructure searches in the QSAR Toolbox may be performed, or similarity searches may be
re-run using a reduced similarity threshold (e.g., 70 or 60%). The compiled list of candidate
analogues is batch run through the CompTox Chemicals Dashboard where QSAR-ready

1 'National Library of Medicine (NLM) retired ChemlDplus in December 2022.

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simplified molecular-input line-entry system (SMILES) notations are collected and toxicity data
availability is determined (e.g., from the Agency for Toxic Substances and Disease Registry
[ATSDR], Office of Environmental Health Hazard Assessment [OEHHA], California
Environmental Protection Agency [CalEPA], U.S. EPA Integrated Risk Information System
[IRIS], PPRTV assessments). The batch output information is then uploaded into the Chemical
Assessment Clustering Engine (C hem ACE) (U.S. HP A. 201 la), which clusters the chemicals
based on chemical fragments and displays the toxicity data availability for each candidate. The
ChemACE output is reviewed by an experienced chemist, who narrows the list of structural
analogues based on known or expected structure-toxicity relationships, reactivity, and known or
expected metabolic pathways.

Toxicokinetic studies tagged as potentially relevant supplemental material during
screening were used to identify metabolic analogues (metabolites and metabolic precursors).
Metabolites were also identified from the two OECD QSAR Toolbox metabolism simulators (in
vivo rat metabolism simulator and rat liver S9 metabolism simulator). Targeted PubMed
searches were conducted to identify metabolic precursors and other compounds that share any of
the observed or predicted metabolites identified for the target chemical. Metabolic analogues are
then added to the pool of candidate analogues and toxicity data availability is determined
(e.g., from ATSDR, OEHHA, CalEPA, U.S. EPA IRIS, PPRTV assessments).

In vivo toxicity data for the target chemical (if available) are evaluated to determine
whether characteristic effects associated with a particular mechanism of toxicity was observed
(e.g., cholinesterase inhibition, inhibition of oxidative phosphorylation). In addition, in vitro
mechanistic data tagged as potentially relevant supplemental material during screening or
obtained from tools including GenRA, ToxCast/Tox21, and Comparative Toxicogenomics
Database (CTD) (CTD. 2022; Davis et al.. 2021) were evaluated for this purpose. Data from
CompTox Chemicals Dashboard ToxCast/Tox21 are collected to determine bioactivity of the
target chemical in in vitro assays that may indicate potential mechanism(s) of action. The
GenRA option within the Dashboard also offers an option to search for analogues based on
similarities in activity in ToxCast/Tox21 in vitro assays. Using the ToxCast/Tox21 bioactivity
data, nearest neighbors identified with similarity indices of >0.5 may be considered potential
candidate analogues. The CTD (CTD. 2022; Davis et al.. 2021) is searched to identify
compounds with gene interactions similar to interactions induced by the target chemical;
compounds with gene interactions similar to the target chemical (with a similarity index >0.5)
may be considered potential candidate analogues. These compounds are then added to the pool
of candidate analogues and toxicity data availability is determined (e.g., from ATSDR, OEHHA,
CalEPA, U.S. EPA IRIS, PPRTV assessments).

The application of a variety of different tools and methods to identify candidate
analogues serves to minimize the limitations of any individual tool with respect to the pool of
chemicals included, chemical fragments considered, and methods for assessing similarity.
Further, the inclusion of techniques to identify analogues based on metabolism and toxicity or
bioactivity expands the pool of candidates beyond those based exclusively on structural
similarity. The specific tools described above used for the expanded analogue approach searches
were selected because they are publicly available, supported by U.S. and OECD agencies,
updated regularly, and widely used.

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Analogue Search Results for Fluorene

Candidate analogues for fluorene were identified based on metabolic relationships,
structural relationships, and toxicity/mechanisms/mode-of-action (MOA) relationships. For
candidates identified through these approaches, the U.S. EPA (IRIS and PPRTV assessments),
ATSDR, and CalEPA sources were searched for subchronic, intermediate, and chronic inhalation
toxicity values. No candidate analogues with inhalation toxicity values were identified. Details
are provided below.

Identification of Structural Analogues with Established Toxicity Values

Fluorene is not a member of an existing OECD or New Chemical category. Candidate
structural analogues for fluorene were identified using similarity searches in the OECD Toolbox,
the U.S. EPA CompTox Chemicals Dashboard, and ChemlDplus tools. A total of 210 unique
structural analogues were identified for fluorene in the Dashboard, OECD QSAR Toolbox, and
ChemlDplus (80% similarity threshold).

The list of potential analogues was reviewed by a chemist with expertise in read-across.
Criteria for including candidates were as follows:

•	Includes one, and only one, five-membered ring.

•	Includes no fewer than two, and no more than three, aromatic rings.

•	Does not include any ring substitutions.

•	Deuterated compounds are excluded because the toxicokinetics may differ relative to

compounds that are not deuterated.

Using these criteria, a total of seven candidate structural analogues for fluorene were
identified, as shown in Table A-l; all structural analogues are benzofluorenes. Two CASRNs
(benzo[a]fluorene, CASRN 30777-18-5 and benz.o[c|fluorene, CASRN 30777-20-9) identified as
analogues by the tools appear to represent general structures (systematic names indicating the
location of the hydrogen could not be verified with readily available sources). However, for
completeness, the names and CASRNs were included in searches for toxicity values.

