EPA 635/R-03/010
www.epa.gov/iris
r/EPA
w
TOXICOLOGICAL REVIEW
OF
2-METHYLNAPHTHALENE
(CAS No. 91-57-6)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
December 2003
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use. Note: This document may undergo revisions in the
future. The most up-to-date version will be made available electronically via the IRIS Home Page at
http://www.epa.gov/iris.
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CONTENTS — TOXICOLOGICAL REVIEW OF 2-METHYLNAPHTHALENE
(CAS No. 91-57-6)
LIST OF TABLES v
LIST OF FIGURES v
FOREWORD vi
AUTHORS, CONTRIBUTORS, AND REVIEWERS vii
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS 3
3. TOXICOKINETICS RELEVANT TO ASSESSMENTS 5
3.1. ABSORPTION 5
3.2. DISTRIBUTION 6
3.3. METABOLISM 8
3.4. ELIMINATION AND EXCRETION 14
3.5. PHYSIOLOGICALLY-BASED TOXICOKINETIC (PBTK) MODELING 15
4. HAZARD IDENTIFICATION 16
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS 16
4.2. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIO AS SAYS IN
ANIMALS—ORAL AND INHALATION 16
4.2.1. Oral Exposure 16
4.2.1.1. Prechronic Toxicity 16
4.2.1.2. Chronic Toxicity 17
4.2.2. Inhalation Exposure 20
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND
INHALATION 20
4.4. OTHER STUDIES 20
4.4.1. Acute Toxicity Data 20
4.4.2. Studies with Methylnaphthalene Mixtures 25
4.4.3. Other Cancer Studies 28
4.4.4. Genotoxicity Studies 29
4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND
MODE OF ACTION—ORAL AND INHALATION 30
4.5.1. Oral Exposure 30
4.5.2. Inhalation Exposure 42
4.5.3. Dermal Exposure 42
4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER
iii
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SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN
CARCINOGENICITY, AND LIKELY MODE OF ACTION 43
4.6.1. Summary of Overall Weight-of-Evidence 43
4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence 43
4.6.3. Mode of Action Information 44
4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 45
4.7.1. Possible Childhood Susceptibility 45
4.7.2. Possible Gender Differences 46
4.7.3. Other 46
5. DOSE RESPONSE ASSESSMENT 47
5.1. ORAL REFERENCE DOSE (RfD) 47
5.1.1. Choice of Principal Study and Critical Effect - with Rationale and Justification 47
5.1.2. Methods of Analysis - Including Models 49
5.1.3. RfD Derivation-Including Application of Uncertainty Factors (UFs) 53
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 55
5.3. CANCER ASSESSMENT 56
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE 58
6.1. HUMAN HAZARD POTENTIAL 58
6.2. DOSE RESPONSE 59
6.2.1. Noncancer/Oral 59
6.2.2. Noncancer/Inhalation 60
6.2.3. Cancer/Oral and Inhalation 60
7. REFERENCES 61
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION
APPENDIX B: BENCHMARK DOSE (BMD) ANALYSIS
IV
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LIST OF TABLES
Table 1. Distribution of radioactivity in guinea pigs after oral administration of
2-[3H]methylnaphthalene 7
Table 2. Incidence of pulmonary alveolar proteinosis in B6C3F1 mice fed 2-methylnaphthalene for 81
weeks 19
Table 3. Oral toxicity studies for 2-methylnaphthalene 32
Table 4. Toxicity studies with mixtures of 2-methylnaphthalene and 1-methylnaphthalene 33
Table 5. Parenteral (single intraperitoneal injection) studies of 2-methylnaphthalene 40
Table Bl. Incidence of pulmonary alveolar proteinosis in B6C3F1 mice fed 2-methylnaphthalene for
81 weeks B-l
Table B2. Benchmark dose modeling (both high and low dose groups compared to concurrent
controls) for critical effect, settings of 10% extra risk, confidence level 0.95 B-2
Table B3. Comparison of benchmark dose modeling results considering 5% and 10% extra risk levels
(with confidence level 0.95) and comparison against historical and concurrent controls .... B-3
LIST OF FIGURES
Figure 1. Structure of 2-methylnaphthalene 3
Figure 2. Metabolism of 2-methylnaphthalene 9
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FOREWORD
The purpose of this lexicological Review is to provide scientific support and rationale for the
hazard and dose-response assessment in IRIS pertaining to chronic exposure to 2-methylnaphthalene.
It is not intended to be a comprehensive treatise on the chemical or lexicological nature of 2-
methylnaphthalene.
In Section 6, EPA has characterized its overall confidence in the quantitative and qualitative
aspects of hazard and dose response. Matters considered in this characterization include knowledge
gaps, uncertainties, quality of data, and scientific controversies. This characterization is presented in an
effort to make apparent the limitations of the assessment and to aid and guide the risk assessor in the
ensuing steps of the risk assessment process.
For other general information about this assessment or other questions relating to IRIS, the
reader is referred to EPA's IRIS Hotline at 202-566-1676.
VI
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER AND AUTHORS
Jamie C. Benedict, Ph.D. (chemical manager)
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Karen Hogan
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Andrew J. McDougal, Ph.D.
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Peter R McClure, Ph.D., DABT
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
REVIEWERS
This document and summary information on IRIS have received peer review both by EPA
scientists and by independent scientists external to EPA. Subsequent to external review and
incorporation of comments, this assessment has undergone an Agency-wide review process whereby
the IRIS Program Director has achieved a consensus approval among the Office of Research and
Development; Office of Air and Radiation; Office of Prevention, Pesticides, and Toxic Substances;
Office of Solid Waste and Emergency Response; Office of Water; Office of Policy, Economics, and
Innovation; Office of Children's Health Protection; Office of Environmental Information; and the
Regional Offices.
vu
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INTERNAL EPA REVIEWERS
George Woodall, Ph.D.
National Center for Environmental Assessment-RTF
Hazardous Pollutant Assessment Branch
U.S. Environmental Protection Agency
Ines Pagan, D. V.M., Ph.D.
National Center for Environmental Assessment-RTF
Hazardous Pollutant Assessment Branch
U.S. Environmental Protection Agency
John Stanek, Ph.D.
National Center for Environmental Assessment-RTF
Hazardous Pollutant Assessment Branch
U.S. Environmental Protection Agency
Roy Smith, Ph.D.
Office of Air and Radiation
Risk and Exposure Assessment Group
U.S. Environmental Protection Agency
Jeffrey Ross, Ph.D.
National Health and Environmental Effects Research Laboratory
Environmental Carcinogenesis Division
U.S. Environmental Protection Agency
Lynn Flowers, Ph.D., DABT
National Center for Environmental Assessment-IO
Integrated Risk Information System
U.S. Environmental Protection Agency
Michael Beringer
Region VU
U.S. Environmental Protection Agency
vui
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EXTERNAL PEER REVIEWERS
James Klaunig, Ph.D.
Indiana University School of Medicine
Indianapolis, IN
Gary D. Stoner, Ph.D.
Ohio State University
Columbus, OH
Hanspeter Witschi, M.D.
University of California
Davis, CA
Yiliang Zhu, Ph.D.
University of South Florida
Tampa, FL
Summaries of the external peer reviewers' comments and the disposition of their
recommendations are in Appendix A.
rx
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1. INTRODUCTION
This document presents background and justification for the hazard and dose-response
assessment summaries in EPA's Integrated Risk Information System (IRIS). IRIS Summaries may
include an oral reference dose (RfD), inhalation reference concentration (RfC) and a carcinogenicity
assessment.
The RfD and RfC provide quantitative information for noncancer dose-response assessments.
The RfD is based on the assumption that thresholds exist for certain toxic effects such as cellular
necrosis but may not exist for other toxic effects such as some carcinogenic responses. It is expressed
in units of mg/kg-day. In general, the RfD is an estimate (with uncertainty spanning perhaps an order of
magnitude) of a daily exposure to the human population (including sensitive subgroups) that is likely to
be without an appreciable risk of deleterious noncancer effects during a lifetime. The inhalation RfC is
analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The inhalation RfC
considers toxic effects for both the respiratory system (portal-of-entry) and for effects peripheral to the
respiratory system (extrarespiratory or systemic effects). It is generally expressed in units of mg/m3.
The carcinogenicity assessment provides information on the carcinogenic hazard potential of the
substance in question and quantitative estimates of risk from oral exposure and inhalation exposure.
The information includes a weight-of-evidence judgment of the likelihood that the agent is a human
carcinogen and the conditions under which the carcinogenic effects may be expressed. Quantitative
risk estimates are presented in three ways. The slope factor is the result of application of a low-dose
extrapolation procedure and is presented as the risk per mg/kg/day. The unit risk is the quantitative
estimate in terms of either risk per |ig/L drinking water or risk per |ig/m3 air breathed. Another form in
which risk is presented is a drinking water or air concentration providing cancer risks of 1 in 10,000; 1
in 100,000; or 1 in 1,000,000.
Development of these hazard identification and dose-response assessments for 2-
methylnaphthalene has followed the general guidelines for risk assessment as set forth by the National
Research Council (1983). EPA guidelines that were used in the development of this assessment may
include the following: Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA,
1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Guidelines for
1
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Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Guidelines for Reproductive Toxicity
Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA,
1998a), Draft Revised Guidelines for Carcinogen Assessment (U.S. EPA, 1999),
Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S.
EPA, 1988), (proposed) Interim Policy for Particle Size and Limit Concentration Issues in
Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the
Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Science Policy Council
Handbook: Peer Review (U.S. EPA, 1998b, 2000a), Science Policy Council Handbook: Risk
Characterization (U.S. EPA, 2000b), Benchmark Dose Technical Guidance Document (U.S.
EPA, 2000c) and Supplementary Guidance for Conducting Health Risk Assessment of Chemical
Mixtures (U.S. EPA, 2000d).
The literature search strategy employed for this compound was based on the CASRNs for 2-
methylnaphthalene (91-57-6) and methylnaphthalene (1321-94-4), and at least one common name. At
a minimum, the following databases were searched: RTECS, HSDB, TSCATS, CCRIS, GENE-
TOX, DART/ETIC, EMIC, TOXLINE, CANCERLIT, MEDLINE, and Current Contents. Any
pertinent scientific information submitted by the public to the IRIS Submission Desk was also
considered in the development of this document. The relevant literature was reviewed through March
2003.
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2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS
2-Methylnaphthalene (CASRN 91-57-6) is a polycyclic aromatic hydrocarbon (PAH),
consisting of two-fused aromatic rings with a methyl group attached on one of the rings at the number
two carbon (Figure 1). Synonyms include • -methylnaphthalene. Certain physical and chemical
properties are shown below (ATSDR, 1995; CRC, 1990).
Figure 1: 2-Methylnaphthalene
Chemical Formula:
Molecular Weight:
Melting Point:
Boiling Point:
Density:
Water Solubility:
LogKow:
Log Koc:
Vapor Pressure:
142.20 g/mol
34.6 °C
241 °C
1.0058g/mL(at20°C)
24.6 mg/L (at 25 °C)
3.86
3.39
0.068 mmHg at 20 °C
Henry's Law Constant: 4.99x10"4 atm-nrYmol
2-Methylnaphthalene is a natural component of crude oil and coal, and is found in pyrolysis and
combustion products such as cigarette and wood smoke, emissions from combustion engines, asphalt,
coal tar residues, and used oils (ATSDR, 1995; HSDB, 2002; Warshawsky, 2001).
Methylnaphthalene (CASRN 1321-94-4) refers to a mixture of approximately two-thirds 2-methyl-
naphthalene and one-third 1-methylnaphthalene (CASRN 90-12-0). Methylnaphthalene is
manufactured from coal tar through the extraction of heteroaromatics and phenols. Distillation of
methylnaphthalene removes 1-methylnaphthalene, leaving 2-methylnaphthalene. Mixtures containing
2-methylnaphthalene are used in the formulation of alkyl-naphthalenesulfonates (used for detergents and
textile wetting agents), chlorinated naphthalenes, and hydronaphthalenes (used as solvents). Pure
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2-methylnaphthalene is a component used in the manufacture of vitamin K and the insecticide carbaryl
(1-naphthyl-N-methylcarbamate) (HSDB, 2002).
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3. TOXICOKINETICS RELEVANT TO ASSESSMENTS
No studies are available regarding the toxicokinetics of 2-methylnaphthalene in humans by any
route of exposure.
The available animal data indicate that 2-methylnaphthalene is absorbed rapidly following
ingestion (approximately 80% within 24 hours). Once absorbed, it is widely distributed among tissues,
reaching peak concentrations in less than 6 hours. It is quickly metabolized by the liver, lungs, and
other tissues. 2-Methylnaphthalene is rapidly excreted (approximately 70-80% within 48 hours in
guinea pigs and 55% in rats), primarily as urinary metabolites (Melancon et al, 1982; Teshima et al.,
1983).
3.1. ABSORPTION
Quantitative evidence of the rapid and extensive absorption of 2-methylnaphthalene is provided
by a study of guinea pigs orally-exposed to 2-methylnaphthalene (Teshima et al., 1983).
Teshima et al. (1983) orally administered 10 mg/kg of 2-[3H]methylnaphthalene in olive oil to
male Hartley guinea pigs. Groups of 3 animals were sacrificed at 3, 6, 24, or 48 hours after exposure
and radioactivity was measured in various organs and tissues. The amount of the radiolabel detected
outside the gastrointestinal tract (i.e., various internal organs, blood, and urine) provides an estimate of
absorbed material, whereas radiolabel found in the gastrointestinal contents and feces provides an
estimate of 2-methylnaphthalene that is not absorbed. Data indicate that at least 25-72% of the
administered dose was absorbed by 3 hours, 44-80% by 6 hours, and 80-86% by 24 hours.
However, the percentages may underestimate the actual amounts absorbed since there may be
significant enterohepatic cycling (see Section 3.4).
Although no quantitative studies are available regarding the rate or extent of 2-
methylnaphthalene absorption by the respiratory tract or skin, findings of systemic toxicity following
exposure by these routes provide qualitative evidence of absorption. Inhalation exposure to
concentrations • 352 mg/m3 2-methylnaphthalene for 4 hours induced a delayed pain response in
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Wistar rats, indicating that some absorption may have occurred (Korsak et al., 1998). Dermal
exposure of B6C3F1 mice to 119 mg/kg of a 1.2% mixture of 2-methylnaphthalene and 1-
methylnaphthalene (approximate 2:1 ratio) in acetone twice weekly for 30 weeks induced pulmonary
toxicity in virtually all exposed mice (Murata et al., 1992). By comparison, oral exposure of B6C3F1
mice to 52.3 mg/kg 2-methylnaphthalene for 81 weeks led to the development of pulmonary toxicity in
approximately half of the exposed mice (Murata et al., 1997). Given that 2-methylnaphthalene is
extensively absorbed following oral exposure (Teshima et al., 1983), when taken together the results
indicate considerable dermal absorption or significant first pass metabolism may have occurred
following oral exposure to 2-methyl-naphthalene.
3.2. DISTRIBUTION
Following oral administration, 2-methylnaphthalene is absorbed from the gastrointestinal tract
into the portal circulation and transported to the liver, where it undergoes oxidative metabolism to form
more polar metabolites. These metabolites are then transported via systemic circulation to the various
organs and tissues, including the kidney. Excretion occurs primarily in the urine. While no human
distribution data are available, two animal studies that measured the distribution of radioactivity
following acute oral (Teshima et al., 1983) and injection dosing (Griffin et al., 1982) were identified.
No distribution studies following inhalation or dermal exposure are available.
Teshima et al. (1983) orally administered single doses of 10 mg/kg 2-[3H]methyl-naphlhalene
to male Hartley guinea pigs (3/group) and observed peak tissue concentrations of radiolabel at 3 hours
in the blood and gallbladder, and at 6 hours in all other tissues (see Table 1). The detection of a
relatively high concentration of radiolabel in the gallbladder at 3 hours suggests that liver concentrations
may have actually peaked before 3 hours. Teshima et al. (1983) reported a clearance half-life of 10.4
hours from the blood, but did not specify the details of the calculation.
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Table 1. Distribution of radioactivity in guinea pigs after oral administration of 2-[3H]methylnaphthalene
Tissue
3 hours
6 hours
24 hours
48 hours
u,g of 3H/g wet tissue
Gallbladder
Kidney
Liver
Blood
Lung
Others (combined)
20.2
5.6
1.7
0.8
0.7
0.8
15.7
7.6
2.7
0.7
0.8
1.1
0.4
0.3
0.2
0.1
0.1
0.2
0.04
0.1
0.1
0.1
0.1
0.1
Percent of total administered dose
Internal organs
Blood
Gastrointestinal contents
Urine
Feces
Total recovery
1.4
0.6
27.9
23.1
0
53
2.1
0.5
20.2
41.3
0
64.2
0.1
0.1
3.1
78.6
10.8
92.7
0.1
0.1
1.0
72.2
11.9
85.2
Source: Adapted from Teshima et al., 1983.
Griffin et al. (1982) administered single intraperitoneal injections of 400 mg/kg [14C]-2-
methylnaphthalene to male C57BL/6J mice. Groups of 4 mice were sacrificed at 0.5, 1, 3, 6, 12, or 24
hours after injection for measurement of radioactivity in fat, kidney, liver, and lung. Blood 2-
methylnaphthalene concentrations decreased with a reported elimination half-life of 3 hours, indicative
of rapid distribution to other tissues or elimination from the body. Peak tissue concentrations of 2-
methylnaphthalene equivalents (nmol/mg wet weight) were attained about 1 hour after injection in the
liver, 2 hours after injection in the fat, and 4 hours after injection in the kidney and the lung (Griffin et al.,
1982). Peak concentrations were highest in fat (13 nmol/mg), followed by lower concentrations in liver
(3.5 nmol/mg), kidney (2.9 nmol/mg), and lung (0.7 nmol/mg). The results demonstrate that 2-
methylnaphthalene did not preferentially accumulate in the lung although the lung was the only site of
toxicity. Histological examination found that the single 400 mg/kg dose induced bronchiolar necrosis
(minimal to prominent sloughing of lining cells in the bronchiolar lumen as revealed by light microscopy)
in all exposed mice (Griffin et al., 1982). No lesions were found in the liver or kidney of exposed mice
at any time point. Consistent with the attainment of peak lung tissue concentration at 4 hours after
injection, no lesions were evident until 8 hours after injection. The authors also evaluated distribution by
measurement of irreversible binding of label from [14C]-2-methylnaphthalene to various tissues over a
dose (0, 50, 100, 300, and 500 mg/kg; intraperitoneal injection) and time course (1, 2, 4, 8, 12, and
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24 hrs). Maximum irreversible binding of 2-methylnaphthalene metabolites was observed in lung, liver,
and kidney tissues at 8 hours post administration. The binding was dose- dependent in all tissues
between 50-500 mg/kg and concentrations of bound radioactivity were higher in the liver and kidney
than in the lung (the only tissue where lesions were found).