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Table A-l. Candidate Structural Analogues Identified for Fluorene based on

Tools and Expert Judgment

Tool (method)3

Analogue (CASRNs) Selected for Toxicity
Value Searches

Structure

Dashboard (Tanimoto), OECD
Toolbox (Dice), and ChemlDplus
(method not described)

1 lH-Benzo[a]fluorene (CASRN 238-84-6)



1 lH-Benzo[6]fluorene (CASRN 243-17-4)



OECD Toolbox (Dice), and
ChemlDplus (method not described)

Benzo[a]fluorene (CASRN 30777-18-5)

crx9

Benzo[6]fluorene (CASRN 30777-19-6)

O-XO

Dashboard (Tanimoto) and
ChemlDplus (method not described)

7H-Benzo[c]fluorene (CASRN 205-12-9)



ChemlDplus (method not described)

Benzo [c]fluorene (CASRN 30777-20-9)



Benzofluorene (CASRN 61089-87-0)



a80% similarity threshold was applied.

OECD = Organisation for Economic Co-operation and Development.

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No inhalation toxicity values were identified for any of the candidate structural
analogues.

Identification of Toxicokinetic Precursors or Metabolites with Established Toxicity

Values

Metabolites of fluorene identified via incubation with rat liver microsomes include
9-hydroxyfluorene (9-fluorenol), 1-hydroxyfluorene, 9-ketofluorene (9-fluorenone), and
hydroperoxides (IARC. 1983). Predicted metabolites were collected from the OECD QSAR
Toolbox. PubMed searches (searching "fluorene" or "86-73-7" and "metabolite") were
conducted to identify metabolic precursors to fluorene. No metabolic precursors were identified.
PubMed was also searched to identify other compounds that are metabolized to any of the
observed or predicted metabolites of fluorene (searching the metabolite name or [CASRN if
available] and "metabolite"). No compounds that share at least one metabolite with fluorene
were identified in these searches.

Table A-2 summarizes the 12 candidate metabolic analogues for fluorene (4 observed
metabolites and an additional 8 unique predicted metabolites). Searches for relevant toxicity
values for the candidate metabolic analogues of fluorene did not identify inhalation toxicity
values for any of the observed or predicted metabolites.

Table A-2. Candidate Metabolic Analogues of Fluorene

Relationship to Fluorene

Compound

Metabolic precursor

None identified

Metabolite

9-Fluorenol (CASRN 1689-64-1)

1-Hydroxyfluorene (CASRN 6344-61-2)

9-Ketofluorene (9-fluorenone, CASRN 486-25-9)

Hydroperoxides3

3-hydroxyfluorene (CASRN 6344-67-8)

2-hydroxyfluorene (CASRN 2443-58-5)

9H-fluoren-4-ol (CASRN 28147-35-5)

9H-fluorene-2,9-diol (CASRN 106593-45-7)

9H-fluorene-2,3 -diola

9H-fluorene-3,4-diol (CASRN 42523-20-6)

9H-fluorene-1,2-diol (CASRN 42523-11-5)

9H-fluorene-3,9-diol (CASRN 1381944-22-4)

Shares common metabolite(s)

None identified

aCASRN not available for this metabolite.

Identification of Analogues on the Basis of ToxicityZMechanistic/MOA Information
and Established Toxicity Values

Available toxicity and mechanistic data for fluorene were evaluated to determine whether
these data could be used to identify candidate analogues. Animal studies on the toxicity of

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fluorene identified through the literature searches were reviewed to determine whether there
were in vivo toxicity data demonstrating characteristic effects associated with a specific
mechanism of toxicity (e.g., cholinesterase inhibition, inhibition of oxidative phosphorylation)
that could be used to identify candidate analogues. Studies of fluorene exposure by oral
administration (Peiffer et al.. 2016; TRL. 1989) reported liver and spleen effects and hematology
changes, while a short-term inhalation study (Peiffer et al.. 2013) reported a lack of
toxicologically relevant neurobehavioral changes. None of these oral or inhalation studies
indicated a specific mechanism of toxicity that could be used to identify candidate analogues.

Fluorene was active in 28 ToxCast/Tox21, 6 EDSP21, and 9 PubChem bioactivity assays
reported in the Dashboard (invitrodb version 3.3; U.S. EPA. 2020a; U.S. EPA. 2020b). The
GenRA option within the Dashboard offers an option to search for analogues based on
similarities in activity in ToxCast/Tox21 in vitro assays. Using the ToxCast bioactivity data,
none of the nearest neighbors identified by GenRA had similarity indices >0.5 (the highest index
was 0.22 for carbamazepine). Using the Tox21 bioactivity data, only one of the nearest
neighbors identified by GenRA had a similarity index of at least 0.5: 4-vinyl-l-cyclohexene
dioxide (similarity index = 0.53) (U.S. EPA. 2020c). This compound does not have an inhalation
toxicity value from the U.S. EPA (IRIS and PPRTV assessments), ATSDR, or CalEPA.

The CTD identified several compounds with gene interactions similar to interactions
induced by fluorene. In the CTD, similarity is measured by the Jaccard index, calculated as the
size of the intersection of interacting genes for chemical A and chemical B divided by the size of
the union of those genes (range 0 [no similarity] to 1 [complete similarity]). Among the
compounds with gene interactions similar to fluorene, the numbers of common gene interactions
ranged from 3 to 7 and similarity indices ranged from 0.19 to 0.27; the compound with the
highest similarity index (0.27) was Sudan III (CASRN 85-86-9). There were no compounds with
a similarity index >0.5.

The methods outlined above identified only 4-vinyl-l-cyclohexene dioxide as a candidate
mechanistic analogue for fluorene, as shown in Table A-3. However, 4-vinyl-l-cyclohexene
dioxide does not have an inhalation toxicity value.

Table A-3. Candidate Mechanistic Analogues Identified for Fluorene

Tool (method)3

Analogue (CASRNs) Selected for Toxicity Value
Searches

Structure

GenRA (Beta), Biology: Tox21
data

4-Vinyl-l-cyclohexene dioxide (CASRN 106-87-6)

<\0

a50% similarity threshold was applied.

GenRA = Generalized Read-Across.

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Summary

Searches for metabolic, structural, and toxicity/mechanistic analogues for fluorene
yielded a total of 20 unique candidate analogues: 12 metabolites, 7 structural analogues12, and
1 mechanistic analogue. None of the candidate analogues have inhalation toxicity values from
the U.S. EPA, ATSDR, or CalEPA. Therefore, no suitable candidate analogues were identified to
calculate screening inhalation provisional toxicity values.

INHALATION NONCANCER TOXICITY VALUES

Derivation of a Screening Subchronic and Chronic Provisional Reference Concentrations
(p-RfCs)

Subchronic and chronic p-RfCs could not be derived due to the lack of an appropriate
analogue having inhalation toxicity values.

12Although seven unique CASRNs were retrieved during structural similarity searches, two of the CASRNs for
structural analogues appear to represent general structures.

Fluorene

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APPENDIX B. DATA TABLES

Table B-l. Effects in Male Wistar Rats Treated with Fluorene via Gavage

for 60 Days"

ADD [HED] in mg/kg-d

Endpoint

0 [0]

1 [0.24]

10 [2.4]

100 [23.6]

Number evaluated (n)

8

8

8

8

Body-weight gain (% compared
to Day 1)









Day 2

0.60bc

0.35 (+0%)

-0.14 (-1%)

-3.10 (-4%)**

Day 7

2.32

1.83 (+0%)

1.34 (-1%)

-5.56 (-8%)**

Day 14

7.25

7.25 (+0%)

7.25 (+0%)

0.35 (-7%)**

Day 21

12.92

12.43 (+0%)

11.69 (-1%)

5.28 (-8%)**

Day 28

17.61

17.11 (+0%)

16.62 (-1%)

10.21 (-7%)**

Relative liver weight (% body
weight)

2.69b,d

2.84 (+6%)*

3.16 (+17%)**

3.69 (+37%)**

aPeiffer et al. (2016).

bData are means based on graphically reported data extracted using the MATLAB tool GRABIT; variance values
were not extracted.

°Value in parentheses is % change relative to control = control mean - treatment mean (for data reported as %).
dValue in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
* Significantly different from control at (p< 0.05) by Dunnett t-test, as reported by the study authors.
**Significantly different from control at (p < 0.01) by Dunnett t-test, as reported by the study authors.

ADD = adjusted daily dose; HED = human equivalent dose.

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Table B-2. Select Clinical Signs of Toxicity in Crl:CD-l Mice Administered
Fluorene via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Effect

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]



Males

Salivation

1/20 (5%)b

16/20 (80%)*

14/20 (70%)*

19/20 (95%)*

Hypoactivity

1/20 (5%)

2/20 (10%)

3/20 (15%)

17/20 (85%)*

Labored respiration

0/20 (0%)

0/20 (0%)

0/20 (0%)

3/20 (15%)

Ptosis

0/20 (0%)

1/20 (5%)

0/20 (0%)

4/20 (20%)

Urine wet abdomen

0/20 (0%)

1/20 (5%)

2/20 (10%)

3/20 (15%)

Unkempt appearance

0/20 (0%)

3/20 (15%)

0/20 (0%)

4/40 (20%)

Females: ADD [HED] in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]c

Salivation

1/20 (5%)

11/20 (55%)*

14/20 (70%)*

16/20 (80%)*

Hypoactivity

2/10 (10%)

1/20 (5%)

1/20 (5%)

15/20 (75%)*

Labored respiration

0/20 (0%)

0/20 (0%)

0/20 (0%)

3/20 (15%)

Ptosis

0/20 (0%)

0/20 (0%)

0/20 (0%)

0/20 (0%)

Urine wet abdomen

0/20 (0%)

0/20 (0%)

0/20 (0%)

0/20 (0%)

Unkempt appearance

0/20 (0%)

1/20 (5%)

0/20 (0%)

2/20 (10%)

aTRL (1989).

bNumber affected/number examined (% incidence).

Two 500 mg/kg-day females (194 and 195) died during week 1 and were replaced with animals from the satellite
group (199S and 200S); 194/199S and 195/200S were counted as single animals for the purpose of tabulating
incidence data.

* Statistically significant from control (p < 0.05) based on one-tailed Fisher's exact test performed for this review.
ADD = adjusted daily dose; HED = human equivalent dose.