In addition, Griffin et al. (1982) evaluated the influence of changes in metabolism on
distribution. Groups of mice (5/group) were treated with inhibitors (piperonyl butoxide or SKF525-A)
or inducers (phenobarbital or 3-methylcholanthrene) of cytochrome P450 (CYP) enzymes, or with
diethylmaleate to deplete tissue levels of glutathione prior to treatment with [14C]-2-methylnaphthalene
(50-500 mg/kg) for the measurement of irreversible binding of label from [14C]-2-methylnaphthalene to
organ macromolecules. The CYP enzyme inhibitor piperonyl butoxide significantly decreased
irreversible binding in the liver, lung, and kidney by approximately 70, 40, and 50%, respectively.
Administration of the CYP enzyme inducer phenobarbital significantly reduced irreversible binding in the
lung by approximately 30% and reduced (not statistically significant) irreversible binding in the liver by
approximately 50%. Depletion of glutathione by treatment with diethylmaleate significantly reduced
irreversible binding in the kidney and lung by approximately 40 and 30%, respectively.
3.3. METABOLISM
The proposed metabolic pathway for 2-methylnaphthalene in mammals is shown in Figure 2.
The pathway has been elucidated through the identification of urinary metabolites eliminated by
laboratory animals following acute exposure (Breger et al., 1983; Teshima et al., 1983; Melancon et
al., 1985), by studies measuring the effects of enzyme modulators on the toxic and biochemical changes
caused by 2-methylnaphthalene exposure in mice (Griffin et al., 1982, 1983), and by in vitro analyses
of purified enzyme preparations (microsomal fractions and recombinant enzymes) from liver, lung, and
kidney tissues (Melancon et al., 1985).
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[ ] putative metabolite
CYP cytochrome P450 enzyme(s)
EH epoxide hydrolase
* Metabolites identified in vitro only
2-Naphthaldehyde* X,
t<
CYP
2-Hydroxymethylnaphthalene
''CYP
I
CYP CYP
« k^N^ *
2-Methylnaphthalene
Hydroxy glutathionyl dihydro-2-methylnaphthalenes*
Hydroxy glutathionyl dihydro-2-methylnaphthalenes*
CYP
1-glutathionyl-7-methylnaphthalene
7-methyl -1-naphthylcysteine
7-methyl-1-naphthol
Hydroxy glutathionyl dihydro-2-methylnaphthalenes*
Figure 2: Metabolism of 2-methylnaphthalene (adapted from Buckpitt and Franklin, 1989; Shultz et al.,
2001; Teshimaetal., 1983).
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CYP enzymes catalyze the first competing steps, involving oxidation at the methyl group (the
predominant path) or oxidation at several positions on the rings (Figure 2). Approximately 50-80% of
2-methylnaphthalene is oxidized at the methyl group to produce 2-hydroxymethyl-naphthalene (Breger
et al, 1983; Melancon et al, 1982; Teshima et al., 1983) which is further oxidized to 2-naphthoic acid
(the carboxylic acid derivative) (Grimes and Young, 1956; Melancon et al., 1982; Teshima et al.,
1983), either directly or through the intermediate, 2-naphthaldehyde. 2-Naphthaldehyde has been
detected only following in vitro incubation of 2-methylnaphthalene with recombinant mouse CYPEF2
(Schultz et al., 2001). CYPEF2, or naphthalene dehydrogenase, has been shown to rapidly metabolize
the structurally related chemical naphthalene (Schultz et al., 1999). Schultz et al.(2001) demonstrated
that CYPEF2 metabolizes 2-methylnaphthalene with a relatively high turnover rate (67.7 min"1) and a
low Km (3.7 • m). 2-Naphthoic acid may be conjugated with glycine or glucuronic acid. Both
reactions can be catalyzed by amino acid transferase (i.e., ATP-dependent acid: CoA ligase and N-
acyltransferase) and uridine diphosphate glucuronosyltranferase, respectively (Parkinson, 2001). The
conjugation of 2-naphthoic acid with glycine forms 2-naphthuric acid, the most prevalent metabolite of
2-methylnaphthalene detected in urine (Grimes and Young, 1956; Melancon et al., 1982; Teshima et
al., 1983).
Approximately 15-20% of 2-methylnaphthalene undergoes ring epoxidation at the 3,4-, 5,6-,
or 7,8- positions (Breger et al., 1983; Melancon et al., 1985). These reactions are catalyzed by CYP
enzymes, including CYPIA and CYPIB. While the epoxides have not been isolated, they are proposed
putative intermediates based on observed metabolites and are thought to be further oxidized by epoxide
hydrolase to produce dihydrodiols (3,4-dihydrodiol, 5,6-dihydrodiol, or 7,8-dihydrodiol) of 2-
methylnaphthalene, or conjugated with glutathione (Griffin et al., 1982; Melancon et al., 1985).
Glutathione conjugation can be catalyzed by isozymes from the large family of glutathione S-
transferases or can proceed spontaneously (Parkinson, 2001). The hydroxy-glutathionyl-dihydro-2-
methylnaphthalenes were detected after 2-methylnaphthalene was incubated with hepatic microsomes
from Swiss-Webster mice or with isolated recombinant mouse CYPIIF2 enzyme and glutathione S-
transferase (Schultz et al., 2001). Figure 2 shows six hydroxy-glutathionyl-2-methylnaphthalenes; two
are formed for each of the epoxide intermediates (3,4-, 5,6-, and 7,8-epoxides), and each can exist in
two enantiomeric forms not shown in Figure 2 (Schultz et al., 2001).
10
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Three additional minor metabolites are formed via the 7,8-epoxide pathway. l-Glutathionyl-7-
methylnaphthalene was identified in the urine of guinea pigs and by in vitro experiments with guinea pig
microsomes (Teshima et al, 1983). 7-Methyl-l-naphthol and 7-methyl-2-naphthol were identified in
the urine of 4 species (rats, mice, guinea pigs, and rabbits)
following oral exposure (Grimes and Young, 1956). The mammalian metabolism of 2-
methylnaphthalene has been analyzed in two quantitative experiments (Melancon et al., 1982; Teshima
et al., 1983). Melancon et al. (1982) administered single subcutaneous injections of 0.3 mg/kg
2-methyl[8-14C]naphthalene to 4 female Sprague-Dawley rats. In collected urine, 3-5% of the
administered dose was unchanged 2-methylnaphthalene, 30-35% was naphthuric acid, 6-8% were
other conjugates of naphthoic acid, 6-8% were dihydrodiols of 2-methylnaphthalene, 4-8% were other
nonconjugated metabolites, and 36-45% were other high-polarity unidentified metabolites. Teshima
et al. (1983) administered single oral doses of 10 mg/kg 2-[3H]methyl-naphthalene to male Hartley
guinea pigs (3/group). At 24 hours, 78.6% of the total administered dose had been excreted in urine as
metabolites. Sixty-one percent of radioactivity in urine was accounted for by 2-naphthuric acid, 11%
by glucuronide conjugates of 2-naphthoic acid, 4% by unconjugated 2-naphthoic acid, 10% by
S-(7-methyl-l-naphthyl)cysteine, and at least 8% by metabolites of 7-methyl-l-naphthol. Additionally,
unquantified glutathione conjugates were detected in the livers of treated guinea pigs (Teshima et al.,
1983). Taken together, these reports indicate that the metabolism of 2-methylnaphthalene is rapid
(approximately 55% in rats within 3 days and approximately 80% in guinea pigs within 1 day) and that
80-85% of the metabolism occurs via oxidation of the 2-methyl group, with ring epoxidation accounting
for only 15-20%.
Standard assays in microsomal preparations (from male Sprague-Dawley rat liver, C57BL/J6
mouse liver and lung, and Swiss-Webster mouse liver, lung, and kidney tissues) demonstrate that the
initial steps of 2-methylnaphthalene metabolism are mediated by CYP enzymes (Breger et al., 1981;
Griffin et al., 1982; Melancon et al., 1985). The experiments further demonstrate that catalysis of 2-
methylnaphthalene metabolism to either dihydrodiols (the ring epoxidation pathway) or 2-
hydroxymethylnaphthalene (the alkyl-group oxidation pathway) required the cofactor NADPH and are
inhibited by heat denaturation or carbon monoxide. Other studies that measured covalent binding of
label from 2-methyl[8-14C]naphthalene to liver, lung, and kidney microsomal proteins of male Swiss-
Webster mice (Buckpitt et al., 1986) or liver slices of male ddY mice (Honda et al., 1990) observed a
similar dependence of binding on CYP activity (i.e., inhibited by cold temperature, nitrogen
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atmosphere, piperonyl butoxide, and SKF 525 A).
Microsomal studies with inducers and inhibitors of CYP activity have likewise demonstrated the
importance of CYP enzymes in 2-methylnaphthalene metabolism, but have not provided clear
mechanistic information. For example, pretreatment of male Sprague-Dawley rats (prior to microsomal
preparation) with the CYP enzyme inducer • -naphthoflavone increased the overall rates of metabolism
4-fold, but the CYP enzyme inducer phenobarbital increased production of only 1 of the 3 dihydrodiol
isomers (also 4-fold; specific isomer not determined) (Breger et al., 1981; Melancon et al, 1985).
Pretreatment of mice (before microsome collection) with 3-methylcholanthrene (an inducer of CYPIA)
reduced the pulmonary (but not hepatic) formation of one dihydrodiol isomer by half (Griffin et al.,
1982). Phenobarbital increased the hepatic formation of a different isomer 3-fold, while neither
piperonyl butoxide (a mixed inhibitor) nor diethylmaleate (depletes glutathione) had significant effects on
metabolite formation (Griffin et al., 1982). Conversely, Griffin et al. (1983) observed no significant
changes in the metabolism of 2-methylnaphthalene in lung and liver microsomes of DBA/2J mice by
pretreatment with 3-methylcholanthrene, piperonyl butoxide, or diethylmaleate. However,
phenobarbital did increase the formation of one of the dihydrodiols (> 4-fold) without decreasing
formation of the other two (Griffin et al., 1983). Taken together, the data suggest that different
isozymes are responsible for different steps in the metabolism of 2-methylnaphthalene and they likely
exhibit tissue- and strain-specificity.
Experiments that tested the effects of CYP enzyme inducers and inhibitors on the distribution
and toxicity of 2-methylnaphthalene in mice (Griffin et al., 1982, 1983) provided suggestive evidence
that CYP enzymes might metabolically activate 2-methylnaphthalene to one (or more) derivatives with
higher toxicity; however, the identities of these putative metabolites are unknown. The studies are
further discussed in Section 4.4.3.
High pressure liquid chromatography (HPLC) was used to determine the metabolism of 14C-2-
methylnaphthalene in rat hepatic microsomes and purified CYP enzymes (Melancon et al., 1985). The
study demonstrated that epoxide hydrolase was rate-limiting for the formation of dihydrodiols
(Melancon et al., 1985). Inhibitors of epoxide hydrolase (cyclohexane oxide and trichloropropylene
oxide) fully inhibited the pulmonary and hepatic formation of all 3 dihydrodiols from 2-
methylnaphthalene in mouse liver and lung microsomes (Griffin et al., 1982).
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Animal studies provide evidence that glutathione conjugation is an important detoxification
pathway. Griffin et al. (1982) assessed reduced glutathione levels following intraperitoneal exposure of
male C57BL/6J mice (4/group) to 400 mg/kg 2-methylnaphthalene at 0.5, 1, 3, 6, 12, 18, or 24 hours
post injection. Compared with controls, exposed mice showed a statistically significant decrease in
levels of glutathione in the liver (32-37% reduction) at 3 and 6 hours after injection with 2-
methylnaphthalene. Glutathione levels in the liver at other time points, and in the lung and kidney at all
time points, were not decreased in exposed mice compared with controls. Results indicate that this
dose of 2-methylnaphthalene led to a short-lived depletion of glutathione levels only in the liver.
Because glutathione does not conjugate directly with 2-methylnaphthalene, it is hypothesized that
glutathione binds to a more reactive metabolite.
Other studies have also observed decreased tissue or intracellular levels of glutathione in
response to exposure to high acute doses of 2-methylnaphthalene, demonstrative of glutathione
conjugation (Griffin et al., 1982, 1983; Honda et al., 1990). Similarly, glutathione depletion (35%
when compared to controls) was detected in primary cultures of female Sprague-Dawley rat
hepatocytes treated with 1 mM of 2-methylnaphthalene (Zhao and Ramos, 1998).
Although many PAHs induce the activity of enzymes that metabolize them, no enzyme induction
by 2-methylnaphthalene has been reported. Fabacher and Hodgson (1977) found no changes in
parameters of enzyme activity in the livers of male inbred North Carolina Department of Health strain
mice (4/group) given daily intraperitoneal injections of 100 mg/kg 2-methyl-naphthalene for 3 days.
Endpoints measured included: O- or N-demethylation of p-nitroanisole and aminopyrene; metabolism
of benzphetamine, piperonyl butoxide, pyridine, and n-octylamine; microsomal protein levels; and
carbon monoxide spectra. Chaloupka et al. (1995) measured hepatic and pulmonary microsomal
ethoxyresorufm O-deethylase activity (EROD) and hepatic methoxyresorufm O-deethylase (MROD)
levels in male B6C3F1 mice (• 4/group) given intraperitoneal injections of a mixture of 2-ring PAHs
containing 23.2% 2-methylnaphthalene, 23.8% naphthalene, 13.3% 1-methylnaphthalene, and 0.22%
indan. MROD is a measure of CYPIA2 while EROD measures CYPIA1 and IA2 enzyme activity.
Doses of the mixture containing 300 mg/kg 2-methylnaphthalene did not induce lung microsomal EROD
activity or hepatic MROD activity, and hepatic EROD activity was only minimally induced by doses
containing 150 and 300 mg/kg 2-methylnaphthalene (2.4- and 6-fold induction, respectively).
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Important differences exist in the metabolism of 2-methylnaphthalene and naphthalene
(ATSDR, 1995; Buckpitt et al., 1986; Buckpitt and Franklin, 1989; NTP, 2000). CYP enzymes
catalyze the initial metabolic step for both compounds, but ring epoxidation is the only initial reaction for
naphthalene. For 2-methylnaphthalene, alkyl-group oxidation is the principal initial reaction and ring
epoxidation is a minor metabolic fate.
No studies evaluating the metabolism of 1-methylnaphthalene in humans or animals are
available. Metabolism of 1-methylnaphthalene may follow a similar pathway as that described for 2-
methylnaphthalene (i.e., side chain oxidation) since the chemicals are structurally related. However, no
studies providing evidence for this common pathway of metabolism were found.
3.4. ELIMINATION AND EXCRETION
No human data are available regarding the elimination or excretion of 2-methyl-naphthalene.
Melancon et al. (1982) and Teshima et al. (1983) indicate that absorbed 2-methylnaphthalene is rapidly
eliminated (approximately 70-80% within 48 hours in guinea pigs and 55% in rats). Approximately
85% of the administered dose is eliminated, approximately 72% in urine, and 11-14% in feces
(Melancon et al., 1982; Teshima et al., 1983). No studies are available describing the elimination of 2-
methylnaphthalene through exhalation or other routes.
Table 1 shows the percent of urinary and fecal elimination of an oral dose of 10 mg/kg
2-[3H]methylnaphthalene from guinea pigs (Teshima et al., 1983). Despite the high initial levels of
radioactivity detected in the gall bladder, urinary excretion exceeded fecal excretion by 7-fold,
suggesting reabsorption of radioactivity from bile in the intestinal tract back into the body (i.e.,
enterohepatic cycling).
Female Sprague-Dawley rats (4/group) given subcutaneous injections of 0.3 mg/kg
2-methyl[8-14C]naphthalene eliminated 54.8% of the administered dose in urine within 3 days (Griffin et
al., 1982).
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Grimes and Young (1956) reported that urinary excretion was qualitatively similar among
rabbits, guinea pigs, and mice given 2-methylnaphthalene by gavage or by intraperitoneal injection, but
did not provide quantitative details.
3.5. PHYSIOLOGICALLY-BASED TOXICOKINETIC (PBTK) MODELING
No human or animal PBTK models were identified for 2-methylnaphthalene.
PBTK rat and mouse models have been developed for naphthalene (Ghanem and Shuler,
2000; NTP, 2000; Quick and Shuler 1999; Sweeney et al., 1996; Willems et al., 2001). The models
were designed for oral, inhalation, intraperitoneal, and intravenous exposure and are based on diffusion
rates and tissue partitioning coefficients as well as in vivo data for distribution, metabolism, and toxicity.
The models assume that naphthalene is metabolized only in the liver and lungs to naphthalene oxide (the
1,2-epoxide of naphthalene) and naphthalene oxide is metabolized only in the liver and lungs by
epoxide hydrolase (to dihydrodiols) or glutathione transferase (to glutathione conjugates).
The PBTK models for naphthalene in rodents are inadequate for predicting the toxicokinetics of
2-methylnaphthalene. An integral feature of the naphthalene models is the metabolism of naphthalene
exclusively to naphthalene oxide. In contrast, only 15-20% of 2-methylnaphthalene undergoes ring
epoxide formation, and 3 different isomers are produced (Melancon et al., 1982; Teshima et al., 1983).
Therefore, the models for naphthalene would not adequately predict the toxicokinetics of 80-85% of
the metabolites of 2-methylnaphthalene.
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS
No epidemiology studies or case reports are available which examine the potential effects of
human exposure to 2-methylnaphthalene by any route of exposure.
4.2. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION
4.2.1. Oral Exposure
4.2.1.1. Prechronic Toxicity
Fitzhugh and Buschke (1949) evaluated the ability of 2-methylnaphthalene to induce cataract
formation in rats. While no cataracts were found in a group of 5 weanling F344 rats fed a diet of 2%
2-methylnaphthalene (equivalent to 2,000 mg/kg-day1) for at least 2 months, cataracts were detected in
rats fed an equivalent concentration of naphthalene. Evaluation of this study is limited by the lack of
experimental details. In this study, 2,000 mg/kg-day was an apparent NOAEL for cataract formation.
Murata et al. (1997) conducted a 13-week range-finding study exposing B6C3F1 mice
(10/sex/group) to diets containing 0, 0.0163, 0.049, 0.147, 0.44, or 1.33% 2-methylnaphthalene for
13 weeks. Estimated doses were: 0, 29.4, 88.4, 265, 794, or 2,400 mg/kg-day for males and 0, 31.8,
95.6, 287, 859, or 2,600 mg/kg-day for females, respectively. Approximate average doses (across
sexes) were 0, 31, 92, 276, 827, or 2,500 mg/kg-day, respectively. The 0.147% 2-methylnaphthalene
diet reduced weight gain in both sexes by 20-21%, while the 0.44 and 1.33% diets reduced weight gain
by 30-38% in both sexes. The authors attributed these effects to food refusal. Only mice in the 0.44
and 1.33% dose groups were examined histologically, and no exposure-related adverse effects were
identified in any organ. Evaluation of the data is limited by inadequate reporting of study results. In this
A daily dose of approximately 2,000 mg/kg, assumes an average body weight of 0.18 kg for subchronically
exposed F344 rats and an average daily food intake of 0.018 kg/day (U.S. EPA, 1988). Calculations: 2% in the diet =
20,000 mg/kg of food. 20,000 mg/kg of food x 0.018 kg of food/day - 0.18 kg of body weight = 2,000 mg/kg-day of 2-
methylnaphthalene.
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study, 92 mg/kg-day and 276 mg/kg-day (averaged between sexes) are the NOAEL and LOAEL,
respectively for reduced weight gain.
4.2.1.2. Chronic Toxicity
Murata et al. (1997) fed B6C3F1 mice (50/sex/group; 10 mice/cage) diets of 0, 0.075, or
0.15% 2-methylnaphthalene for 81 weeks. The average intakes were reported as 0, 54.3 or 113.8
mg/kg-day for males and 0, 50.3, or 107.6 mg/kg-day for females, respectively. Mice were monitored
daily for clinical signs of toxicity. For the first 16 weeks, food consumption and body weight were
measured weekly, and every other week thereafter. Blood was collected at sacrifice for leukocyte
classification and comprehensive biochemical analyses. Organ weights were measured for the brain,
heart, kidney, liver, individual lobes of the lung, pancreas, salivary glands, spleen, and testis.
Histopathology was performed for these tissues and the adrenal glands, bone (sternal, vertebral, and
rib), eye, harderian glands, mammary gland, ovary, seminal vesicle, skeletal muscle, skin, small and
large intestine, spinal cord, stomach, trachea, uterus, and vagina. Pulmonary function was not evaluated
in the control or treated groups. Quantitative differences between groups were statistically analyzed
using Fisher's exact test and analysis of variance (ANOVA) with a multiple comparison post-test by
Dunnett; p • 0.05% was used as the threshold for statistical significance.
Both 2-methylnaphthalene and 1-methylnaphthalene were tested simultaneously under the same
experimental conditions and protocols (Murata et al., 1993, 1997)2. A shared group of control mice
(50 males and 50 females) was used in both of these studies. All dose and control groups were housed
in the same room. Quantitative details regarding the control animals as well as some of the
methodology utilized for the analysis of non-neoplastic endpoints and the qualitative description of these
endpoints (for both studies) were provided in the Murata et al. (1993) study. They were also omitted
Mice exposed to 0.075% or 0.15% 1-methylnaphthalene showed increased incidences of pulmonary
alveolar proteinosis in males and females and total lung tumors in males only (Murata et al., 1993). Daily doses
calculated from reported total intakes were 75.1 and 143.7 mg/kg-day 1-methylnaphthalene for females and 71.6 and
140.2 mg/kg-day for males. For male mice exposed to dietary concentrations of 0.075% or 0.15% 1-
methylnaphthalene, incidences were 13/50 and 15/50 for total lung tumors, and 23/50 and 19/50 for pulmonary
alveolar proteinosis (Murata et al., 1993). For female mice, respective incidences were 2/50 and 5/49 for total lung
tumors, and 23/50 and 17/49 for pulmonary alveolar proteinosis. No other exposure-related adverse effects were
observed in any other organs or tissues.
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from the later study (Murata et al., 1997).
Survival and food consumption were not affected by exposure to 2-methylnaphthalene at 0.075
or 0.15% dietary levels for 81 weeks (Murata et al., 1997). While body weight data were presented
graphically as mean growth curves for males and females in the control and exposed groups, group
means and standard deviations were not presented. The study report specified that the reduction in
final mean body weight was statistically significant for the high-dose male group. The reported mean
final body weights for the male and female high-dose groups were reduced by 7.5 and 4.5%,
respectively, when compared with controls. The decrease in body weight was not considered to be
biologically significant for the 2-methylnaphthalene assessment.
As shown in Table 2, dietary exposure to 2-methylnaphthalene was associated with a
statistically significant (p<0.05; Cochran-Armitage trend tests performed by Syracuse Research
Corporation) increased incidence of pulmonary alveolar proteinosis in male and female mice in both
exposure groups, when compared with controls (Murata et al., 1997). Pulmonary alveolar proteinosis
was characterized by the authors as being similar to those lesions described in an earlier study (Murata
et al., 1993; 1997). According to the Murata et al. (1993) report, pulmonary alveolar proteinosis was
characterized by an accumulation of phospholipids in the alveolar lumens and, upon gross inspection,
white protuberant nodules approximately 1-5 mm in diameter were also observed. Histologically, there
was visible filling of alveolar lumens with cholesterol crystals, foamy cells, and an amorphous acidophilic
material. No prominent fibrosis, edema, alveolitis, or lipidosis were observed in alveolar walls or in
epithelial cells. No evidence of bronchiolar Clara cell necrosis or sloughing was reported in the Murata
et al., (1997) study nor was there histopathological evidence of non-neoplastic effects in any other
tissue.
In humans, pulmonary alveolar proteinosis has been associated with increased serum lactate
dehydrogenase (LDH) (Goldstein et al., 1998; Wang et al., 1997). However, no changes in serum
LDH were reported in mice exposed to 2-methylnaphthalene (Murata et al., 1997).
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Table 2. Incidence of pulmonary alveolar proteinosis in B6C3F1 mice fed 2-methylnaphthalene for 81
weeks
Dose (% diet)
Dose (mg/kg-day)
Pulmonary alveolar proteinosis
Lung adenoma
Lung adenocarcinoma
Total lung tumors
Female
0
0
5/50
4/50
1/50
5/50
0.075
50.3
27/49*
4/49
0/49
4/49
0.15
107.6
22/48*
5/48
1/48
6/48
Male
0
0
4/49
2/49
0/49
2/49
0.075
54.3
21/49*
9/49*
1/49
10/49*
0.15
113.8
23/49*
5/49
1/49
6/49
* Statistically significant by Fisher's exact test (p<0.05)
Source: Adapted from Murata et al., 1997.
The authors indicated that the control non-zero incidence of pulmonary alveolar proteinosis
(9/99) for males and females was unusual because pulmonary alveolar proteinosis has not appeared
spontaneously in more than 5,000 B6C3F1 mice housed in the same room in the past. The appearance
of pulmonary alveolar proteinosis in the control mice was similar, but less pronounced than that seen in
2-methylnaphthalene-exposed mice. Murata et al. (1997) speculated that the increased incidence of
pulmonary alveolar proteinosis in controls may have resulted from the inhalation of volatilized 1- or 2-
methylnaphthalene due to insufficient room ventilation.
Serum neutral fat levels were elevated in exposed males and females, and relative and absolute
brain and kidney weights were increased among exposed males. In exposed females, counts of stab
(immature) and segmented (mature) neutrophils were significantly decreased, and lymphocyte counts
were increased when compared to controls (Murata et al., 1997). Although statistical significance was
indicated for some of the effects, the biological significance of these differences is unclear, due to the
lack of reported data (i.e., response magnitude and exposure level).
Table 2 also shows the incidence of lung adenomas, lung adenocarcinomas, and total lung
tumors (adenomas plus adenocarcinomas) in mice exposed to 2-methylnaphthalene. No significant
differences were observed in the total tumor bearing mice between controls and 2-methylnaphthalene-
exposed groups for either sex. While, the male low dose group (54.3 mg/kg-day) had a statistically
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significant increased incidence of lung adenomas and total lung tumors when compared with controls,
the incidence of lung tumor in the higher dose male group was not increased in a statistically significant
manner. Analysis of the male total lung tumor data by the Cochran-Armitage trend test at the p • 0.05
level did not find a statistically significant trend with increasing dose (performed by Syracuse Research
Corporation). The study provides only limited evidence of a carcinogenic response in male mice to 2-
methylnaphthalene in the diet. No significant elevations in tumor incidence were observed for exposed
male mice at other (non-lung) sites or in exposed female mice at any site. It is not explicitly stated
whether the total lung tumor incidences cited in the study refer to the number of lung-tumor bearing
mice or to the number of lung tumors found in a group. The study authors also noted that the lung
tumors were mostly single incidences.
4.2.2. Inhalation Exposure
No studies are available in which health effects were evaluated in animals following prechronic
or chronic inhalation exposure to 2-methylnaphthalene.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
No studies are available regarding the effects of 2-methylnaphthalene on reproduction or
development in humans or animals via any route of exposure.
4.4. OTHER STUDIES
4.4.1. Acute Toxicity Data
No acute oral toxicity studies were identified for 2-methylnaphthalene.
There are two acute inhalation studies with 2-methylnaphthalene: one examining neurobehavior
in rats and sensory/respiratory irritation in mice (Korsak et al, 1998) and one examining hematologic
endpoints in dogs (Lorber, 1972).
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Korsak et al. (1998) evaluated acute neurotoxicity in rats and sensory/respiratory irritation in
mice immediately following whole-body exposure to 2-methylnaphthalene. Male Wistar rats were
placed on a hot-plate (54.5°C) to measure latency of paw-lick response immediately after exposure to
0, 229, 352, or 525 mg/m3 2-methylnaphthalene for 4 hours (20, 10, 10, and 20 rats/group,
respectively). The Kruskal-Wallis statistical test was used to evaluate pain sensitivity, with p« 0.05
considered significant. Mean latencies (measured in seconds) to the paw lick response were 10.5 ±
2.6, 13.9 ± 3.3, 25.7 ± 6.3, and 33.3 ± 19.9 for the control through high-dose groups, respectively.
Mean latencies in the 2 highest exposure groups were higher than the control mean (statistically
significant), indicating a decreased sensitivity to pain when compared with controls. Defining latency
elongation • 60 seconds as a 100% decrease in pain sensitivity, exposure to the low- through high-dose
groups decreased pain sensitivity by 6.8, 30.7, and 46.0%, respectively. Rotarod performance (the
trained ability to maintain balance on a rotating rod for 2 minutes) was tested in groups of 10 rats
immediately after cessation of exposure to the same concentrations used in the pain sensitivity test. No
failures occurred in the control, low-, or mid-concentration groups. In the high concentration group,
only 1/10 rats failed to stay on the rod. Thus, no significant effect on rotarod performance was
observed.
To assess sensory/respiratory irritation of 2-methylnaphthalene, male Balb/C mice (8-10/group)
were exposed to 0, 28, 58, 125, or 349 mg/m3 of 2-methylnaphthalene for 6 minutes. Respiratory rates
were measured before, during, and 12 minutes after exposure (Korsak et al., 1998). Respiratory rate
decreased most rapidly in the first 2 minutes of exposure. Immediately after 6 minutes of exposure,
respiratory rates decreased by approximately 8, 30, 70, and 80% at the low through high
concentrations, respectively, but returned to 75-95% of normal within 12 minutes after cessation of
exposure. The calculated concentration depressing respiratory rate in mice by 50% (RD50) was
67 mg/m3 (95% upper confidence interval of 81 mg/m3). The authors considered irritation to be the
cause of these respiratory changes.
Lorber (1972) did not observe hematotoxicity in intact or splenectomized dogs following acute
whole-body exposure to 2-methylnaphthalene. The Lorber (1972) study was conducted because an
earlier unpublished study of exposure to a pyrethrin-based pesticide dissolved in a 3% mixture of
methylnaphthalenes reportedly affected blood counts in intact and splenectomized dogs. Accordingly,
Lorber (1972) tested the individual napthalenes to determine if they could account for the
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hematotoxicity individually. Therefore, on 4 consecutive days, a pesticide fogger was used to bathe
dogs (4-6 intact dogs and 4-12 splenectomized dogs/group) in a mist of 1 liter of kerosene containing
2-methylnaphthalene or practical-grade 2-methylnaphthalene for four 5-minute periods, with pauses
lasting 7-10 minutes during which the mist settled. The strains and genders of the dogs were not
reported. The amounts of 2-methylnaphthalene fogged could not be determined from the information
provided3; therefore, no accurate exposure concentration could be estimated4. Blood was collected
prior to first exposure, prior to last exposure, and 7 and 10 days after first exposure. Iliac bone
marrow aspirates were collected under anesthesia before and after exposure. Endpoints measured
were mean levels of leukocytes, reticulocytes, platelets, and red blood cell survival. Post-exposure
values were compared to pre-exposure values using student's t test at the p • 0.05 significance level.
No statistically significant differences were observed for any of the endpoints evaluated. Because
exposure levels experienced by the dogs could not be reliably estimated, the study does not identify a
reliable inhalation NOAEL for hematologic effects from acute exposure to 2-methylnaphthalene.
Although no acute oral or inhalation studies evaluated the effects of 2-methylnaphthalene on
lung histopathology, data supporting the fact that the lung is a target of 2-methylnaphthalene exposure
has been provided by acute injection studies. In mice, histological changes and sloughing of Clara cells
(a type of nonciliated cell that lines the bronchioles of the lungs) have been reported at doses as low as
100 mg/kg (Buckpitt et al., 1986; Griffin et al., 1981, 1982, 1983; Honda et al, 1990; Rasmussen et
al, 1986). In these studies, higher doses of 2-methylnaphthalene also produced bronchiolar and
pulmonary necrosis.
Lorber (1972) reported that dogs were fogged with one liter of refined, deodorized kerosene either by itself
or containing one of the chemicals in amounts similar to what might be found in liter or gallon quantities of
commercial insecticides. The latter will be termed simulated gallons. 2-Methylnaphthalene and practical-grade 2-
methylnaphthalene were mixed in 1 liter volumes of kerosene in a concentration similar to the three percent mixture
often employed commercially. The proportion of 2-methylnaphthalene in the mixture was not reported. Therefore, a
liter would have had some quantity less than 30 g of 2-methylnaphthalene. Given that 1 gallon = 4.545 liters, a
simulated gallon would have had an approximate quantity less than 100 g of 2-methylnaphthalene.
Lorber (1972) reported that dogs were exposed in cages as far as possible from the fogger, in a 10 x 9 x 8
foot room. Given that 1 foot = 0.3048 meters, the volume was approximately 20 m3. Homogenous dispersion of 30 or
100 g into 20 m3 would have produced atmospheres of 1,000 or 5,000 mg/m3 of 2-methylnaphthalene for 41-50
minutes/day for 4 days. Inhaled concentrations were likely to have been substantially less because the amount of 2-
methylnaphthalene in the test solutions were less than 30 or 100 g, as discussed in footnote 3. Additionally, the
rapid settling of the fogged mixtures may have resulted in substantially reduced inhaled concentrations. The
potential for dermal absorption due to deposits of the mixture on fur of the animals also exists.
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Griffin et al. (1981) administered single intraperitoneal injections of 0, 0.1, 1, 10, 100, 200,
400, 600, 800, or 1,000 mg/kg 2-methylnaphthalene in corn oil to male C57BL/6J mice (5/group),
with sacrifice 24 or 48 hours later. Endpoints measured included: survival; liver, kidney, and lung
histopathology by light microscopy; and electron microcopy of lung tissue. One death (1/5) was seen
at the highest dose. No liver or kidney lesions were detected by light microscopy. No lung toxicity
was seen in mice exposed to concentrations up to 10 mg/kg by light or electron microscopy. However,
at 100 mg/kg and above, the incidence and severity of bronchiolar necrosis continued to increase with
increasing dose. At 100 mg/kg, pulmonary necrosis was observed in 2/5 mice and was limited to
irregularities of cells lining the bronchioles, with cells present in the lumen. More severe pulmonary
necrosis was seen in all mice exposed to doses • 200 mg/kg, with minimal-to-prominent sloughing of
nonciliated cells (Clara cells) lining the bronchioles. At 1,000 mg/kg, all mice exhibited complete
sloughing of all bronchiolar lining cells. The extent of necrosis was reduced in all treated groups
sacrificed 48 hours after dosing compared to those sacrificed 24 hours after dosing. For example,
following the administration of 200 mg/kg, 5/5 mice showed bronchiolar necrosis at 24 hours, but at 48
hours 3/5 mice showed necrosis.
Griffin et al. (1982) sacrificed male C57BL/6J mice (4-5/group) 1, 2, 4, 8, 12, or 24 hours
after administering intraperitoneal injections of 0 or 400 mg/kg of 2-methylnaphthalene. Liver, kidney,
and lung tissue were collected for histopathology. No liver or kidney damage was observed. While no
pulmonary necrosis was observed between 1 and 4 hours, all mice exhibited some evidence of necrosis
beginning at 8 hours, that ranged from irregularity of the bronchiolar lining with normal areas to
prominent sloughing of the bronchiolar lining.
Griffin et al. (1983) examined the pulmonary toxicity of 2-methylnaphthalene in DBA/2J mice,
which are considered less responsive to inducers of CYPIA and CYPffi than C57BL/6J mice.
Male mice (5/group) were injected intraperitoneally with 0, 0.1, 1, 10, 100, 200, 400, 600, 800, or
1,000 mg/kg 2-methylnaphthalene in corn oil and were sacrificed 24 hours later. Mortality was
observed in 2/5 mice in the 1,000 mg/kg dose-group. Histopathology of the liver, kidney, and lungs
detected no damage to the liver or kidney at any dose, and no pulmonary toxicity was observed at
doses up to 10 mg/kg. Slight evidence of pulmonary necrosis was detected in 4/5 mice receiving 100
mg/kg, and severe pulmonary effects were observed in all mice given higher doses. At 100 mg/kg
level, 2 mice showed irregularities of cells lining one or two bronchioles with sloughed cells in the lumen
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(score of 1+ on a 0, 1+, 2+, 3+, or 4+ severity scale), 2 mice showed minimal sloughing of lining cells
into the lumen of some bronchioles (score of 2+), and 1 mouse showed complete sloughing of all
bronchiolar lining cells (score of 4+). In the 200 mg/kg group, pulmonary necrosis was scored as 1+ in
2 mice and 2+ in 3 mice. Prominent sloughing of bronchiolar lining cells into the lumen (scored as 3+)
was observed in all mice at 400 mg/kg. All mice at 600 and 800 mg/kg showed complete sloughing of
the bronchiolar lining (score of 4+). Mortality was reported for 2/5 mice in the 1,000 mg/kg group.
Honda et al. (1990) administered single intraperitoneal injections of 0, 100, 200, 400, or
600 mg/kg of 2-methylnaphthalene to male ddY mice and sacrificed them 24 hours later. No lung
damage was seen at 100 or 200 mg/kg. However, electron microscopic analysis detected bronchiolar
damage at 400 mg/kg and exfoliated Clara cells in the bronchiolar lumen at 600 mg/kg. The number of
animals per group was not reported. Additional intraperitoneal injection experiments in male ddY mice
(3-5/group) observed statistically significant (p < 0.05) decreases in pulmonary glutathione levels at 6
and 12 hours post injection with doses as low as 100 mg/kg of 2-methylnaphthalene (20 and 32%,
respectively), but plasma glutathione levels were not decreased in doses as high as 400 mg/kg.
Rasmussen et al. (1986) administered single intraperitoneal injections of 0, 1, or 2 mmol/kg of
2-methylnaphthalene (0, 142 or 284 mg/kg) in peanut oil to male Swiss-Webster mice (2/group) with
sacrifice at 24 hours, 3 days, 7 days, or 14 days. Lung, liver, and kidney tissues were examined with
light microscopy, and lung cells were analyzed by electron microscopy. Lung cell proliferation was
measured in the control and 284 mg/kg groups only. Doses of 0.5 or 3 mmol/kg (71 or 427 mg/kg)
were also administered, but only electron microscopy results were reported for these mice. Statistical
analyses of collected data were not performed. Cytotoxic effects on the epithelium of the lung airways
examined by light microscopy were scored on a 0-5 scale (0 = no effect; 1 = swelling of Clara cells
with occasional sloughed cells in terminal bronchioles; 2 = sloughed cells evident in bronchioles, but
ciliated cells intact and minimal effects in bronchi and trachea; 3 = sloughed Clara cells throughout
airways; 4 = sloughed Clara cells and ciliated cells in bronchioles with some damage in bronchi and
trachea; and 5 = sloughed cells throughout all airways, including trachea, leaving large areas of bare
basement membrane). Tissue samples were scored without knowledge of the treatment group.