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Table B-3. Food Consumption on Select Weeks in Crl:CD-l Mice
Administered Fluorene via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Endpoint

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]

Food consumption (g/animal/week)

Week 1

30.5 ± 3.29b,c

34.2 ± 2.76 (+12%)*

33.6 + 4.64 (+10%)

31.2 + 5.26 (+2%)

Week 2

29.1 ±4.02

32.2 ±2.92 (+11%)*

35.3+ 7.18 (+21%)**

33.6 + 4.84 (+15%)**

Week 3

28.4 ±3.72

30.9 ± 1.89 (+9%)*

32.3 + 4.54 (+14%)*

31.9 + 2.72 (+12%)**

Week 6

30.5 ±2.60

31.6 ±2.10 (+4%)

32.9 + 2.64 (+8%)*

33.8 + 2.86 (+11%)**

Week 8

31.5 ±3.21

32.7±1.89 (+4%)

32.2 + 3.09 (+2%)

37.7 + 2.41 (+20%)**

Week 9

29.4 ±3.88

32.2 + 2.11 (+10%)*

35.9+ 3.18 (+22%)**

32.0 + 2.79 (+9%)*

Week 10

31.6 ±2.83

32.3 + 1.87 (+2%)

34.8 + 2.91 (+10%)**

35.0 + 2.59 (+11%)**

Week 11

28.7 ±2.53

30.3 + 2.08 (+6%)

32.5 + 2.93 (+13%)**

31.9 + 2.57(+ll%)**

Week 12

31.0 ± 2.31

32.5 + 2.16(+5%)

34.1 + 2.29 (+10%)**

34.3 + 2.85 (+11%)**

Week 13

25.7 ±2.61

26.7 + 1.29 (+4%)

28.3 + 3.51 (+10%)*

28.0 + 2.98 (+9%)

Females: ADD [HED

in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]

Food consumption (g/animal/week)

Week 1

27.6 ±2.16

27.5 + 2.55 (+0%)

29.3 + 3.20 (+6%)

31.8 + 4.67 (+15%)**

Week 2

28.0 ±2.02

30.0 + 3.91 (+7%)

31.6 + 3.28 (+13%)**

33.3 + 4.26 (+19%)**

Week 3

29.5 ±2.48

28.7 + 2.28 (-3%)

29.3 + 2.38 (-1%)

31.8 + 2.53 (+8%)*

Week 6

30.3 ±2.08

31.7 + 2.43 (+5%)

30.8 + 2.51 (+2%)

34.8 + 6.55(+15%)*

Week 8

30.9 ±3.08

31.4 + 2.95 (+2%)

32.0 + 2.52 (+4%)

33.6 + 3.68 (+9%)*

Week 9

29.8 ±3.42

31.7 + 3.43 (+6%)

31.8 + 2.49 (+7%)

32.9 + 3.03 (+10%)*

Week 10

31.7 ±2.80

31.7 + 2.63 (+0%)

33.2 + 2.96 (+5%)

34.1 + 2.38 (+8%)*

Week 11

29.5 ±3.57

29.6 + 2.63 (+0%)

31.1 + 2.77 (+5%)

31.5+1.88 (+7%)

Week 12

31.6 ±2.88

31.3 +2.24 (-1%)

33.8+ 3.16 (+7%)*

34.4+ 2.17 (+9%)**

Week 13

26.5 ±2.99

26.2 + 2.01 (-1%)

27.2 + 2.13 (+3%)

28.1 + 2.19 (+6%)

aTRL (1989).
bData are means ± SD.

°Value in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
* Significantly different from control by Dunnett's test (p < 0.05), as reported by the study authors.
**Significantly different from control by Dunnett's test (p < 0.01), as reported by the study authors.

ADD = adjusted daily dose; HED = human equivalent dose; SD = standard deviation.

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Table B-4. Body-Weight Effects on Select Weeks in Crl:CD-l Mice
Administered Fluorene via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Endpoint

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]

Body weight (g)









Week 1

28.8 ± 2.87b,c

29.8 ± 1.72 (+3%)

29.7 ± 1.88 (+3%)

28.9 ± 2.09 (+0%)

Week 2

29.9 ±2.76

30.2 ± 1.85 (+1%)

30.1 ± 1.83 (+1%)

30.1 ± 1.46 (+1%)

Week 5

31.7 ± 3.17

32.5 ± 1.63 (+3%)

32.7 ± 1.84 (+3%)

32.5 ± 1.69 (+3%)

Week 6

31.8 ± 3.11

32.5 ± 1.81 (+2%)

32.7 ± 1.70 (+3%)

33.2 ± 1.57 (+4%)

Week 7

32.2 ±3.15

33.0 ± 1.71 (+2%)

33.2 ± 1.85 (+3%)

33.7 ± 1.69 (+5%)

Week 9

32.7 ±3.22

33.3 ± 1.81 (+2%)

33.5 ± 1.95 (+2%)

34.3 ± 1.76 (+5%)

Week 10

33.2 ±3.33

33.5 ± 1.72 (+1%)

33.9 ±2.04 (+2%)

34.5 ± 1.79 (+4%)

Week 11

33.7 ±3.38

34.2 ± 1.79 (+1%)

34.4 ±2.19 (+2%)

35.0 ± 1.87 (+4%)

Week 12

33.7 ±3.32

34.2 ± 1.88 (+1%)

34.0 ±2.81 (+1%)

34.7 ± 1.91 (+3%)

Week 13

33.7 ±3.24

34.3 ± 1.86 (+2%)

34.5 ±2.11 (+2%)

34.8 ± 2.30 (+3%)

Body-weight change
(Weeks l-13)d (g)

4.9

4.5 (-8%)

4.8 (-2%)

5.9 (+20%)

Females: ADD [HED] in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]

Body weight (g)









Week 1

23.2 ± 1.80

23.6 ± 1.57 (+2%)

23.3 ± 1.24 (+0%)

23.8 ±2.17 (+3%)

Week 2

23.6 ± 1.73

24.3 ± 1.36 (+3%)

24.1 ± 1.59 (+2%)

25.1 ±2.01 (+6%)*

Week 5

25.6 ± 1.84

26.1 ± 1.76 (+2%)

26.3 ± 1.80 (+3%)

27.6 ±2.11 (+8%)**

Week 6

25.7 ±2.12

26.2 ± 1.44 (+2%)

26.2 ± 1.55 (+2%)

27.6 ± 1.97 (+7%)**

Week 7

26.2 ± 1.99

26.4 ± 1.33 (+1%)

26.6 ± 1.59 (+2%)

27.8 ± 2.09 (+6%)*

Week 9

26.3 ±2.00

27.1 ± 1.68 (+3%)

27.1 ± 1.58 (+3%)

28.3 ± 1.83 (+8%)**

Week 10

27.2 ±2.31

27.0 ± 1.39 (-1%)

27.2 ± 1.68 (+0%)

28.7 ± 2.22 (+6%)*

Week 11

27.5 ±2.40

27.6 ± 1.74 (+0%)

27.9 ± 1.89 (+1%)

29.4 ± 1.96 (+7%)*

Week 12

27.2 ±2.10

27.2 ± 1.68 (+0%)

27.8 ± 1.95 (+2%)

29.2 ± 1.98 (+7%)**

Week 13

27.6 ± 1.97

27.5 ± 1.44 (+0%)

28.1 ± 1.88 (+2%)

29.5 ± 1.94 (+7%)**

Body-weight change
(Weeks l-13)d (g)

4.4

3.9 (-11%)

4.8 (+9%)

5.7 (+30%)

aTRL (1989).
bData are means ± SD.

°Value in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
dBody-weight change calculated for this review as mean body weight at week 13 - mean body weight at Week 1.
* Significantly different from control by Dunnett's test (p < 0.05), as reported by the study authors.
**Significantly different from control by Dunnett's test (p < 0.01), as reported by the study authors.

ADD = adjusted daily dose; HED = human equivalent dose; SD = standard deviation.

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Table B-5. Select Hematological Effects in Crl:CD-l Mice Administered
Fluorene via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Endpoint

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]

Number evaluated (n)

9b

10

10

10

RBC count (xl06)/nL

8.23 +0.53c-d

7.68 ± 0.77 (-7%)

7.52 ± 0.54 (-9%)

6.47±0.65 (-21%)**

PCV (%)

43.9 ±3.60

41.4 ±3.85 (-6%)

39.7 ±3.66 (-10%)*

34.4 ±3.41 (-22%)**

Hemoglobin (g/dL)

13.4 ±0.71

13.4 ±0.79 (+0%)

12.9 ± 0.52 (-4%)

11.3 ±0.90 (-16%)**

MCV (fL)

53.8 ±2.6

54.3 ± 0.9 (+1%)

53.2 ± 1.8 (-1%)

53.7 ± 0.7 (+0%)

MCH (pg)

16.3 ±0.75

17.6 ± 0.94 (+8%)**

17.2 ± 0.90 (+6%)

17.5 + 0.81 (+7%)*

MCHC (g/dL)

30.6 ± 1.78

32.5 ± 1.51 (+6%)

32.6 ± 2.05 (+7%)*

33.0+ 1.40 (+8%)*

WBC count (xl03/nL)

7.0 ±2.37

6.8 ± 1.54 (-3%)

6.2 ± 1.56 (-11%)

7.8 + 3.03 (+11%)

Females: ADD [HED] in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]

Number evaluated (n)

10

10

10

10

RBC count (><106)/nL

8.35 ±0.54

8.01 ±0.73 (-4%)

7.52 ± 0.43 (-10%)*

6.86 + 0.83 (-18%)**

PCV (%)

45.2 ±2.85

43.9 ±5.07 (-3%)

40.7 ± 2.68 (-10%)*

36.8 + 2.66 (-19%)**

Hemoglobin (g/dL)

14.2 ±0.69

14.0 ± 1.01 (-1%)

13.6 ±0.68 (-4%)

12.4 + 0.90 (-13%)**

MCV (fL)

54.5 ± 1.1

55.1 ± 1.7 (+1%)

54.7 ± 1.2 (+0%)

56.3 + 1.3 (+3%)*

MCH (pg)

17.0 ±0.67

17.5 ± 0.72 (+3%)

18.1 ±0.98 (+6%)*

18.3 + 2.32 (+8%)

MCHC (g/dL)

314 ± 1.19

32.1 ± 1.76 (+2%)

33.4 ± 1.70 (+6%)*

33.8+ 1.20 (+8%)**

WBC count (xl03/nL)

6.3 ±2.46

7.0 ± 1.90 (+11%)

7.1 ±1.69 (+13%)

9.2 + 3.62 (+46%)*

aTRL (1989).

bOne specimen clotted and was not used for analysis.

Data are means ± SD.

dValue in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
* Significantly different from control by Dunnett's test (p < 0.05), as reported by the study authors.
**Significantly different from control by Dunnett's test (p < 0.01), as reported by the study authors.

ADD = adjusted daily dose; HED = human equivalent dose; MCH = mean corpuscular hemoglobin;

MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; PCV = packed cell
volume; RBC = red blood cell; SD = standard deviation; WBC = white blood cell.