Maximal average scores for lung cytotoxic effects were observed 3 days after injection. The maximal
average scores were 1.4 and 3.0 for 142 and 284 mg/kg mice, compared with an average score of 0
for control mice. At day 14, cytotoxic effects were still evident and average scores were 1.5 and 2.0
24
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for the 142 and 284 mg/kg mice, compared with 0.4 for control mice.
Electron microscopy of lung tissue collected from exposed mice at 6, 12, or 24 hours after
injection showed Clara cell flattening, cytoplasmic vacuolization, loss of smooth endoplasmic reticulum,
reduced number of microvilli, prominent ribosomes, and electron-dense mitochondria. Cytoplasmic
vacuolization was reported to have occurred in control mice, but not as extensively as in exposed mice.
Clara cell ultrastructural changes were reported to have increased in severity with increasing dose, from
71 to 427 mg/kg. Airways in mice from the highest dose group (427 mg/kg) were reported to be the
most severely affected showing, in addition to Clara cell effects, flattened and vacuolated ciliated cells
with dilated cisternae of the granulated endoplasmic reticulum, electron-dense mitochondria, and
prominent ribosomes. At 1, 3, and 7 days after injection, cell proliferation indices in bronchiolar
epithelial cells from the 284 mg/kg dose group increased by 3-, 32-, and 3-fold, compared with vehicle
control values. Cell proliferation indices in alveolar cells from the 284 mg/kg dose group showed a
similar response over time, but were not as greatly increased as in bronchiolar cells. Examination of
liver and kidney sections from exposed mice revealed minimal changes in the liver and no changes in the
kidney. The study report did not further describe these changes or specify the dose levels at which they
occurred.
Buckpitt et al. (1986) administered single doses of 0 or 300 mg/kg 2-methylnaphthalene to
male Swiss-Webster mice (5/group) by intraperitoneal injection, with sacrifice 24 hours later.
Histological examinations identified bronchiolar necrosis in all treated animals, and no lesions among
controls. Pulmonary necrosis was considered moderate (bronchiolar epithelial cell swelling,
vacuolization, and exfoliation) for 3/5 mice and severe (extensive sloughing in terminal and larger
airways with widespread exfoliation) for 2/5 mice. For this study, the LOAEL for bronchiolar necrosis
in male Swiss Webster mice is 300 mg/kg 2-methylnaphthalene.
Female Wistar rats (numbers not provided) given single intraperitoneal injections of 0 or 1
mmol/kg (142 mg/kg) of 2-methylnaphthalene showed no evidence of pulmonary necrosis (Dinsdale
and Verschoyle, 1987).
4.4.2. Studies with Methylnaphthalene Mixtures
25
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Methylnaphthalene mixtures are used as industrial solvents, coolants, and dye carriers.
Methylnaphthalene mixtures are composed of 2-methylnaphthalene and 1-methylnaphthalene in an
approximate ratio of 2:1. Animal studies with methylnaphthalene mixtures provide supporting evidence
that the lung is a sensitive target organ for 2-methylnaphthalene exposure.
Evidence of lung toxicity was observed in acute oral and dermal lethality testing with a
methylnaphthalene mixture (Union Carbide, 1982). Wistar rats (5 females and 3-5 males/group)
exposed by gavage to single doses of 4.0 mL/kg (4,000 mg/kg)5 or greater developed dark red and
mottled lungs. Female (but not male) rats also exhibited labored breathing. The calculated oral LD50
values were 4.29 mL/kg (4,200 mg/kg) for males and 3.25 mL/kg (3,180 mg/kg) for females. The
same report also indicated that female New Zealand white rabbits (4/group) exposed dermally to
8.0 ml/kg (8,000 mg/kg) developed dark red lungs and blanched livers. The calculated dermal LD50
value for females was 5.38 mL/kg (4,660 mg/kg). No signs of toxicity or gross pathology were
observed in Wistar rats (5/sex) exposed to a saturated vapor of a methylnaphthalene mixture for
6 hours; the methodology reported was insufficient to estimate the exposure concentration (Union
Carbide, 1982). Acute dermal and eye irritation studies with a methylnaphthalene mixture in rabbits
found that it was irritating, but not corrosive (Carnegie Mellon, 1974; Union Carbide, 1982). Because
these studies were designed to measure lethality, lung pathology in surviving animals was assessed after
a 14-day recovery period.
Murata et al. (1992) exposed female B6C3F1 mice (15/group) to 119 mg/kg of a
methylnaphthalene mixture by applying an acetone solution containing 1.2% methylnaphlhalene to their
backs twice weekly for 30 weeks. Lung tissue samples were analyzed using light and electron
microscopy. Exposure to the mixture resulted in a 14% reduction in final body weight (compared to
controls) that was not statistically significant. All mice (15/15) exposed to the methylnaphthalene
mixture developed pulmonary alveolar proteinosis. Lung surfaces grossly contained multiple grayish
white nodules. Ffistologically, the alveoli appeared to be filled with cholesterol crystals, an amorphous
eosinophilic material, and many mononucleated giant cells with foamy cytoplasm. The alveolar spaces
in areas were proteinosis was present were also filled with free myelinoid structures. The authors
5Based on a density of 0.978 g/ml for methylnaphthalene (NTP, 2002a). Example calculation: 4.0 ml/kg x
0.979 g/ml x 1,000 mg/g = 4,000 mg/kg.
26
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reported that most myelinoid structures appeared to originate from hyperplastic and hypertrophic type
n pneumocyte meocrine secretions. The enlarged mononucleated giant cells contained myelinoid
structures similar to those observed in the alveolar space, along with lipid droplets. The myelinoid
structures consisted of concentrically arranged and multilayered membranes interspersed with
amorphous materials. Various numbers and sizes of needle-like crystals were also observed in the
mononucleated giant cells cytoplasm. Alveolar walls were partially thickened but there was no
prominent fibrosis. The thickening was due to hyperplasia and hypertrophy of type n pneumocytes, or
focal hyperplasia of cells resembling type I pneumocytes in appearance. Ultrastructural analyses
verified these observations, and detected numerous necrotic cells in areas of proteinosis. Murata et al.
(1992) concluded that the mononucleated giant cells were type n pneumocytes overfilled with
myelinoid structures (rather than macrophages that might have engulfed lamellar bodies) and that some
of these cells ruptured into the alveolar lumens. The authors reported that a higher dermal dose (238
mg/kg, twice weekly) induced a 100% incidence of pulmonary alveolar proteinosis in a shorter time
period (20 weeks), but noted that this was unpublished data (Murata et al., 1992). Murata et al.
(1992) stated that the incidence of pulmonary alveolar proteinosis observed in mice exposed to a
methylnaphthalene mixture via the dermal route had been demonstrated in an earlier study conducted by
their laboratory (Emi and Konishi, 1985).
Emi and Konishi (1985) painted the shaved backs of female B6C3F1 mice with 0, 29.7, or
118.8 mg/kg of a methylnaphthalene mixture in acetone twice weekly for 61 weeks. The control
through high-dose groups contained 4, 11, and 32 mice, respectively. At sacrifice, animals were
necropsied, and histology was performed on the skin and principal organs (not identified). Although
survival information was not provided, a reported peak in mortality at 38 weeks was attributed to lipid
pneumonia. Lipid pneumonia was observed (in animals that died) as early as 10 weeks in which Emi
and Konishi (1985) described the condition as severe. The final incidences of lipid pneumonia were
0/4, 3/11, and 31/32 for the control, low, and high dose groups, respectively. Lipid pneumonia was
characterized grossly by multiple delocalized white spots and soft clearly-demarcated nodules. The
predominant histological feature was hypertrophy and hyperplasia of type n pneumocytes in the lung.
Additional observations included slight alveolar wall thickening, multinucleated giant cell reaction, and
the presence of foamy cells and cholesterol crystals in the alveolar lumen. Evidence of focal alveolar
dilation and emphysema was also observed but was considered a compensatory reaction by the
authors.
27
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A subsequent study was performed to analyze the types of lipids present in the lung following
exposure to a methylnaphthalene mixture (Taki et al, 1986). Female B6C3F1 mice received doses of
0, 118.8, or 237.6 mg/kg of the methylnaphthalene mixture (3, 8, or 7/group, respectively) in acetone
on the shaved skin of their backs twice a week for 50 weeks (equivalent to 0, 33.9 or 67.9 mg/kg-
day). Lung tissue was collected at 50 weeks for quantitation of lipid content. Lung histopathology was
not reported. Cholesteryl ester was observed in the lungs of all exposed animals, but not in controls.
Exposure to the mixture also increased lung triglyceride, cholesterol, and phospholipid levels. The most
dramatically increased phospholipids were phosphatidylcholine (increased 1.5- to 5-fold in low-dose
animals and 3- to 5.7-fold in high-dose animals) and phosphatidylglycerol (increased 1.5- to 5.8-fold in
low-dose animals and 3- to 5.8-fold in high-dose animals). The authors considered these changes to
be evidence of lipid pneumonia.
T-cell-independent and T-cell-dependent immunity were suppressed in mice injected with a
mixture containing 2-methylnaphthalene (Harper et al., 1996). Female B6C3F1 mice (5/group) were
given single intraperitoneal injections of 0, 24, 47, 188, or 754 mg/kg of a mixture containing 2-ring
PAHs (consisting of 38.3% 2-methylnaphthalene, 39.3% naphthalene, 22.0% 1-methylnaphthalene,
and 0.36% indan). Two days later, the mice were challenged with injections of T-cell-independent or
T-cell-dependent antigens (trinitrophenyl-lipopolysaccharide [TNP] or TNP-haptenated sheep red
blood cells, respectively). Mice were sacrificed 2 days after the challenge. Levels of serum anti-TNP
IgM and the ability of spleen cells to form plaque in the presence of the administered antigen and
complement were measured as a determinant of immune function. Decreased plaque formation
following the T-cell dependent and T-cell independent challenge and increased anti-TNP IgM levels
were observed. Similar immunosuppression was observed for a mixture containing the 2-ring, 3-ring,
and • 4-ring PAHs. The study was inconclusive regarding the possible effects of 2-methylnaphthalene
on the immune system, due to the potentially confounding influence of other chemicals present in the test
mixture.
4.4.3. Other Cancer Studies
No evidence of cocarcinogenic activity was found in female ICR/Ha Sprague-Dawley mice
(30/group) dermally exposed to 0 or 25 • g (32 • g/kg-day) 2-methylnaphthalene plus 300 ng of
28
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benzo[a]pyrene (BaP) in acetone 3 times per week for 78 weeks (Schmeltz et al, 1978). While
negative (acetone only) and positive (BaP plus 12-o-tetradeconoyl phorbol-13-acetate) controls were
included, 2-methylnaphthalene was not tested alone. Compared to positive controls, the exposure
increased the time-to-first-tumor (52 versus 58 weeks) and decreased the number of tumor-bearing
animals (44% versus 20%). The statistical significance of these findings could not be determined from
the data presented. Similar inhibitory effects (compared to BaP alone) regarding the number of tumor-
bearing animals were found with mixtures of BaP with naphthalene, 1-methylnaphthalene,
1,2-dimethylnaphthalene, 2-ethylnaphthalene, or the naphthalene-fraction of cigarette smoke.
4.4.4. Genotoxicity Studies
No genotoxicity studies in humans or animals are available. No studies investigating potential
germline mutations are available. Data from in vitro short-term tests provide limited evidence for
genotoxic activity of 2-methylnaphthalene (Florin et al., 1980; Harvey and Halonen, 1968; Hermann,
1981; Kopper Co. Inc., 1982; Kulka et al., 1988; Weis et al., 1998).
No mutagenicity was observed in Salmonella typhimurium strains TA98, TA100, TA1535,
or TA1537 treated with 2-methylnaphthalene (Florin et al., 1980; Hermann, 1981) or
methylnaphthalene mixtures (Kopper Co. Inc., 1982), with or without metabolic activation by S9
hepatic microsomal fractions. In these studies, S9 hepatic microsomal fractions were prepared from
male Sprague-Dawley, Fischer 344, or Wistar rats induced with either Aroclor 1254 or 3-
methylcholanthrene. In vitro exposure of human lymphocytes to 2-methylnaphthalene with metabolic
activation by S9 fractions produced statistically significant increases in the incidence of sister chromatid
exchanges (• 22%) at all concentrations tested (0.25 to 4 mM) and of chromatid breaks (6.5-fold) only
at the highest concentration tested (4 mM) (Kulka et al., 1988). No differences were observed
following exposure without metabolic activation. The authors considered the sister chromatid response
to be negative because the magnitude of the response was less than a 2-fold increase. They also
considered the chromatid breaks to be minor because no damage was observed at concentrations • 2
mM.
In vitro assays in WB-F344 rat liver epithelial cells indicated that 2-methylnaphthalene, as well
as naphthalene and 1-methylnaphthalene, inhibits gap junctional intercellular communication (Weis et al.,
29
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1998). The inhibition of intracellular communication has been postulated by the authors to be an
epigenetic mechanism of tumor promotion by preventing intercellular transport of regulatory molecules.
Harvey and Halonen (1968) showed that 2-methylnaphthalene binds to four nucleic acids
(adenosine, thymidine, uridine, and guanosine), as well as 3 structurally analogous compounds (caffeine,
tyrptophan and riboflavin) in a silica gel matrix. The physical conditions of the experiment were not
provided (e.g., temperature, pH). While the experiment provides suggestive evidence that 2-
methylnaphthalene may interact with DNA (even in the absence of metabolic activation), more recent
corroborating studies are not available.
4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND MODE
OF ACTION—ORAL AND INHALATION
4.5.1. Oral Exposure
There are no studies examining possible associations between acute or repeated oral exposure
to 2-methylnaphthalene and noncancer health effects in humans. A number of studies in laboratory
animals indicate pulmonary toxicity following exposure to 2-methylnaphthalene. One study in mice
provides evidence of the development of pulmonary alveolar proteinosis following near-lifetime
exposure to 2-methylnaphthalene at dose levels of approximately 50 mg/kg-day (Murata et al., 1997).
In this study, male and female B6C3F1 mice were exposed to 0, 0.075, or 0.15% 2-methylnaphthalene
in the diet for 81 weeks. Average daily doses were 0, 54.3 or 113.8 mg/kg-day for males and 0, 50.3
or 107.6 mg/kg-day for females. There was a statistically significant increase in the incidence of
pulmonary alveolar proteinosis in both exposure groups when compared to controls. Incidences for the
control through high-dose groups were 4/49, 21/49, and 23/49 for male mice and 5/50, 27/49, and
22/48 for female mice, respectively. Histological examination of major tissues and organs revealed no
other exposure-related non-neoplastic effects at other sites (including the bronchiolar regions of the
lung). Pulmonary function in the control and exposed mice was not measured in this study. The
findings indicate that the alveolar region of the lung is the critical target of chronic oral exposure to 2-
methylnaphthalene.
30
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In addition to pulmonary alveolar proteinosis, other effects observed in the study included
changes in brain and kidney weights, blood variables (decreased differential counts of neutrophils and
increased lymphocytes), and serum levels of neutral fat, total lipids, and phospholipids (Murata et al.,
1997). The biological significance of these findings is difficult to assess due to the lack of reporting
regarding the magnitude of the changes and the dose levels at which they occurred. The authors
proposed that additional research is warranted to determine whether the elevated serum levels of fat is
related to the induction of pulmonary alveolar proteinosis or is a subsequent effect of this condition.
The authors were contacted and the data on these variables was requested. The authors provided the
data concerning the number of neutrophils and lymphocytes along with the serum levels of fats and
lipids. The authors did not provide brain and kidney weight data. Currently, no U.S. EPA guidance
exists detailing the biological significance of changes in immunological parameters and their use as
critical effects. Thus, the altered number of neutrophils and lymphocytes was deemed inappropriate for
use as the critical effect. However, it should be recognized that decreased neutrophils, also referred to
as leukopenia, may alter immune function and promote infection. In addition, altered activity of the
hematopoietic growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF), a growth
factor responsible for the proliferation and differentiation of neutrophils and macrophage lineage
hemopoietic cells, has been suggested to be involved in the pathogenesis of pulmonary alveolar
proteinosis in humans (Mazzone et al., 2001). Finally, there was no definitive evidence indicating
whether changes in the serum levels of neutral fats, lipids, and phospholipids are related to the induction
of pulmonary alveolar proteinosis or are a result of this condition following exposure to 2-
methylnaphthalene.
The oral toxicity data base for 2-methylnaphthalene is sparse (Table 3). A poorly reported
study in rats that found no evidence for cataracts after • 2 months exposure to 2,000 mg/kg-day 2-
methylnaphthalene (Fitzhugh and Buschke, 1949). Murata et al. (1997) conducted a preliminary dose-
selection study in which B6C3F1 mice (10/sex/group) were fed diets containing 0, 0.0163, 0.049,
0.147, 0.44, or 1.33% 2-methylnaphthalene for 13 weeks. The two highest dose groups were without
histologically-visible non-neoplastic adverse effects in any organs when compared with controls, but
showed growth retardation (tissues from mice in the lower dose groups were not evaluated). The
finding that pulmonary alveolar proteinosis did not develop in mice after 13 weeks of exposure to
dietary concentrations of 0.44 or 1.33%, coupled with the finding that 81 weeks of exposure to 0.075
or 0.15% 2-methylnaphthalene increased the incidence of this effect, suggest that the development of
31
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pulmonary alveolar proteinosis requires chronic-duration oral exposure at the dose levels tested. There
are no oral exposure studies examining the possible developmental, reproductive, or neurologic toxicity
of 2-methyl-naphthalene in animals.