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Table B-6. Select Clinical Chemistry Effects in Crl:CD-l Mice Administered
Fluorene via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Endpoint

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]

Number evaluated (n)

9b

10

10

10

Cholesterol (mg/dL)

139.9 ±23.87c-d

148.9 ± 13.12 (+6%)

175.5 ± 27.32 (+25%)**

174.4 ± 22.89 (+25%)**

BUN (mg/dL)

24.6 ± 4.08#

20.4 ± 2.75 (-17%)

19.7 ± 5.32 (-20%)*

19.2 ±4.11 (-22%)*

Total bilirubin (mg/dL)

0.28 ± 0.10#

0.20 ±0.10 (-29%)

0.33 ±0.10 (+18%)

0.45 ±0.09 (+61%)**

ALP (U/L)

38.8 ± 10.18

43.9 ± 17.07 (+13%)

43.5 ±38.64 (+12%)

36.3 ± 16.56 (-6%)

ALT (U/L)

24.0 ±6.10

20.6 ± 5.22 (-14%)

21.4 ±5.27 (-11%)

25.6 ± 14.66 (+7%)

AST (U/L)

72.2 ±30.25

43.7 ±6.21 (-39%)

56.1 ±21.44 (-22%)

50.2 ± 14.10 (-30%)

LDH (U/L)

121.9 ±88.44

84.3 ±21.92 (-31%)

84.5 ±26.98 (-31%)

89.1 ± 47.70 (-27%)

Potassium (meq/L)

10.12 ± 1.12

9.86 ± 1.35 (-3%)

10.07 ± 1.50 (+0%)

8.76 ± 0.84 (-13%)



Females: ADD [HED] in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]

Number evaluated (n)

10

9b

10

9b

Cholesterol (mg/dL)

104.2 ±27.38

124.8 ± 32.17 (+20%)

143.6 ±25.34 (+38%)*

162.4 ± 33.22 (+56%)**

BUN (mg/dL)

20.1 ±5.10#

17.0 ± 2.42 (-15%)

17.0 ±3.19 (-15%)

15.3 ± 1.65 (-24%)*

Total bilirubin (mg/dL)

0.21 ±0.10#

0.23 ±0.11 (+10%)

0.27 ±0.12 (+29%)

0.36 ±0.08 (+71%)*

ALP (U/L)

54.9 ± 10.00

43.0 ± 10.18 (-22%)*

47.2 ± 8.66 (-14%)

39.1 ±8.99 (-29%)**

ALT (U/L)

19.1 ±4.40

19.4 ±3.13 (+2%)

21.8 ±5.59 (+14%)

24.9 ±5.16 (+30%)*

AST (U/L)

60.6 ± 20.72

53.3 ± 12.50 (-12%)

56.8 ± 15.93 (-6%)

55.5 ± 10.48 (-8%)

LDH (U/L)

122.6 ± 56.72

100.7 ± 17.18 (-18%)

89.8 ±31.64 (-27%)

86.0 ± 36.52 (-30%)

Potassium (meq/L)

10.00 ±0.95

9.57 ± 1.01 (-4%)

9.41 ± 0.64 (-6%)

8.65 ± 0.95 (-14%)**

aTRL (1989).

bA specimen could not be obtained from one animal.

Data are means ± SD.

dValue in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
* Significantly different from control by Dunnett's test (p < 0.05), as reported by the study authors.
**Significantly different from control by Dunnett's test (p < 0.01), as reported by the study authors.

"Significant trend test (not further specified; p < 0.01), as reported by the study authors.

ADD = adjusted daily dose; ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate
aminotransferase; BUN = blood urea nitrogen; HED = human equivalent dose; LDH = lactate dehydrogenase;
SD = standard deviation.

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Table B-7. Select Organ Weights in Crl:CD-l Mice Administered Fluorene

via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Endpoint

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]

Number evaluated
(«)

19b

20

20

20

Necropsy body
weight (g)

33.6 ± 3.22c,d

34.1 ± 1.86 (+1%)

34.5 ±2.13 (+3%)

34.8 ± 2.09 (+4%)

Absolute liver (g)

1.689 ±0.223

1.838 ±0.146 (+9%)

2.028 ± 0.242 (+20%)**

2.288 ±0.219 (+35%)**

Relative liver
(% body weight)

5.020 ±0.413

5.389 ±0.313 (+7%)*

5.865 ± 0.442 (+17%)**

6.588 ±0.601 (+31%)**

Absolute kidney (g)

0.608 ±0.108

0.628 ± 0.052 (+3%)

0.658 ± 0.072 (+8%)

0.681 ±0.077 (+12%)*

Relative kidney
(% body weight)

1.807 ±0.253

1.843 ±0.160 (+2%)

1.908 ±0.173 (+6%)

1.958 ±0.180 (+8%)*

Absolute spleen (g)

0.084 ± 0.029

0.083 ±0.018 (-1%)

0.110 ±0.025 (+31%)*

0.173 ±0.044
(+106%)**

Relative spleen
(% body weight)

0.249 ± 0.075

0.244 ± 0.049 (-2%)

0.318 ±0.074 (+28%)*

0.497 ±0.13 (+99%)**

Absolute brain (g)

0.501 ±0.027

0.498 ± 0.024 (-1%)

0.512 ±0.036 (+2%)

0.505 ± 0.024 (+1%)

Relative brain
(% body weight)

1.501 ±0.130

1.466 ±0.117 (-2%)

1.487 ±0.100 (-1%)