Table 3. Oral toxicity studies for 2-methylnaphthalene
Species
Rat
(5 rats of
unspecified
sex)
Mouse
(10/sex/
group)
Mouse
(50/sex/
group)
Dose/Duration
• 2 months in diet;
2,000 mg/kg-day
1 3 weeks in diet;
average doses: 0, 31,
92,276, 827, or 2,500
mg/kg-day
81 weeks in diet;
doses: 0, 54.3, or
113. 8 (M); 0,50. 3, or
107. 6 (F) mg/kg-day
NOAEL
2,000 mg/
kg-day
92 mg/kg-
day
LOAEL
276 mg/
kg-day
54.3 (M)
50.3 (F)
mg/kg-day
Effect
No cataractogenesis
Decreased weight gain;
no non-neoplastic
effects identified
histologically in any
organs at 827 or 2,500
mg/kg-day
Increased pulmonary
alveolar proteinosis at
both doses in both
sexes
Reference
Fitzhugh and
Buschke, 1949
Murata et al,
1997
Murata et al.,
1997
Additional support that the lung is a critical toxicity target of 2-methylnaphthalene comes from
studies of animals exposed to a mixture of methylnaphthalenes. Table 4 summarizes the results from
available animal toxicity studies examining methylnaphthalene mixtures. The strongest supporting
evidence comes from a report that twice weekly application of 119 mg/kg of a mixture of 1- and 2-
methylnaphthalene to the skin of B6C3F1 mice for 30 weeks or 238 mg/kg for 20 weeks produced a
100% incidence of pulmonary alveolar proteinosis (Murata et al., 1992). Murata et al. (1992) stated
that their results were consistent with those previously observed in their laboratory (Emi and Konishi,
1985). Emi and Konishi (1985) identified lipid pneumonia in 0/4 control mice and in 3/11 mice treated
dermally with 29.7 mg/kg doses of a methyl-naphthalene mixture for 61 weeks. Emi and Konishi
(1985) also observed lipid pneumonia in 31/32 female B6C3F1 mice exposed to 118.8 mg/kg doses of
a methylnaphthalene mixture applied dermally twice per week for 61 weeks. The authors of the
Murata et al. (1992) study were contacted concerning the discrepancy in the classification of these
similar lesions following dermal exposure to methylnaphthalene mixtures (i.e., lipid pneumonia versus
pulmonary alveolar proteinosis). The authors stated that the lesions seen in both dermal studies with a
mixture of methylnaphthalenes were basically the same and that during the earlier study they were
32
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unfamiliar with such lesions and wavered on their classification. The authors also indicated that in
studies subsequent to Emi and Konishi (1985), where consistent and similar pulmonary effects were
observed (Murata et al., 1992, 1993, 1997) following either dermal exposure to a mixture of
Table 4. Toxicity studies with mixtures of 2-methylnaphthalene and 1-methylnaphthalene
Route
Oral
Inhalation
Dermal
Dermal
Dermal
Dermal
Dermal
Specie
s
Rat
Rat
Rabbit
Mouse
Mouse
Mouse
Mouse
Duration
Single dose
6 hours
Single dose
20 weeks,
two times
weekly
30 weeks,
two times
weekly
50 weeks,
two times
weekly
61 weeks,
two times
weekly
NOAEL
2,000 mg/kg
Substantially
saturated vapor
LOAEL
4,000 mg/kg
4,000 mg/kg
8,000 mg/kg
238 mg/kg per
application
119 mg/kg per
application
119 mg/kg per
application
29.7 or 118. 8
mg/kg per
application
Effect
Lung discolora-
tion, labored
breathing, and
death
No clinical signs,
mortality, or gross
lung pathology
Death and
discolored lung
and liver
Pulmonary
alveolar protein-
osis
Pulmonary
alveolar protein-
osis & decreased
final body weight
Changes in lung
lipids indicative of
lipid pneumonia
Pulmonary lipid
pneumonia &
decreased survival
Referenc
e
Union
Carbide,
1982
Union
Carbide,
1982
Union
Carbide,
1982
Murata
etal, 1992
Murata
etal., 1992
Takiet al.,
1986
Emi and
Konishi,
1985
NOAEL = no-observed-adverse-effect level; LOAEL = lowest-observed-adverse-effect level.
methylnaphthalenes or dietary exposure to 2-methylnaphthalene, they determined that pulmonary
alveolar proteinosis was a more appropriate description of pulmonary toxicity. A subsequent study
33
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reported that 119 or 238 mg/kg of methylnaphthalene, applied twice weekly (dermal) to female
B6C3F1 mice for 50 weeks, produced changes in lung lipid contents that were indicative of lipid
pneumonia (Taki et al., 1986).
Lipid pneumonia is characterized by inflammation and fibrotic changes in the lungs resulting
from the inhalation of oils or fatty substances (exogenous lipid pneumonia) or the accumulation of
endogenous lipid material, typically cholesterol and/or lipids (endogenous lipid pneumonia). Lipid
pneumonia often develops from obstructive pneumonitis and typically is observed in the vicinity of lung
tumors. The disorder is characterized by the alveolar accumulation (without conclusive evidence of the
involvement of epithelial cells) of foamy macrophages that contain lipid droplets in their cytoplasm.
While lipid pneumonia and pulmonary alveolar proteinosis usually occur independently, they are often
observed simultaneously. The disorders correspond to two separate and morphologically distinct
presentations of lipid accumulation in the alveoli of the lung. Pulmonary alveolar proteinosis is
characterized by the accumulation of lamellar bodies in the alveoli (described later in Section 4.5.1.).
The lamellar bodies are composed of apoproteins and lipids that appear to be surfactant related. Few
foamy macrophages are present in the alveoli. Altered function of the type n pneumocytes (epithelial
cells of the pulmonary alveoli) is believed to be involved in the development of pulmonary alveolar
proteinosis (Mazzone et al., 2001; Seymour and Presneill, 2002). In contrast, lipid pneumonia is
characterized by the accumulation of foamy macrophages that are filled with lipid droplets. There is
suggestive, but not definitive evidence that type n pneumocytes may be involved in the development of
lipid pneumonia (Sulkowska et al., 1997; Sulkowski and Sulkowska, 1999). Specifically, Sulkowska
et al. (1997) and Sulkowski and Sulkowska (1999) found evidence of type II pneumocyte proliferation
in late stage or fully advanced forms of lipid pneumonia, but not in the early stages of this disorder upon
histological examination of lung fragments from patients with non-small cell lung carcinomas and in
rodents administered cyclophosphamide intraperitoneally to induce lung damage. Thus, it is unclear
whether lipid pneumonia and pulmonary alveolar proteinosis share a common pathogenesis or etiology.
The suggested mode of action in animals is consistent with what is generally known regarding
the etiology of pulmonary alveolar proteinosis in humans. Available evidence in animals supports the
hypothesis that type n pneumocytes may be a specific cellular target for the development of 2-
methylnaphthalene-induced pulmonary alveolar proteinosis. Light microscopic examination of lung
34
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tissue from mice that were repeatedly exposed to dermal doses of a methylnaphthalene mixture (119
mg/kg methylnaphthalene mixture twice a week for 30 weeks) showed hyperplasia and hypertrophy of
type II pneumocytes in alveolar regions with proteinosis (Murata et al., 1992). Electron microscopic
examination showed that alveolar spaces were filled with numerous myelinoid structures resembling
lamellar bodies of type n pneumocytes (Murata et al., 1992). Associated with this extracellular
material were mononucleated giant cells (balloon cells) containing numerous myelinoid structures, lipid
droplets, and electron dense needle-like crystals. Murata et al. (1992) hypothesized that, in response
to a mixture containing 2-methylnaphthalene, type n pneumocytes produce increased amounts of
lamellar bodies due to hyperplasia and hypertrophy, and eventually transform into mononucleated giant
cells. The rupture of these cells is hypothesized to lead to the accumulation of the myelinoid structures
in the alveolar lumen. No in-depth ultrastructural studies of the pathogenesis of pulmonary alveolar
proteinosis from chronic exposure to 2-methylnaphthalene alone were available. However, Murata et
al. (1997) suggested that the adverse pulmonary effects detected by light microscopy following chronic
oral exposure to 2-methylnaphthalene alone were very similar to those detected following chronic
dermal exposure to the methyl-naphthalene mixture. These similarities suggest that the mode of action
(i.e., specific cellular targeting of type n pneumocytes in the alveolar region of the lung) prompted by
observations following exposure to the methylnaphthalene mixture are relevant to 2-methylnaphthalene.
Pulmonary alveolar proteinosis (also referred to as alveolar lipoproteinosis, alveolar
phospholipidosis, alveolar proteinosis, and pulmonary alveolar lipoproteinosis) is a disorder in humans
that is characterized by the accumulation of surfactant lipids and proteins in the alveoli. The condition
develops most commonly between the ages of 20-50 and more often in males then females (3:1,
respectively) and in smokers when compared to nonsmokers. The primary symptom associated with
this condition is dyspnea that may be accompanied with cough. Altered serum lactate dehydrogenase
(LDH) levels have been observed in few patients. Patients examined physically may appear normal,
but may have nonspecific pulmonary symptoms such as sporadic reduction in diffusing capacity to
modest reduction in vital capacity. In the majority of cases, pulmonary alveolar proteinosis is diagnosed
by the presence of a milky bronchiolar lavage fluid containing large amounts of granular acellular
eosinophilic proteinaceous material with abnormal foamy macrophages filled with periodic acid-Schiff
base (PAS) positive intracellular material. Upon examination of the bronchiolar lavage fluid by electron
microscopy, concentrically laminated phospholipid structures, known as lamellar bodies, may be
present and are used to confirm pulmonary alveolar proteinosis. Histopathologically, pulmonary
35
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alveolar proteinosis is diagnosed by the almost complete filling of the alveolar space with PAS positive
surfactant material while the architecture of the alveoli is well preserved. Studies indicate that treatment
with whole lung lavage may improve symptoms and pulmonary function in the majority of patients with
this condition (Shah et al., 2000; Mazzone et al., 2001; Seymour and Presneill, 2002). During the
whole lung lavage procedure, the patient is anesthetized and intubated. While one lung is ventilated the
other is lavaged with saline. The lung is infused with 3-5 mL increments of saline until the drained
effluent is clear. The second lung is either lavaged the same day or 3-7 days later. Persons with
pulmonary alveolar proteinosis may require several whole lung lavage treatments for recovery, but a
small proportion require lavage to maintain functional status or are not responsive. The overall
prognosis for pulmonary alveolar proteinosis treated by lavage is excellent, with few incidence of
reported death (Mazzone et al., 2001). In addition, cases of this condition (approximately 8% of
patients as reported in the available literature) have been reported to spontaneously resolve (Shah et al.,
2000; Mazzone et al., 2001; Seymour and Presneill, 2002). Development of rare secondary infections
from organisms such asAspergillus, Nocardia, or Mycobacterium is the major complication
associated with this condition.
It was initially suggested that the occurrence of pulmonary alveolar proteinosis in humans was
the result of inhaled irritant particles. However, inhalation toxicity studies in animals failed to produce
the clinical features associated with pulmonary alveolar proteinosis and human lung biopsy samples did
not contain actual particulate matter. Advances in the understanding of the pathogenesis of pulmonary
alveolar proteinosis in humans have led to the realization that there are three distinct forms of this
condition (primary acquired, secondary, and congenital pulmonary alveolar proteinosis) each of which
have similar histologic presentations. Approximately 80% of pulmonary alveolar proteinosis cases
occur as the primary acquired disorder of unknown etiology, and are not associated with a familial
predisposition. Primary acquired pulmonary alveolar proteinosis is thought to involve the accumulation
of surfactant in the alveolar spaces due to altered clearance by dysfunctional macrophages in the alveoli
(Seymour and Presneill, 2002; Mazzone et al., 2001; Lee et al., 1997; Wang et al., 1997). Surfactant
is synthesized, secreted, and recycled by type n pneumocytes in the alveoli. Surfactant catabolism
involves contribution from the type n pneumocytes and macrophages. Studies in humans and knockout
mice suggest that clearance of surfactant by macrophages is reduced due to altered activity of GM-
CSF which is responsible for the transformation of monocytes into mature macrophages in the lungs
(Shah et al., 2000; Mazzone et al., 2001; Seymour and Presneill, 2002). These mature macrophages
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degrade surfactant. Altered activity of the growth factor (GM-CSF) may be due to the production of a
neutralizing antibody to GM-CSF. The inhibition of GM-CSF activity leads to immature macrophages,
undegraded surfactant, and surfactant buildup in the lung (Shah et al, 2000; Mazzone et al, 2001;
Seymour and Presneill, 2002).
In rare instances, several underlying conditions such as lysinuric protein intolerance, acute
exposure to silica dust or other inhaled environmental or industrial chemicals, immunodeficiency
disorders, malignancies and hematopoietic disorders rarely lead to the development of secondary
acquired proteinosis in humans. The low occurrence of pulmonary alveolar proteinosis following
exposure to silica and other inhaled environmental and industrial chemicals, reported in a few selected
case studies is most likely due to improved occupational health and safety standards (Seymour and
Presneill, 2002).
Congenital pulmonary alveolar proteinosis is an autosomal recessive genetic disorder that may
develop at birth or later in life. This form of pulmonary alveolar proteinosis is primarily believed to be
due to a mutation in the surfactant-associated protein B (SP-B) gene. In addition, a proportion of
infants affected with this form of the disorder are thought to have abnormalities in the receptor for GM-
CSF. Infants with congenital pulmonary alveolar proteinosis are affected with severe lung failure shortly
after birth and have a poor prognosis for survival (Shah et al., 2000; Seymour and Presneill, 2002).
Since whole lung lavage is difficult to perform on neonates, the most promising treatment for infants is
lung transplantation (Seymour and Presneill, 2002; Vaughan and Zimmerman, 2002). Difficulties
associated with whole lung lavage are magnified by the difficulty in passing the intubation tube and
instruments used to perform the lavage through the glottis of infants. Children that develop pulmonary
alveolar proteinosis later in life generally require repeated lavage treatments, but have greater chance of
survival (Seymour and Presneill, 2002; Vaughan and Zimmerman, 2002). In addition, children
heterozygous for the mutation in the SP-B gene most likely develop respiratory symptoms later in life
and have a more positive prognosis than children that are homozygous recessive for this disorder
(Seymour and Presneill, 2002).
It is unknown whether 2-methylnaphthalene by itself or its metabolites are responsible for the
development of pulmonary alveolar proteinosis. The higher incidence of pulmonary alveolar proteinosis
in mice exposed dermally to mixtures of 1- and 2-methylnaphthalene described above (Murata et al.,
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1992), compared with the incidence in mice exposed orally to 2-methyl-naphthalene alone at
comparable doses (Murata et al., 1997), suggests that first pass hepatic metabolism associated with
oral exposure may limit parent compound reaching the lung. Conversely, type n pneumocytes in the
alveoli (the possible specific cellular target following oral exposure to 2-methylnaphthalene) are
enriched in CYP enzymes (Castranova et al., 1988) and these enzymes are involved in metabolizing 2-
methylnaphthalene (see Section 3.3.).
It is evident that the primary effect of 2-methylnaphthalene exposure is pulmonary toxicity.
However, it is unknown whether 2-methylnaphthalene by itself or one or more of its metabolites are
responsible for the development of pulmonary alveolar proteinosis. Several metabolism studies have
evaluated the effect of CYP enzyme inducers and inhibitors or glutathione depletion on 2-
methylnaphthalene-induced toxicity. The studies provide equivocal evidence indicating whether 2-
methylnaphthalene or potentially reactive metabolites are responsible for lung toxicity. For example, as
described in Section 3.2., Griffin et al. (1982) pretreated male C57BL/6J mice with CYP enzyme
inducers or inhibitors prior to intraperitoneal injection with 2-methylnaphthalene (200 or 400 mg/kg) to
assess the role of metabolism in 2-methylnaphthalene-induced pulmonary toxicity. None of the
pretreatments alone nor any of the pretreatments plus 200 mg/kg-day 2-methylnaphthalene resulted in
pulmonary toxicity or lethality when compared to controls. Exposure to 400 mg/kg 2-
methylnaphthalene alone resulted in the induction of bronchiolar necrosis in all exposed mice compared
to controls (Griffin et al., 1982). On the other hand, pretreatment with the CYP enzyme inducers
phenobarbital and 3-methylcholanthrene appeared to provide some protection from 2-
methylnaphthalene-induced pulmonary toxicity, indicating that 2-methylnaphthalene, rather than its
metabolites, were responsible for the toxicity.
In contrast to the effects observed in C57BL/6J mice (Griffin et al., 1982), pretreatment of
male DBA/2J mice with the same CYP enzyme inducers or inhibitors prior to intraperitoneal exposure
to 2-methylnaphthalene (as described in Sections 3.2. and 4.4.1.) did not decrease the severity of 2-
methylnaphthalene-induced bronchiolar lesions (Griffin et al., 1983).
Griffin et al. (1982) suggested that glutathione conjugation of reactive metabolites may play a
detoxifying role in response to the acute toxicity of 2-methylnaphthalene. Pretreatment of male
C57BL/6J mice (5/group) with 625 mg/kg diethylmaleate (to deplete glutathione) 30 minutes before
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treatment with 400 mg/kg 2-methylnaphthalene resulted in mortality for 4/5 mice. The surviving mouse
exhibited prominent sloughing of the bronchiolar lining, but a description of lung histopathology was not
reported for the nonsurvivors. In contrast, the same dose (400 mg/kg) of 2-methylnaphthalene without
glutathione depletion was not fatal, but resulted in the development of bronchiolar necrosis.
No bronchiolar necrosis was observed in male ddY mice given single intraperitoneal injections
of 200 mg/kg 2-methylnaphthalene. Pretreatment with diethylmaleate (600 • I/kg) 1 hour prior to
injection caused extensive sloughing and exfoliation of bronchiolar epithelial cells in all animals (5/5)
(Honda et al., 1990).
These observations suggest that metabolism of 2-methylnaphthalene may play a role in the
pathogenesis of pulmonary alveolar proteinosis in type n pneumocytes.
Additional evidence on whether 2-methylnaphthalene or its metabolites are responsible for lung
toxicity comes from intraperitoneal injection studies with 2-methylnaphthalene (Table 5). Castranova et
al. (1988) noted that two types of cells in the lung (type n pneumocytes in the alveoli and the
nonciliated Clara cells lining the bronchioles) exhibit substantial CYP enzyme activity and are expected
to metabolize foreign chemicals. Both cell types are secretory and are located in different parts of the
lung (Cho et al., 1995; Junqueira et al., 1995). While chronic exposure of B6C3F1 mice to 2-
methylnaphthalene in the diet appeared to target the type n pneumocytes, inducing pulmonary alveolar
proteinosis (Murata et al., 1992, 1997), acute intraperitoneal exposure of B6C3F1 mice to 2-
methylnaphthalene targeted the Clara cells, inducing bronchiolar necrosis characterized by Clara cell
abnormalities, focal or complete sloughing of Clara cells, and complete sloughing of the entire
bronchiolar lining (Buckpitt et al., 1986; Griffin et al., 1981, 1982, 1983; Honda et al., 1990;
Rasmussen et al., 1986). These observations provide indirect evidence that the development of both
types of toxic response may involve metabolism of 2-methylnaphthalene.
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Table 5. Parenteral (single intraperitoneal injection) studies of 2-methylnaphthalene
Species/Strain
Rat, Wistar
Mouse, C57BL/6J
Mouse, DBA/2J
Mouse, ddY
Mouse, Swiss-Webster
Mouse, Swiss-Webster
Mouse, C57BL/6J
NOAEL
142 mg/kg
10 mg/kg
10 mg/kg
200 mg/kg
LOAEL
100 mg/kg
100 mg/kg
400 mg/kg
142 mg/kg
300 mg/kg
400 mg/kg
Effect
No lung lesions
Bronchiolar necrosis
Bronchiolar necrosis
Bronchiolar necrosis
Bronchiolar necrosis,
bronchiolar epithelial cell
proliferation, and minimal
liver histopathology
Bronchiolar necrosis
Bronchiolar necrosis
Reference
Dinsdale and
Verschoyle, 1987
Griffin etal, 1981
Griffin etal, 1983
Honda etal., 1990
Rasmussen et al.,
1986
Buckpitt etal., 1986
Griffin etal., 1982
NOAEL = no-observed-adverse-effect level; LOAEL = lowest-observed-adverse-effect level.