1.457 ±0.10 (-3%)



Females: ADD [HED] in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]

Number evaluated
(«)

19b

19b

20

19b

Necropsy body
weight (g)

27.3 ± 1.86

27.4 ± 1.93 (+0%)

27.9 ± 2.09 (+2%)

29.4 ± 2.00 (+8%)**

Absolute liver (g)

1.371 ±0.167

1.490 ±0.170 (+9%)

1.708 ±0.223 (+25%)**

1.986 ±0.195 (+45%)**

Relative liver
(% body weight)

5.025 ±0.439

5.432 ± 0.450 (+8%)*

6.101 ±0.465 (+21%)**

6.759 ± 0.374 (+35%)**

Absolute kidney (g)

0.414 ±0.032

0.418 ±0.048 (+1%)

0.387 ± 0.074 (-7%)

0.433 ± 0.039 (+5%)

Relative kidney
(% body weight)6

1.519 ±0.097

1.524 ±0.134 (+0%)

1.388 ±0.257 (-9%)f

1.474 ±0.101 (-3%)

Absolute spleen (g)

0.091 ±0.028

0.100 ±0.018 (+10%)

0.123 ±0.027 (+35%)**

0.181 ±0.052 (+99%)**

Relative spleen
(% body weight)

0.333 ±0.098

0.365 ± 0.068 (+10%)

0.443 ± 0.103 (+33%)**

0.612 ±0.163 (+84%)**

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Table B-7. Select Organ Weights in Crl:CD-l Mice Administered Fluorene

via Gavage for 13 Weeks"

Absolute brain

0.507 ± 0.040

0.507 ± 0.042 (+0%)

0.490 ± 0.028 (-3%)

0.498 ± 0.034 (-2%)

Relative brain
(% body weight)

1.865 ±0.157

1.855 ±0.141 (-1%)

1.764 ±0.157 (-5%)

1.700 ±0.125 (-9%)**

aTRL (1989).

bAn animal died prior to terminal sacrifice (death was attributed by the study authors to gavage error).

Data are means ± SD.

dValue in parentheses is % change relative to control = ([treatment mean - control mean] control mean) x 100.
"Variance data were not legible in the study report. Variance data were calculated for this review from individual
kidney weight data.

Includes data for one animal (141) with kidney weight ~3 times lower than the others in this group (0.13 g,
compared to a range of 0.35-0.49 g for other animals).

* Significantly different from control by Dunnett's test (p < 0.05), as reported by the study authors.
**Significantly different from control by Dunnett's test (p < 0.01), as reported by the study authors.

ADD = adjusted daily dose; HED = human equivalent dose; SD = standard deviation.

Table B-8. Select Histopathological Effects in Crl:CD-l Mice Administered
Fluorene via Gavage for 13 Weeks"

Males: ADD [HED] in mg/kg-d

Effect

0 [0]

125 [18.3]

250 [36.6]

500 [73.7]

Liver









Pigment; Kupffer cells

0/20 (0%)b

0/20 (0%)

0/20 (0%)

12/20 (60%)*

Cytomegaly; centrilobular

0/20 (0%)

0/20 (0%)

3/20 (15%)

14/20 (70%)*

Cytologic alteration; centrilobular

0/20 (0%)

0/20 (0%)

1/20 (5%)

7/20 (35%)*

Spleen; hemosiderosis









Minimal

0/20 (0%)

1/20 (5%)

7/20 (35%)*

3/20 (15%)

Mild

0/20 (0%)

0/20 (0%)

7/20 (35%)*

12/20 (60%)*

Moderate

0/20 (0%)

0/20 (0%)

0/20 (0%)

5/20 (25%)*

Total

0/20 (0%)

1/20 (5%)

14/20 (70%)*

20/20 (100%)*

Spleen; hematopoietic cell proliferation









Minimal

0/20 (0%)

0/20 (0%)

7/20 (35%)*

2/20 (10%)

Mild

0/20 (0%)

0/20 (0%)

1/20 (5%)

14/20 (70%)*

Moderate

0/20 (0%)

0/20 (0%)

0/20 (0%)

2/20 (10%)

Total

0/20 (0%)

0/20 (0%)

8/20 (40%)*

18/20 (90%)*

Testes









Degeneration

0/20 (0%)

0/20 (0%)

0/20 (0%)

5/20 (25%)*

Hypospermia

0/20 (0%)

0/20 (0%)

0/20 (0%)

2/20 (10%)

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Table B-8. Select Histopathological Effects in Crl:CD-l Mice Administered
Fluorene via Gavage for 13 Weeks"

Females: ADD [HED] in mg/kg-d



0 [0]

125 [17.4]

250 [34.7]

500 [70.1]c

Liver









Pigment; Kupffer cells

0/20 (0%)

0/20 (0%)

0/20 (0%)

4/20 (20%)

Cytomegaly; centrilobular

0/20 (0%)

0/20 (0%)

0/20 (0%)

1/20 (5%)

Cytologic alteration; centrilobular

NR

NR

NR

NR

Spleen; hemosiderosis









Minimal

0/20 (0%)

7/20 (35%)*

10/20 (50%)*

3/20 (15%)

Mild

0/20 (0%)

3/20 (15%)

6/20 (30%)*

13/20 (65%)*

Moderate

0/20 (0%)

0/20 (0%)

0/20 (0%)

4/20 (20%)

Total

0/20 (0%)

10/20 (50%)*

16/20 (80%)*

20/20 (100%)*

Spleen; hematopoietic cell proliferation









Minimal

1/20 (5%)

3/20 (15%)

8/20 (40%)*

5/20 (25%)

Mild

0/20 (0%)

0/20 (0%)

3/20 (15%)

13/20 (65%)*

Moderate

0/20 (0%)

0/20 (0%)

0/20 (0%)

2/20 (10%)

Total

1/20 (5%)

3/20 (15%)

11/20 (55%)*

20/20 (100%)*

aTRL (1989).

bNumber affected/number examined (% incidence). Includes animals that died prior to terminal sacrifice.
Two 500 mg/kg-day females (194 and 195) died during week 1 and were replaced with animals from the satellite
group (199S and 200S); 199S and 200S were evaluated for histopathological effects.