Studies of the mode of action by which acute intraperitoneal injections of 2-methylnaphthalene
cause bronchiolar necrosis in mice indicate the possible involvement of reactive metabolites produced
via CYP enzymes, but the mode of action at the molecular level has not been elucidated and the
ultimate toxicant has not been identified.
The mode of action of acute Clara cell toxicity of 2-methylnaphthalene may be similar to that of
naphthalene. The mode of action of naphthalene toxicity is hypothesized to involve metabolism by
CYPIA1 and other enzymes via ring epoxidation to reactive species such as 1,2-epoxides and 1,2-
quinones (Cho et al., 1995; Greene et al., 2000; Lakritz et al., 1996; Van Winkle et al., 1999). The
reactive species then interact with cellular components. The observation that 2-methylnaphthalene is
less acutely toxic than naphthalene (Buckpitt and Franklin, 1989; Cho et al., 1995) supports this
hypothesis, since only a small fraction of 2-methylnaphthalene (15-20%) undergoes ring epoxidation
(Breger et al., 1983; Melancon et al., 1985).
Findings from mode of action studies regarding the acute response in mice to intraperitoneal
injection with 2-methylnaphthalene support the understanding that the lung is a critical toxicity target, but
may only be partially related to the pathogenesis of pulmonary alveolar proteinosis from chronic oral or
dermal exposure to 2-methylnaphthalene. In mice chronically exposed to 2-methylnaphthalene for 81
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weeks, no evidence for exposure-related bronchiolar lesions (Clara cell toxicity) were found (Murata et
al, 1993, 1997). The finding may be related to observations suggesting that Clara cells can develop
resistance to naphthalene toxicity (Lakritz et al., 1996). Pretreatment of male Swiss-Webster mice with
a nontoxic initial dose of 200 mg/kg naphthalene for 7 days made the Clara cell lining of the bronchioles
more resistant to a subsequent dose of 300 mg/kg naphthalene compared with mice given only 300
mg/kg naphthalene without pretreatment (Lakritz et al., 1996). The authors suggested that reduced
expression of CYPIffi, CYPIIF, CYP reductase, and secretory protein led to this increased resistance
in the Clara cells. However, the possible development of Clara cell resistance to the acute toxicity of
2-methylnaphthalene has not been studied.
There are limited data to suggest that rats may be less sensitive than mice to lung damage
caused by acute exposure to 2-methylnaphthalene. Wistar rats given intraperitoneal doses of 140
mg/kg 2-methylnaphthalene did not lead to pulmonary toxicity (Dinsdale and Verschoyle, 1987). In
contrast, bronchiolar necrosis was induced in Swiss-Webster mice injected with approximately the
same dose (Rasmussen et al., 1986) and C57BL/6J and DBA/2J mice injected with 100 mg/kg 2-
methylnaphthalene (Griffin et al., 1981, 1982, 1983). The data are consistent with findings that rats are
more resistant than mice to the acute Clara cell toxicity of naphthalene (NTP, 2000; O'Brien et al.,
1985). No data are available for interspecies comparisons of the chronic toxicity of 2-
methylnaphthalene.
2-Methylnaphthalene does not appear to target the liver or kidneys. No histopathological
damage in these organs was reported in mice following oral exposure to doses as high as 114 mg/kg-
day for 81 weeks (Murata et al., 1997) or following acute intraperitoneal injections to doses associated
with mortality (1,000 mg/kg) (Griffin et al., 1981, 1983). Additionally, no changes in clinical chemistry
markers of liver or kidney damage were seen in the 81-week study (Murata et al., 1997). Rasmussen
et al. (1986) reported minimal changes in the livers of mice intraperitoneally injected with 2-
methylnaphthalene, but did not further describe these changes or specify the dose levels at which they
occurred. In addition, in vitro assays have demonstrated cytotoxicity caused by 2-methylnaphthalene
exposure in Sprague-Dawley rat cortical tubular epithelial cells and glomerular mesangial cells (Bowes
and Ramos, 1994; Parrish et al., 1998; Zhao and Ramos, 1998), but the relevance of these changes is
suspect given the absence of kidney changes in the acute and chronic in vivo exposure studies with 2-
methylnaphthalene in mice.
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4.5.2. Inhalation Exposure
No human studies regarding the inhalation toxicity of 2-methylnaphthalene are available.
In addition, no chronic or subchronic animal inhalation studies with 2-methylnaphthalene are
available.
Several acute inhalation toxicity studies are available. Signs of nervous system depression were
observed in rats exposed for 4 hours, and a transient decrease in respiratory rate was observed in mice
exposed for 6 minutes (Korsak et al., 1998). No signs of hematotoxicity in dogs were found after
exposure to mists of an unknown concentration of 2-methylnaphthalene for 50 minute periods over 4
consecutive days (Lorber, 1972). An acute inhalation study with a methylnaphthalene mixture
(exposure concentration unknown) reported that no clinical signs, mortality, or gross pathology were
found in rats (Union Carbide, 1982).
4.5.3. Dermal Exposure
The pulmonary toxicity of 2-methylnaphthalene appears dependent on the route of exposure.
Dermal exposure to a methylnaphthalene mixture induced pulmonary alveolar proteinosis in all exposed
mice within 30 weeks, compared to oral exposure to 2-methylnaphthalene, which induced pulmonary
alveolar proteinosis in roughly half of exposed animals within 81 weeks at approximately equal
administered dose levels (Murata et al., 1992, 1997). The findings suggest that first pass hepatic
metabolism associated with oral exposure may limit the amount of parent material reaching the lung.
Moreover, focal interstitial fibrosis in restricted areas and decreased survival were observed following
dermal exposure to a methylnaphthalene mixture (Emi and Konishi, 1985), but not following oral
exposure to 2-methylnaphthalene (Murata et al., 1997). These observations suggest that toxicity
differences may exist across oral and dermal routes. It should be noted that the higher incidence of
disease following dermal exposure to methylnaphthalene mixtures may be partly attributed to increased
absorption of methylnaphthalene in an acetone solution rather than methylnaphthalene in an aqueous
solution.
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4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER
SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN CARCINOGENICITY,
AND LIKELYMODE OF ACTION
4.6.1. Summary of Overall Weight-of-Evidence
Under EPA's Draft Revised Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999),
the data are inadequate for an assessment of human carcinogenic potential, based on the absence
of data concerning the carcinogenic potential of 2-methylnaphthalene in humans and limited equivocal
evidence in animals as discussed below.
4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence
No epidemiological studies or case reports regarding the carcinogenic potential of 2-methyl-
naphthalene in humans are available. Animal cancer bioassays are limited to an 81-week dietary study
(Murata et al, 1997). Murata et al. (1997) observed a statistically significant increase in the incidence
of lung adenomas and total lung tumors (adenomas and carcinomas combined) in male mice orally
exposed to 54.3 mg/kg-day 2-methylnaphthalene, but not in males orally exposed to 113.8 mg/kg-day
or in females exposed to either 50.3 or 107.6 mg/kg-day 2-methylnaphthalene. The incidences of lung
carcinomas alone were not significantly different from controls for any exposure group. No increased
incidence was seen for other tumor types. The study (Murata et al., 1997) was conducted in
conjunction with a study testing 0.075 and 0.15% 1-methyl-naphthalene in the diet (Murata et al.,
1993). Both studies shared a common control group of mice, and all mice were housed in the same
room. While Murata et al. (1993, 1997) did not quantitate the concentration of 1- or 2-
methylnaphthalene in the air, it should be noted that 2-methylnaphthalene is slightly more volatile than 1-
methylnaphthalene (vapor pressure of 0.068 and 0.087 mm Hg, respectively). Potential confounding
from possible inhalation exposure to 1- and 2-methylnaphthalene adds some uncertainty to the
relationship between oral exposure to 2-methylnaphthalene and the increased incidence of lung tumors.
Historical controls for B6C3F1 male and female control mice typically develop lung adenomas and
carcinomas spontaneously at an incidence of 19-24.8% and 7-8.5%, respectively ( NTP, 2002b, c).
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In addition, in a skin painting study where female mice were exposed to 2-methylnaphthalene
(equivalent to 32 • g/kg-day) plus BaP for 78 weeks, 2-methylnaphthalene plus BaP increased the
time-to-first-tumor (52 versus 58 weeks) and decreased the number of tumor-bearing animals
(44 versus 20%) when compared to mice treated only with BaP (Schmeltz et al., 1978). In this study
2-methylnaphthalene was not tested alone. The statistical significance of these findings could not be
determined from the data presented. The incidences of non-skin tumors were also not reported. No
mutagenicity was observed in tests using Salmonella typhimurium or cultured human lymphocytes
(Florin et al., 1980; Hermann, 1981; Kopper Co. Inc., 1982; Kulka et al., 1988).
There are no data indicating that the metabolism of 2-methylnaphthalene and the structurally-
related 1-methylnaphthalene are similar. This lack of information precludes the use of evidence for 1-
methylnaphthalene carcinogenicity (see Footnote 3, Section 4.2.1.2) as supporting evidence for 2-
methylnaphthalene carcinogenicity. It should be noted that evidence of carcinogenicity of the
structurally-related PAH naphthalene has been hypothesized to be due to, at least in part, to
metabolism via CYP-mediated ring epoxidation to reactive metabolites such as the 1,2-epoxide or 1,2-
quinone derivatives (Cho et al., 1995; Greene et al., 2000; Lakritz et al., 1996; NTP, 2000; Van
Winkle et al., 1999). The metabolic formation of ring epoxides is a relatively minor pathway for 2-
methylnaphthalene, whereas it is the principal pathway for naphthalene (NTP 2000; U.S. EPA, 1998c)
and, thus, the use of naphthalene carcinogenicity data as supporting evidence for 2-methylnaphthalene
carcinogenicity is of limited value.
4.6.3. Mode of Action Information
The mode of action for tumor formation in mid-dose male mice in the Murata et al. (1997)
study is not known. No evidence of bronchiolar necrosis or Clara cell damage was seen in the mice
exhibiting lung tumors after 81 weeks of dietary exposure to 2-methylnaphthalene (Murata et al.,
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1997). In addition, the available data do not support the hypothesis that pulmonary alveolar proteinosis
might be a precursor to lung tumor formation (Murata et al, 1993, 1997). For example, compared
with 1-methylnaphthalene, 2-methylnaphthalene induced equal or slightly higher incidences of
pulmonary alveolar proteinosis, but lower incidences of lung tumors. In addition, Murata et al. (1993)
reported that the numbers of mice developing pulmonary alveolar proteinosis and lung tumors following
exposure to 1-methylnaphthalene were not statistically correlated, and the sites of development of
alveolar proteinosis and lung tumors were not always clearly linked.
4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
4.7.1. Possible Childhood Susceptibility
No studies are available regarding the adverse effects of 2-methylnaphthalene in children or
prenatal, neonatal, or postnatal animals.
When compared to adults, infants and children who develop pulmonary alveolar proteinosis
have more severe symptoms and a poor prognosis for survival. Whole lung lavage, the standard
treatment for this disorder, is unavailable to infants because of the difficulty in performing intubation and
lavage techniques on their small airways. In addition, studies in children with pulmonary alveolar
proteinosis provide suggestive evidence that congenital deficiencies in the expression of some proteins,
such as surfactant protein B, may contribute to this disease (Mazzone et al., 2001; Mildenberger et al.,
2001; Wang et al., 1997). Such individuals may be more sensitive than the general population to the
toxic effects of repeated exposure to 2-methylnaphthalene.
The toxicokinetics of xenobiotics can vary widely between children and adults due to
immaturity of the phase I and phase n enzyme systems and clearance mechanisms in children (Ginsberg
et al., 2002). Studies in animals indicate an equivocal effect of metabolism on the toxicity following 2-
methylnaphthalene exposure (Griffin et al., 1982, 1983; Honda et al., 1990). The importance and
identity of the specific isozymes and/or metabolites responsible for these adverse effects are not well
understood.
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4.7.2. Possible Gender Differences
Gender-specific susceptibility to 2-methylnaphthalene toxicity is not known. While clinical
cases of pulmonary alveolar proteinosis are 3-fold more common in men than in women (Mazzone et
al, 2001), no data are available regarding gender sensitivity to 2-methylnaphthalene in humans.
The available animal data do not provide definitive information for gender differences in
susceptibility to 2-methylnaphthalene toxicity. Acute animal testing data suggested that females were
somewhat more sensitive to 2-methylnaphthalene toxicity than males (Union Carbide, 1982). For
example, gavage studies in rats determined LD50 values of 4.29 mL/kg for males and 3.73 mL/kg for
females (4,310 and 3,270 mg/kg, respectively) and dermal studies in rabbits calculated LD50 values of
6.1 mL/kg in males and 4.8 mL/kg in females (6,130 and 4,790 mg/kg, respectively). Although no
significant differences in the incidence of pulmonary alveolar proteinosis were observed between male
and female B6C3F1 mice given equivalent dietary doses of 2-methylnaphthalene for 81 weeks, only
exposed male (rather than female) mice showed an increased incidence of lung tumors (Murata et al.,
1993, 1997).
4.7.3. Other
No data are available regarding the effects of 2-methylnaphthalene on other potentially
susceptible populations. Individuals with existing clinical pulmonary alveolar proteinosis may be more
susceptible to the effects of 2-methylnaphthalene than healthy individuals. In addition, individuals with
risk factors for pulmonary alveolar proteinosis include persons with myeloid leukemias, pulmonary
infection, a history of smoking, or inhalation of silica or certain heavy metals may be more susceptible
(Mazzone et al., 2001; Seymour and Presneill, 2002; Wang et al., 1997).
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5. DOSE RESPONSE ASSESSMENT
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical Effect - with Rationale and Justification
No epidemiology studies or case reports are available which examine the potential effects of
human exposure to 2-methylnaphthalene by oral exposure.
Only one chronic study is available in which animals were orally exposed to 2-methyl-
naphthalene (Murata et al., 1997). This study was chosen as the principal study. Male and female
B6C3F1 mice (50/sex/group) were fed diets containing 0, 0.075 or 0.15% of 2-methylnaphthalene for
81 weeks. Numerous endpoints were evaluated, including histology for more than 24 tissues,
hematology, and serum chemistry. Pulmonary function was not evaluated in either control or exposed
mice. Mean growth curve data showed reduced weight gain in males at both doses and in females
exposed to the high dose. While a statistically significant reduction in final body weight was observed
only in the high-dose male group, the decrease was not considered a biologically significant effect in this
assessment. Several other statistically significant differences in blood parameters and organ weights
between control and exposure groups were reported. However, the biological significance of these
differences is unclear because no data were provided regarding the magnitude of the response or
exposure levels at which they occurred. Affected variables included relative and absolute brain and
kidney weights, serum neutral fat levels, and differential counts of neutrophils and lymphocytes. A
statistically significant increase in the incidence of pulmonary alveolar proteinosis was observed at both
doses for males (54.3 and 113.8 mg/kg-day) and females (50.3 and 107.6 mg/kg-day). Incidences for
control through high-dose groups were: 4/49, 21/49, and 23/49 for males, and 5/50, 27/48, and 22/48
for females, respectively. No other incidence of non-neoplastic effects were identified in exposed
groups of male and female mice. For these reasons, pulmonary alveolar proteinosis is chosen as the
critical effect.
The selection of pulmonary alveolar proteinosis as the critical effect following oral exposure to
2-methylnaphthalene is supported by dermal studies with a methylnaphthalene mixture containing both
2- and 1-methylnaphthalene, in an approximate 2:1 ratio, respectively. All female B6C3F1 mice
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dermally exposed to 119 mg/kg methylnaphthalene twice weekly for 30 weeks, or 238 mg/kg twice
weekly for 20 weeks exhibited pulmonary alveolar proteinosis (Murata et al., 1992). Similarly, a 61-
week study reported lipid pneumonia in B6C3F1 mice dermally exposed to 118.8 mg/kg-day
methylnaphthalene mixture (Emi and Konishi, 1985).
Other available oral toxicity studies of 2-methylnaphthalene are of prechronic duration.
Fitzhugh and Buschke (1949) conducted a study on cataract formation in rats fed 2-methyl-naphthalene
for at least 2 months. No cataracts were observed and no other endpoints were studied. Murata et al.
(1997) conducted a range-finding study in which groups of B6C3F1 mice (10/sex/group) were fed
diets containing approximate average daily doses of 0, 31, 92, 276, 827, or 2,500 mg/kg-day 2-
methylnaphthalene for 13 weeks. No histopathological effects were observed in tissues and organs of
male or female mice exposed to 827 or 2,500 mg/kg-day. Decreased weight gain was observed at the
three highest dose levels in both males and females, and was attributed to food refusal (Murata et al.,
1997). The absence of pulmonary alveolar proteinosis in prechronically exposed mice, which were
exposed to much higher doses than those used in the chronic study, suggests that the development of
pulmonary alveolar proteinosis may require chronic duration exposure.
Although Rasmussen et al. (1986) reported minimal liver damage in male Swiss-Webster mice
injected with 142 or 284 mg/kg 2-methylnaphthalene, no histological evidence of liver or kidney
damage was seen in male C57BL/6J (Griffin et al., 1981, 1982) or male DBA/2J mice (Griffin et al.,
1983) at doses up to 1,000 mg/kg. An acute dose of 1,000 mg/kg 2-methylnaphthalene was frankly
toxic, as evidenced by mortality observed in 3/10 mice dosed at this concentration (Griffin et al., 1981,
1983).
A limitation of the principal study by Murata et al. (1997) is the occurrence of pulmonary
alveolar proteinosis in control mice. The authors described the condition as being less pronounced but
similar to the adverse lung effects observed in the 2-methylnaphthalene exposed mice. The authors also
indicated that pulmonary alveolar proteinosis had not been observed in more than 5,000 B6C3F1
control mice, and speculated that the background incidence may have been elevated by the inhalation of
volatilized test chemicals and poor room ventilation. The study was conducted in conjunction with a
study testing 0.075 and 0.15% 1-methylnaphthalene in the diet (Murata et al., 1993). Both studies
shared a common control group of mice, and all mice were housed in the same room (Murata et al.,
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1993, 1997). While Murata et al. (1993, 1997) did not quantitate the concentration of either 1- or 2-
methylnaphthalene in the air, it should be noted that 2-methylnaphthalene is slightly more volatile than 1-
methylnaphthalene (vapor pressure of 0.068 mm Hg compared to 0.087 mm Hg, respectively).
Potential confounding from possible inhalation exposure to 1- and 2-methylnaphthalene adds some
uncertainty to the dose-response relationship between oral exposure to 2-methylnaphthalene and
pulmonary alveolar proteinosis.
5.1.2. Methods of Analysis - Including Models
The data were analyzed using benchmark dose (BMD) modeling for the derivation of the point
of departure. Based on the Murata et al. (1997) study, the critical effect is pulmonary alveolar
proteinosis. While the principal study for 2-methylnaphthalene shows a dose-response relationship
between oral exposure to 2-methylnaphthalene and pulmonary alveolar proteinosis (Murata et al.,
1997), the data are somewhat uncertain for characterizing risk at lower exposures. First, the potential
confounding from possible inhalation exposure to 1- and 2-methylnaphthalene by all animals
complicates the quantitative assessment of the dose-response relationship, at least in how to interpret
the incidence of pulmonary alveolar proteinosis in control animals. No data were found which
characterize the response of mice to either chemical alone by inhalation. Since the incidence of
pulmonary alveolar proteinosis was reported to be unusually high compared with historical controls, it
may not be a relevant baseline. Moreover, the similar degree of pulmonary alveolar proteinosis in the
two exposed groups, both averaging about 45%, provides little information concerning the shape of the
dose-response relationship expected at lower exposures (see Table Bl and the BMDS graph in the
model output in Appendix B). Nevertheless, some judgments about these issues can be made which
allow estimating an RfD from these data, as discussed below.
Given the possible simultaneous inhalation exposure to 1- and 2-methylnaphthalene during the
oral exposure study, consideration of concurrent and historical control information may provide some
bounds on the degree of effects that can be associated with oral exposure to 2-methylnaphthalene. The
concurrent control group is generally the most relevant comparison group, unless there is documentation
that the control group was treated differently than the exposed groups. There is no reason to believe
that the control group was treated any differently than the exposure groups in the principal study. Even
if there were secondary exposure to volatilized test materials and it can be assumed that all animals
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were similarly exposed, the concurrent control group provides the most relevant baseline for assessing
adverse effects in the exposed groups.
Alternatively, use of the historical control information may provide an upper bound on the
magnitude of effects associated with oral exposure to 2-methylnaphthalene. Several hypothetical
situations can be used to characterize the contribution of 1-methylnaphthalene to the observed effects.
In the simplest case, if coexposure to 1-methylnaphthalene has no adverse effects, and 2-
methylnaphthalene exposure by inhalation is an unavoidable consequence of exposure to 2-
methylnaphthalene in the diet, then the appropriate control group would be one which could have been
isolated from any possible inhalation exposure of 2-methylnaphthalene. The historical control group
would then be considered the best available comparison group. This comparison would yield the
largest difference in effect level between control and exposed groups.
It is not clear, however, that inhalation exposure to 1-methylnaphthalene has no association with
pulmonary alveolar proteinosis. The animals exposed simultaneously to 1-methylnaphthalene in their
diet demonstrated incidences of pulmonary alveolar proteinosis similar to that of the 2-
methylnaphthalene-exposed animals (see Footnote 2, Section 4.2.1.2.), indicating that oral 1-
methylnaphthalene exposure is associated with pulmonary alveolar proteinosis. Therefore, use of the
concurrent control is important to adjust for any effect of simultaneous 1-methylnaphthalene exposure.
If the effect of 1-methylnaphthalene is additive and constant across the experimental groups, the
concurrent control incidence effectively accounts for this. If, however, 1-methylnaphthalene and 2-
methylnaphthalene interact, then the concurrent control incidence may be a low estimate of the
contribution of 1-methylnaphthalene in the groups purposely exposed to 2-methylnaphthalene. In that
case, use of the concurrent control is more relevant than the historical control, but would still lead to an
overestimate of the effect attributable to 2-methylnaphthalene at a given dose level, due to
underestimating the contribution of 1-methylnaphthalene in the 2-methylnaphthalene-exposed groups.
In other words, the benchmark dose (BMD) estimate would be lower than it should be. Without data
to clarify whether there is an interaction (or how large it may be), there is no way to estimate the impact
on the BMD.
In summary, use of the historical control provides an upper bound on the degree of effect
associated with oral exposure to 2-methylnaphthalene, while the concurrent control accounts for any
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additive effects of simultaneous inhalation exposure to 1-methylnaphthalene in this study. If
simultaneous inhalation exposure to 1-methylnaphthalene is associated with an interactive effect with 2-
methylnaphthalene, use of the concurrent control would also provide a high-end estimate of the risk of
pulmonary alveolar proteinosis at a given exposure level. Both control groups are considered below in
characterizing the point of departure.
Both commonly used approaches for identifying a point of departure for low-dose
extrapolation, the LOAEL/NOAEL methodology and BMD modeling, have some relevance for this
data set. The LOAEL/NOAEL methodology is not as dependent on the level of response in the
control group as BMD modeling, as long as the response level in the exposed group is significantly
different from the control. The lower dose in the Murata et al. (1997) data set is easily identified as the
LOAEL, regardless of whether it is compared with the concurrent or the historical control groups. In
addition, the similarity of responses in the orally exposed groups has very little impact on identifying the
LOAEL. The NOAEL/LOAEL approach would yield a LOAEL of 52.3 mg/kg-day, using the
combined male and female data.
On the other hand, BMD modeling can provide a point of departure which is consistent with
more of the observed data than the LOAEL/NOAEL approach uses, by taking into account the degree
of response at the point of departure, and addressing the variability inherent in the data. In this case,
the shape of the dose-response at lower exposures would still be somewhat uncertain, however.
Similar responses in the dose groups suggest that the observed plateau may continue somewhat into the
lower exposure range, but not much more can be inferred about the low-dose behavior of the
relationship. The incidence data for males and females were fit using all dichotomous variable models
available in the BMDS Version 1.3.2. software (U.S. EPA, 2002); the results are shown in Appendix
B. Note that the incidence of pulmonary alveolar proteinosis for male and females were not different or
statistically significant from each other (p • 0.05, using Fisher's exact test), indicating neither sex was
clearly more sensitive. Consequently, the BMD modeling analysis also considers the combined
incidences for each exposure group to strengthen the quantitative results.
Much of the BMD modeling did not provide adequate fits, as indicated by chi-square
goodness-of-fit p-values less than 0.1 (see Appendix B). None of the models fit the female mouse data
well, nor the combined female and male data, due to the non-monotonic response pattern in female
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mice. For the male mouse data, the application of the log-logistic model provided the best fit, as
indicated by the lowest Akaike Information Criterion (AIC) among the models with adequate fits
(p>0.1), according to the criteria in the draft BMDS guidance (U.S. EPA, 2000c). The BMD10 and
BMD05 from the log-logistic model were 13.7 and 6.9 mg/kg-day, respectively, for pulmonary alveolar
proteinosis in male mice exposed to 2-methylnaphthalene in the diet for 81 weeks (Murata et al, 1997).
The lower 95% confidence limit on the BMD10 and BMD05 (i.e., BMDL10 and BMDL05 ) were 9.1
and 4.5 mg/kg-day, respectively. However, this model does not fit the data well since the largest
deviation in the fit occurs at the low dose response where it is especially important to have an adequate
prediction. Also, even though the male and female responses were not different or statistically
significant from each other, the female response (55%) was somewhat higher than the male response
(43%) at the low dose (see Table Bl). The biological significance of this difference is not clear,
especially because of the uncertain nature of the background incidence of pulmonary alveolar
proteinosis in this study, as noted earlier (see Sections 5.1.1. and 5.1.2.). Therefore, reliance on the
combined male and female incidence data appears to be the most appropriate approach.
Another approach to fit the low dose responses observed in male and female mice is to exclude
the high dose groups from the combined data set. This practice is justified by the following
considerations (U.S. EPA, 2000c). Without a mechanistic understanding of how pulmonary alveolar
proteinosis results from exposure to 2-methylnaphthalene, data from exposures much higher than that
associated with the benchmark response do not provide very much information about the shape of the
response in the region of the benchmark response. The lack of fit for the full data set appears to be due
to the characteristics of the high dose groups, where the response plateaus. Although dropping the high
dose groups ignores some of the data and decreases the degrees of freedom for modeling, it is a
reasonable approach because the focus of BMD analysis is on the low dose and response region (U.S.
EPA, 2000c). The BMDS quantal-linear model provides a model that is closest to a straight line,
which is all that can be justified for modeling essentially two data points (the combined control groups
and the similar male and female low dose groups). The resulting model parameters are provided in
Appendix B. Note that goodness-of-fit measures are irrelevant in this case, since a straight line is
defined by two points.
A benchmark response level of 5% extra risk of the critical effect, pulmonary alveolar
proteinosis, was selected for this assessment. This effect is similar to a disorder of unknown etiology
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that has been identified in humans. If this disorder were to occur in humans following exposure to 2-
methylnaphthalene, it is anticipated that children may be more susceptible especially since children
affected with the disorder often experience more severe symptoms than adults. Thus, a 5% extra risk
of pulmonary alveolar proteinosis was judged to be an appropriate level of extra risk for this critical
effect. The BMD10 and BMD05 from the quantal-linear model was 9.6 and 4.7 mg/kg-day for
pulmonary alveolar proteinosis in male and female mice exposed to 2-methylnaphthalene in the diet for
81 weeks respectively (Murata et al, 1997). The lower 95% confidence limit on the BMD10 and
BMD05 (i.e., BMDL10 and BMDL05) was 7.3 and 3.5 mg/kg-day respectively.
Limited modeling was carried out using the reported historical incidence of 0 cases of
pulmonary alveolar proteinosis in -5,000 control mice and the combined male and female incidence
data from the exposed groups. The fits paralleled those using the concurrent control, with none
providing an adequate fit. The BMD10 was 8.2 mg/kg-day, and the BMDL10 was 6.5 mg/kg-day. This
BMDL10 is marginally lower than the value of 7.3 mg/kg-day derived using the concurrent control
group. Given the lack of information confirming any confounding exposures and whether or not an
interaction would be expected, the concurrent control appears to provide the most suitable baseline for
estimating the extra risk of developing pulmonary alveolar proteinosis. Therefore, the high dose groups
were dropped and a quantal-linear model was fit to the low-dose male and female data, as described
below.
5.1.3. RfD Derivation - Including Application of Uncertainty Factors (Ufs)
Using benchmark dose modeling, the BMDL05 of 3.5 mg/kg-day for 5% extra risk of
pulmonary alveolar proteinosis in mice exposed to 2-methylnaphthalene in the diet for 81 weeks
(Murata et al., 1997) was selected as the point of departure for the RfD. To calculate the RfD using
the BMDL05, several uncertainty factors (UFs) were applied.
A total UF of 1000 was applied to this effect level: 10 for extrapolation for interspecies
differences (UFA: animal to human); 10 for consideration of intraspecies variation (UFH: human
variability); and 10 for deficiencies in the database (UFD). Uncertainty factors for subchronic to chronic
exposure extrapolation and for LOAEL to NOAEL extrapolation were not considered necessary.
These decisions are described in greater detail below.
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A 10-fold UF was used to account for uncertainty in extrapolating from laboratory animals to
humans (i.e., interspecies variability). No information was available regarding the toxicity of 2-
methylnaphthalene in humans exposed orally. No information was available to assess toxicokinetic
differences between animals and humans.
A 10-fold UF was used to account for variation in sensitivity among members of the human
population (i.e., interindividual variability). This UF was not reduced due to a lack of human oral
exposure data.
A 10-fold UF was used to account for uncertainty associated with deficiencies in the data base.
One chronic duration oral toxicity study in one animal species (mice) is available (Murata et al, 1997).
The data base lacks adequate studies of oral developmental toxicity, reproductive toxicity, and
neurotoxicity. The data base also lacks a 2-generation reproductive toxicity study.
An UF was not needed to account for subchronic to chronic extrapolation because a chronic
study (81 weeks) was used to derive the RfD.
An UF for LOAEL-to-NOAEL extrapolation was not considered as such, since benchmark
dose modeling was used to determine the point of departure. While the 5% extra response level used
to derive the RfD is not a no-response level, some consideration of what level of extra risk of
pulmonary alveolar proteinosis constitutes a minimal health risk is appropriate.
The RfD for 2-methylnaphthalene was calculated as follows:
RfD = BMDL05 -UF
= 3.5mg/kg-day- 1000
= 0.004 mg/kg-day
In addition to the uncertainties noted above, there is model uncertainty owing to the lack of
actual dose-response information or mode of action information in the region of the dose-response
where the point of departure is estimated. As noted earlier, the responses in 2-methylnaphthalene-
exposed animals suggest a continuation of the plateau into the lower exposure region, so using a linear
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model may provide a higher benchmark dose than is appropriate. In addition, while BMDS was used
to generate a lower bound on the estimated benchmark dose, the lower bound probably describes too
narrow a confidence limit on the benchmark dose. This is because the uncertainty in the data set cannot
be adequately described without the high dose responses.
In comparison, the NOAEL/LOAEL approach would yield a LOAEL of 52.3 mg/kg-day,
using the combined male and female data. This dose would be adjusted by a LOAEL-to-NOAEL
extrapolation uncertainty factor of up to 10 in order to estimate an RfD. The observed response at the
LOAEL, relative to the concurrent control, was approximately 44%, in terms of extra risk: ER = [P(d)-
P(0)]/[1-P(0)] = [48/98 - 9/99]/[l - 9/99] = 0.44, where P(d) is the proportion responding at dose d
(here the low dose), and P(0) is the proportion responding at dose 0 (control). Use of the full LOAEL-
to-NOAEL uncertainty factor of 10 appears justified, and it might be argued that a factor of 10 is not
enough. However, the default of 10 would contribute to a total UF of 10,000 given the other
uncertainty factors already considered. The RfD/RfC Technical Panel Report (U.S. EPA, 2002)
recommended "limiting the total uncertainty factor applied for any particular chemical to no more than
3000 and avoiding the derivation of a reference value that involves application of the full 10-fold
uncertainty factor in four or more areas of extrapolation."
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
No epidemiology studies or case reports are available which examined the potential effects of
human inhalation exposure to 2-methylnaphthalene.
No chronic or prechronic studies are available that exposed animals by inhalation to 2-
methylnaphthalene.
Two reports are available on acute exposure of animals to 2-methylnaphthalene; neither are
suitable for RfC derivation. Lorber (1972) investigated hematoxicity endpoints in intact and
splenectomized dogs exposed to mists of 2-methylnaphthalene (at unknown concentrations) for 41-50
minutes for 4 consecutive days. No clear evidence of hematoxicity was observed. Korsak et al.
(1998) exposed rats by inhalation to 2-methylnaphthalene for 4 hours to evaluate neurotoxicity, and
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mice for 6 minutes to evaluate sensory/respiratory irritation (Korsak et al., 1998). In rats, none of the
concentrations tested affected a neuromuscular test (rotarod performance) but two concentrations
decreased pain sensitivity (measured by latency of paw-lick response to a heated surface). In mice,
rapid but reversible decreases in respiratory rate were
observed with the response magnitude increasing with increasing exposure concentration.
An RfC for 2-methylnaphthalene cannot be derived in the absence of an inhalation study of
sufficient duration that evaluates a comprehensive array of endpoints to establish a NOAEL or
LOAEL. A route-to-route extrapolation is not currently possible. No toxicokinetic models are
available for 2-methylnaphthalene, but there is evidence to suggest that its ability to induce pulmonary
alveolar proteinosis in mice may vary across routes of exposure (as discussed in Section 4.5.2.).
5.3. CANCER ASSESSMENT
As discussed in Section 4.6.1., the available data base for 2-methylnaphthalene is inadequate
to assess human carcinogenic potential. Limited evidence of carcinogenicity in animals was provided
by an 81-week dietary study in B6C3F1 mice (Murata et al., 1997). A statistically significant increase
in the incidence of lung adenomas and total lung tumors (adenomas and carcinomas combined) for the
low-dose male group (54.3 mg/kg-day) was observed when compared to controls. However, no
evidence of carcinogenicity was observed in male mice exposed to the high dose (113.8 mg/kg-day) or
in female mice (50.3 or 107.6 mg/kg-day). No evidence of a trend of increasing tumor incidence with
increasing dose was seen for males or females. Lack of an apparent dose-response relationship makes
the data unsuitable for quantitative assessment of carcinogenic potential. No statistically significant
elevations in other tumor incidences were seen in any exposure group.
A dermal cocarcinogenicity study was an unsuitable test of 2-methylnaphthalene carcinogenicity
because 2-methylnaphthalene was tested only in a mixture with benzo[a]pyrene (BaP) (Schmeltz et al.,
1978).
In addition, no genotoxicity studies in humans or animals and studies investigating potential
germ-line mutations are available. Data from in vitro short-term assays provide limited evidence for
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genotoxic activity of 2-methylnaphthalene (Florin et al, 1980; Harvey and Halonen, 1968; Hermann,
1981; Kopper Co. Inc., 1982; Kulka et al., 1988; Weis et al., 1998).
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
2-Methylnaphthalene (CAS No. 91-57-6) is a natural component of crude oil and coal and is
found as a pyrolytic byproduct from the combustion of tabacco, wood, petroleum-based fuels and coal.
It is also used as a chemical intermediate in the synthesis of vitamin K.
No data are available regarding the potential toxicity of 2-methylnaphthalene in exposed
humans via the oral route. However, the available animal data indicate that the lung is a sensitive target
organ. The critical effect observed in mice following chronic oral exposure to 2-methylnaphthalene
(Murata et al, 1997) and chronic dermal exposure to methylnaphthalene mixtures (Emi and Konishi,
1985; Murata et al., 1992) was pulmonary alveolar proteinosis. This effect was characterized by
accumulation of foamy cells, cholesterol crystals, and proteinaceous materials rich in lipids in the lumen
of the pulmonary alveoli (Murata et al., 1997). Since the effect is similar to a disorder of unknown
etiology that has been observed in humans, it is anticipated that humans exposed to 2-
methylnaphthalene may develop pulmonary alveolar proteinosis.
In humans, pulmonary alveolar proteinosis is characterized by symptoms such as dyspnea and
cough with possible decreased pulmonary function, identified by decreased functional lung volume and
reduced diffusing capacity. It has not been associated with airflow obstruction (Lee et al., 1997;
Mazzone et al., 2001; Wang et al., 1997). Cases of pulmonary alveolar proteinosis in humans have not
been directly associated with exposure to 2-methylnaphthalene.
The effects of prechronic or chronic inhalation exposure to 2-methylnaphthalene have not been
studied in humans or animals. No suitable toxicokinetic models are available to extrapolate between
routes of exposure. Since chronic exposure to 2-methylnaphthalene by oral and dermal routes targets
the lung causing pulmonary alveolar proteinosis, it is plausible that similar adverse effects may be seen
after chronic inhalation exposure to 2-methylnaphthalene. However, no conclusions can be drawn from
the current data regarding potential exposure-response relationships for chronic inhalation exposure.
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Under the Draft Revised Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999), the
available data for 2-methylnaphthalene are inadequate to assess human carcinogenic potential.
There are no studies of the potential carcinogenicity of 2-methylnaphthalene in humans, and only one
adequate cancer animal bioassay is available (Murata et al, 1997). While the study found an increased
incidence of total lung tumors and adenomas in male mice, but not female mice, exposed to 2-
methylnaphthalene in the diet for 81 weeks, the incidence was only increased at the lower of two
exposure levels. The relevance of these observations to humans is uncertain. Other animal species have
not been tested and results from short-term genotoxicity tests provide no supporting evidence for the
carcinogenicity of 2-methylnaphthalene. As such, the available evidence of 2-methylnaphthalene
carcinogenicity is limited and insufficient to determine that 2-methylnaphthalene is carcinogenic to
humans.
6.2. DOSE RESPONSE
6.2.1. Noncancer/Oral
The RED of 0.004 mg/kg-day was calculated from a BMDL05 of 3.5 mg/kg-day for 5% extra
risk for pulmonary alveolar proteinosis in mice exposed to 2-methylnaphthalene in the diet for 81
weeks. A total UF of 1000 was used: 10 for interspecies variability, 10 for interindividual variability,
and 10 for data base deficiencies.