* Statistically significant from control (p < 0.05) based on one-tailed Fisher's exact test performed for this review.
ADD = adjusted daily dose; HED = human equivalent dose; NR = not reported.

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Table B-9. Measured Concentrations of Fluorene in a Study of Male Wistar
Han Rats Exposed Nose-Only to Fluorene for 14 Days"

Target concentration in mg/m3 [HEC]

Endpoint

0 [0]
(freely moving)

0 [0]
(restrained)

0.01 [0.0025]

1 [0.25]

Measured concentration
(mg/m3)









Day 1

ND

ND

0.0097 ± 0.0004 (-3%)b,c,d

1.069 ±0.0170 (+7%)*

Day 4

ND

ND

0.0097 ± 0.0002 (-3%)

0.9810 ±0.136 (-2%)*

aPeiffer et al. (2013).
bData are means ± SEM.

°Values provided in the study report as ppb were converted to mg/m3 using the following equation: exposure in
mg/m3 = exposure in ppm x MW of fluorene + 24.45, using a MW of fluorene of 166.22 g/mol (U.S. EPA. 202lb).
dValue in parentheses is % change relative to target concentration = ([measured concentration - target
concentration] + target concentration) x 100.

* Significantly different from the target concentration at (p < 0.01) by Dunnett t-test, as reported by the study
authors.

HEC = human equivalent concentration; MW = molecular weight; ND = not detected; SEM = standard error of the
mean.

Table B-10. Restraint Effects in Control Male Wistar Han Rats Exposed to

Air for 14 Days"

Target concentration in mg/m3 [HEC]

Endpoint

0 [0]
(freely moving)

0 [0]
(restrained)

Number evaluated (n)

12

12

Body weight (g)





Day before habituation

243.22 ± 2.0lb-c

246.89 ± 2.48 (+2%)

Day prior to initiation of exposure

282.56 ±3.45

270.61 ±2.95 (-4%)*

After 7 days exposure

303.00 ±4.56

288.56 ±4.10 (-5%)*

After 14 days exposure

317.83 ±5.41

299.00 ± 4.97 (-6%)*

Open-field test





Total number of crossed cases

117.8 ± 10.5

149.2 ±8.1 (+27%)*

Y-maze





Total arm entries

25.3 ± 1.1

32.6 ± 1.6 (+29%)**

aPeiffer et al. (2013).
bData are means ± SEM.

°Value in parentheses is % change relative to freely-moving control = ([restrained control mean - freely-moving
control mean] + freely-moving control mean) x 100.

* Significantly different from control at (p< 0.05) by Dunnett t-test, as reported by the study authors.
**Significantly different from control at (p< 0.01) by Dunnett t-test, as reported by the study authors.

HEC = human equivalent concentration; SEM = standard error of the mean.

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Table B-ll. Behavioral Effects in Male Wistar Han Rats Exposed Nose-Only

to Fluorene for 14 Days"

Target concentration in mg/m3 [HEC]

Endpoint

0 [0]
(restrained)

0.01 [0.0025]

1 [0.25]

Number evaluated (n)

12

12

12

Open-field test







Number of crossed squares; central area

4.60b,c

8.60 (+87%)**

8.20 (+78%)*

Time; central area (s)

12.5

21.1 (+69%)*

11.4 (-9%)

Total number of crossed squares

149.5

159.6 (+7%)

163.3 (+9%)

Elevated-plus maze







Central area time (s)

63.8 ± 6.3d

76.8 ± 5.4 (+20%)*

79.5 ± 4.6 (+25%)*

Open arm time (s)

87.5 ±9.7

75.4 ± 12.7 (-14%)

71.7 + 9.9 (-18%)

Closed arm time (s)

148.7 ± 11.2

147.8 ± 14.8 (-1%)

148.8 ± 10.7 (+0%)

Head dipping in open arms (%)

62.3 ± 5.7e

54.7 ± 7.4 (-8%)

40.0 ± 7.1 (-22%)f

Total head dipping

7.7 ±0.8

8.3 ± 0.9 (+8%)

8.2 ± 1.2 (+6%)

aPeiffer et al. (2013).

bData are means based on graphically reported data extracted using the MATLAB tool, GRAB IT; variance values
were not extracted.

°Value in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
dData are means ± SEM.

"Value in parentheses is % change relative to control = control mean - treatment mean (for data reported as %).
fp = 0.08 (marginal statistical significance) based on Dunnett t-test, as reported by the study authors.
* Significantly different from control at (p< 0.05) by Dunnett t-test, as reported by the study authors.
**Significantly different from control at (p < 0.01) by Dunnett t-test, as reported by the study authors.

HEC = human equivalent concentration; SEM = standard error of the mean.

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