No data are available regarding the potential toxicity of 2-methylnaphthalene in exposed
humans via the oral route and no suitable toxicokinetic or toxicodynamic models have been developed
to reduce uncertainty in extrapolating from mice to humans.
The extent of variability in susceptibility to 2-methylnaphthalene among humans is unknown;
representing another important area of uncertainty in the RfD. Chronic experiments relevant to 2-
methylnaphthalene exposure have only been performed in one strain of one species, B6C3F1 mice.
Subpopulations expected to be more susceptible to 2-methylnaphthalene toxicity include those with
limited or altered capacity to metabolize and detoxify 2-methylnaphthalene, and people with existing
pulmonary alveolar proteinosis or those with risk factors for developing the disease.
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The principal study for the RfD (Murata et al, 1997) examined a comprehensive number of
endpoints, including extensive histopathology, and tested two dose levels using sufficient numbers
(50/group) of both sexes of B6C3F1 mice. Potential confounding from possible inhalation exposure of
controls to 1- and 2-methylnaphthalene in this study adds some uncertainty to the dose-response
relationship. Aside from this study, the oral data base is sparse. No information is available for the
testing of 2-methylnaphthalene in assays of developmental toxicity, reproductive toxicity, and
neurotoxicity.
Relative to a NOAEL/LOAEL approach for RfD derivation, the use of BMD modeling reduces
the uncertainty associated with the RfD by incorporating information available for the control and high-
exposure groups in addition to the LOAEL. Additional uncertainties arise when extrapolating from the
relatively high exposure levels used in the study (Murata et al., 1997) to lower exposure levels, and a
lack of empirical data identifying a NOAEL. The BMDS quantal-linear model was selected because it
provided the best fit to the data (Appendix B).
6.2.2. Noncancer/Inhalation
The data base for inhalation exposure is limited to several acute studies and therefore, was
unsuitable for calculating an RfC value.
6.2.3. Cancer/Oral and Inhalation
Under the Draft Revised Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999), the
data base for 2-methylnaphthalene is inadequate to assess human carcinogenic potential. As such,
the data are unsuitable to calculate quantitative cancer risk estimates for humans.
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION
-------
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION
The support document and IRIS summary for 2-methylnaphthalene have undergone both internal peer
review performed by scientists within EPA and a more formal external peer review performed by
scientists in accordance with EPA guidance on peer review (U.S. EPA, 1998b, 2000a). Comments
made by the internal reviewers were addressed prior to submitting the documents for external peer
review and are not part of this appendix. The four external peer reviewers were tasked with providing
written answers to general questions on the overall assessment and on chemical-specific questions in
areas of scientific controversy or uncertainty. A summary of significant comments made by the external
reviewers and EPA's response to these comments follows:
The reviewers made several editorial suggestions to clarify specific portions of the text. These changes
were incorporated in the document as appropriate and are not discussed further.
COMMENTS FROM EXTERNAL PEER REVIEW
Overall Document Quality
Questions 1 and 2: How well were the data from individual studies characterized and are the
conclusions that are drawn from each study valid? How well are the data integrated into an overall
conclusion and characterization of hazard as presented in Sections 4.5, 4.6, 5, and 6?
Comment: The reviewers agreed that the data from individual studies was well characterized and
properly incorporated into an overall conclusion and characterization of hazard. One reviewer noted
that despite the availability of previous studies evaluating the toxicity of 2-methylnaphthalene, the data
base for this chemical has deficiencies such as the lack of human exposure and carcinogenicity studies.
The reviewer felt that as a result of the sparse data base, data integration in the dose-response was
lacking.
Response: The absence of additional long-term carcinogenicity and inhalation studies, in addition to
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deficiencies in the data base is discussed in Sections 5.1.3., 5.2., and 5.3. In addition, all available data
on 2-methylnaphthalene (oral, limited inhalation, intraperitoneal studies, and studies on
methylnaphthalene mixtures containing 2-methylnaphthalene) has been described in depth and
integrated in the dose-response assessment.
Question 3: What is the overall quality of the document?
Comment: All four reviewers commented on the high overall quality of the document.
General Questions and Issues
Question 1: Are any other data/studies available that are relevant (i.e., useful for the hazard
identification or dose-response assessment) to the assessment of the adverse health effects, both cancer
and noncancer, of this chemical?
Comments: All reviewers agreed that there were no other available studies that were relevant to the
hazard identification or dose-response assessment for 2-methylnaphthalene (both cancer and
noncancer)
RfD Derivation
Question 1: Under Section 5.1.1., Choice of Principal Study and Critical Effect, the RfD is based
on an 81-week study in mice fed 2-methylnaphthalene (Murata et al, 1997). The critical effect
observed was pulmonary alveolar proteinosis. This study was conducted concurrently with an 81-week
study in mice fed 1-methylnaphthalene (Murata et al., 1993) with a shared control group between the
1- and 2-methylnaphthalene exposure groups. All animals were housed in the same room. While the
incidence of pulmonary alveolar proteinosis in controls was increased (for the particular strain of mouse
utilized in both studies), the authors suggested it may have been due to volatilized 1- and 2-
methylnaphthalene. Is use of the Murata et al. (1997) study justified and is the rationale for this study
adequately explained in the Toxicological Review (Section 5.1.1.) in light of incidence of the critical
effect in the control group?
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Comments: All reviewers agreed that the use of the Murata et al. (1997) study was justified and that
the rationale for this study was adequately explained in the document.
One reviewer expressed interest in why the principal study was published after a study evaluating the
toxicity of 1-methylnaphthalene, even though these studies were conducted concurrently and by the
same laboratory. The reviewer also questioned why the principal study was published as a short
communication. The reviewer suggested that the study authors could be contacted, but that the
information was not necessary for inclusion in the document. This reviewer felt that the information
would be helpful in determining the confidence in the principal study.
Another reviewer noted that the possibility of interactive effects was not throughly explained in relation
to the hypothesized simultaneous exposures to airborne 1- and 2-methylnaphthalene in the Murata et al.
(1997) study and the subsequent incidence of pulmonary alveolar proteinosis in the control group.
Response: The principal study authors were contacted and questioned about their choices concerning
the type of publication and the delay in publication of the 2-methylnaphthalene data. The authors
indicated that the delay in publishing the 2-methylnaphthalene data (Murata et al., 1997) following the
publication of 1-methylnaphthalene data (Murata et al., 1992) was simply a matter of time constraints.
The study authors also indicated that the short communication was used as a result of the existence of
an extensive and detailed publication on 1-methylnaphthalene that was conducted concurrently under
the same protocol and conditions.
Although the possibility of interactive effects was not thoroughly explained in the document, an
expanded discussion of this issue has been added to the document.
Question 2: Under Section 5.1.1., Choice of the Principal Study and the Critical Effect, the critical
effect is identified as pulmonary alveolar proteinosis. Is this the correct critical effect and is it adequately
described? Is this critical effect biologically significant? Finally, does the information presented from
animal studies mirror what is know about the disease in humans and is this information adequately
described?
Comment: All reviewers agree that pulmonary alveolar proteinosis is the correct critical effect and it
A-3
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has been throughly described. The reviewers also agreed that this effect is biologically significant. One
reviewer indicated that the disease state in humans had not been adequately described in terms of cell
types involved in reducing pulmonary function and histopathology of pulmonary alveolar proteinosis,
and indicated that it was unclear as to whether the pathology of this disorder in humans is similar to that
in rodents. The reviewer also commented that it has been shown that pulmonary alveolar proteinosis
may lead to decreased pulmonary function in humans. The reviewer also noted that changes in type n
pneumocytes could provided a logical explanation for reduced pulmonary function in mice treated with
2-methylnaphthalene. Another reviewer requested that the relationship of the critical effect to drug-
induced phospholipidosis be addressed in this section of the assessment.
One reviewer noted that the dose-response assessment focused only on the incidence of the critical
effect in males and this decision should either be explained more clearly and transparently or that both
the male and female incidence data should be utilized in the dose-response assessment.
Response: Section 5.5.1. has been augmented and revised for clarity. The description of the cell
types involved in the pathogenesis of primary acquired and the congenital forms of pulmonary alveolar
proteinosis in humans (less is known concerning the pathogenesis of the secondary acquired form of the
disorder) has been more extensively discussed. This section also provides evidence that the
pathogenesis of pulmonary alveolar proteinosis may be similar for rodents and humans.
The concept that, changes in type n pneumocytes offers a logical explanation for reduced pulmonary
function in mice treated with 2-methylnaphthalene is somewhat equivocal. Human pulmonary alveolar
proteinosis has been associated with reduced pulmonary function. This association has not been shown
in rodents. In addition, pulmonary function was not an endpoint measured in the Murata et al. (1997)
study.
Drug-induced phospholipidosis is a condition that develops as the result of exposure to drugs that have
a cationic lipophilic structure and is characterized by the: accumulation of phospholipids in cells,
appearance of lamellar inclusion bodies, accumulation of the inducing drug in association with the
increased phospholipids, and reversibility of alterations after removal of exposure to the drug (Reasor
and Kacew, 2001). The disorder shares some common characteristics with pulmonary alveolar
proteinosis (also known as pulmonary phospholipidosis), but is not identical. While both involve the
A-4
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accumulation of lipid in the alveoli, pulmonary alveolar proteinosis is not characterized by the
accumulation of an inducing agent in the alveoli of the lung and there is no evidence that removal of
exposure to the inducing agent is associated with reversibility of the disease. Inhalation toxicity studies
in animals have failed to produce the clinical features associated with pulmonary alveolar proteinosis
and human lung biopsy samples from patients with pulmonary alveolar proteinosis typically do not
contain actual particulate matter (Seymour and Presneill, 2002). In addition, acute exposure to silica
dust or other inhaled environmental or industrial chemicals rarely leads to the development of secondary
acquired proteinosis and has only been documented in a few case studies (Seymour and Presneill,
2002).
Since it is recognized that the female mouse incidence data have utility in the assessment, incidence data
for both the males and females have been considered and used in the dose-response assessment.
Additional modeling and explanatory text has been added to the document.
Question 3: Under Section 5.1.2, Methods of Analysis, Including Models, is the point of departure
determined appropriately (i.e., benchmark dose approach)? Is the 10% response level appropriate
and is the use of this response level supported adequately?
Comments: All reviewers felt that the point of departure had been determined appropriately and 10%
was an adequate level of response.
One reviewer commented that the discussion of choice of a benchmark dose model in relation to the
shape of the dose-response curve (Appendix B) was inaccurate. The reviewer disagreed with part of
the description of the dose-response characterization, that models with a concave shape were clearly
not relevant for these data.
Response: The dose-response characterization in the document was intended to describe fits of
particular models to the data, not that a concave shape could not be consistent with the available data.
Additional language has been added to the document for clarity.
Question 4: Under Section 5.1.3, RfD Derivation-Including the Application of Uncertainty
Factors, are the appropriate uncertainty factors applied? Is the explanation for each transparent?
A-5
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Specifically, is the recommendation for not applying an effect level extrapolation factor justified
adequately?
Comments: All reviewers felt that the proper uncertainty factors had been applied and were
adequately justified in the assessment.
Cancer Weight-of-Evidence Designation
Question 1: The weight-of-evidence and cancer characterization are discussed in Section 4.6. Have
appropriate criteria been applied from EPA's Draft Revised Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 1999)?
Comment: All reviewers agreed that the data were inadequate to assess human carcinogenic potential
of 2-methylnaphthalene and felt that the appropriate criteria from EPA's Draft Revised Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 1999) had been applied.
One reviewer noted that while agreeing with the overall conclusion that the Murata et al. (1997) study
did not provide adequate evidence for determining the carcinogenic potential of 2-methylnaphthalene,
additional carcinogenicity studies should be performed in order to evaluate the possibility that 2-
methylnaphthalene toxicity masks any potential carcinogenic response. The reviewer suggested that
since lung tumors in mice are typically derived from type n pneumocytes and Murata et al. (1997)
speculated that the type n pneumocytes may be the specific cellular target of 2-methylnaphthalene
toxicity, the potential exists that 2-methylnaphthalene-induced toxicity masks the carcinogenic potential
of this chemical in the mouse lung. This reviewer suggested that 2-methylnaphthalene may be
carcinogenic in the mouse lung at lower doses, administered for shorter durations and recommended
that further experiments be conducted to determine whether this may or may not be the case.
One reviewer noted the need for further long-term carcinogenicity and inhalation studies to determine
the potential carcinogenicity of 2-methylnaphthalene. The absence of additional long-term
carcinogenicity and inhalation studies is recognized in Sections 5.1.3., 5.2., and 5.3.
Response: It is recognized that further studies should be conducted to better evaluate the pathogenesis
and carcinogenicity of 2-methylnaphthalene. However, since there is currently no definitive evidence to
suggest that 2-methylnaphthalene may be carcinogenic in the mouse lung at lower doses and
administered for shorter durations, a discussion of this reviewer's hypothesis is not included in the
document. The specific molecular mode of action of 2-methylnaphthalene-induced pulmonary alveolar
proteinosis is not completely understood. The suggestion by the principal study authors that 2-
methylnaphthalene may target type n pneumocytes is consistent with the pathogenesis of the disease in
humans and is substantiated by the available evidence in mice exposed to a mixture of
methylnaphthalenes. Specifically, the lungs of mice chronically exposed to a mixture of
methylnaphthalenes (containing both 1- and 2-methylnaphthalene) show hypertrophy and hyperplasia of
A-6
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type II pneumocytes (Murata et al, 1992).
PUBLIC COMMENTS (RECEIVED SEPTEMBER 2003)
Comment: A commenter did not consider the Murata et al. (1997) study adequate for the derivation of
an RfD based on the possible simultaneous exposure of the animals to 1- and 2-methylnaphthalene. In
addition, the commenter indicated that the BMD modeling did not adequately use all of the data (i.e.,
the female data was omitted from the analysis). Also, the commenter suggested that BMD modeling of
the study's data set may be technically inappropriate.
Response: An expanded discussion of the possible simultaneous exposure to 1- and 2-
methylnaphthalene was added to the document following external peer review. In addition, both male
and female data were used in the BMD modeling for derivation of the RfD. An added discussion of the
BMD modeling has also been added to the document.
A-7
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APPENDIX B. BENCHMARK DOSE (BMD) ANALYSIS
-------
APPENDIX B: BENCHMARK DOSE (BMD) ANALYSIS
Using BMDS version 1.3.2., the modeled data included the incidence of pulmonary alveolar proteinosis
observed in male and female B6C3F1 mice exposed to 2-methylnaphthalene in the diet (Murata et al.,
1997) as shown in Table Bl.
Table Bl. Incidence of pulmonary alveolar proteinosis in B6C3F1 mice fed 2-methylnaphthalene for 81
weeks
Dietary dose (%)
0
0.075
0.15
Females
Dose
(mg/kg-day)
0
50.3
107.6
Incidence
5/50(10%)
27/49 (55%)
22/48 (46%)
Males
Dose
(mg/kg-day)
0
54.3
113.8
Incidence
4/49 (8%)
21/49 (43%)
23/49 (47%)
Source: Adapted from Murata et al., 1997.
The benchmark response (BMR) was defined as a 5% increase in extra risk for the critical effect,
pulmonary alveolar proteinosis. Tables B2 and B3 show the statistical results used to evaluate the
goodness-of-fit. Models which were clearly not relevant, that is, those which completely missed any of
these dose-response points and their confidence intervals, such as the quantal-quadratic model, are not
included in the summary. The BMD05 and BMD10 and a BMDL05 and BMDL10 were estimated as a
consistent point of comparison across chemicals as recommended by the Benchmark Dose Technical
Guidance Document (U.S. EPA, 2000c).
For each model, the software performed residual and overall chi-square goodness-of-fit tests, and
determined the Akaike's Information Criterion (AIC). The chi-square p-value is a measure of the
closeness between the observed data and the predicted data (predicted using the modeled fit). Models
with chi-square p-values • 0.1 were considered adequate fits. The AIC is a measure of the model fit
based on the log-likelihood at the maximum likelihood estimates for the parameters. Models with lower
AIC values among those with adequate chi-square p-values were identified. Based on these criteria,
the fit of male data to the log-logistic model is the best fitting model. Output from the software for the
log-logistic model run (of the male mouse incidence data) follows Tables B2 and B3.
Since female mice demonstrated a somewhat higher response at the low-dose than male mice, modeling
B-l
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of both low-dose groups was also carried out (Table B3).
Since there is some validity to using the historical control data as a reference group (assuming 0
responses from 5,000 mice), additional runs were considered using the male and female incidence data
from the low dose group. The results are shown for comparison in Table B3.
Table B2. Benchmark dose modeling (both high and low dose groups compared to concurrent controls)
for critical effect, settings of 10% extra risk, confidence level 0.95
Model
Log-Logistic
Log-Probit
Probit
Quantal-
linear a
Model results
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
Females
176.7
0.0099
10.7
7.0
182.4
0.001
25.7
19.3
183.8
<0.001
28.3
22.3
179.2
0.002
15.5
11.2
Males
167.8
0.228
13.7
9.1
171.0
0.029
30.1
22.9
172.4
0.014
32.1
25.9
168.9
0.106
18.0
13.1
Combined
(males + females)
341.6
0.027
12.1
9.0
350.1
0.001
27.8
22.7
350.8
0.001
27.9
22.7
345.2
0.005
16.7
13.2
a The output for the gamma, quantal-linear (one-stage multistage), and weibull model fits were identical.
B-2
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Table B3. Comparison of benchmark dose modeling results considering 5% and 10% extra risk levels
(with confidence level 0.95) and comparison against historical and concurrent controls
Data Set
Male mice,
low and high dose groups
(concurrent controls)
Male and female mice,
low-dose only
(concurrent controls)
Male and female mice,
low-dose only
(historical controls)
Model
Log-Logistic
Quantal-lineai3
Quantal-linear3
Model results
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
BMD05 (mg/kg-day)
BMDL05 (mg/kg-day)
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
BMD05 (mg/kg-day)
BMDL05 (mg/kg-day)
AIC
Chi Square p-value
BMD10 (mg/kg-day)
BMDL10 (mg/kg-day)
167.8
0.228
13.7
9.1
6.9
4.5
200.7
0.15
9.6
7.3
4.7
3.5
138.5
0.34
8.2
6.5
' The quantal-linear model provides a fit closest to a straight line.
-------
Output Bl: Log-logistic model, male mouse incidence data for pulmonary alveolar proteinosis, with concurrent
control (10% BMR)
Log-Logistic Model with 0.95 Confidence Level
0.6
0.5
13
T5 0.4
I
o
05
0.2
0.1
0
Log-Logistic
20
40
60
dose
80
100
120
11:53 04/03 2003
BMDS MODEL RUN
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
B-4
-------
Asymptotic Correlation Matrix of Parameter Estimates
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Log(likelihood) Deviance Test DF
-81.189
-81.9066 1.43511 1
-92.8591 23.3401 2
P-value
Scaled
Residual
Chi-square = 1.45 DF = 1
Benchmark Dose Computation
Specified effect =
Risk Type =
Confidence level =
HMD =
BMDL =
0.1
Extra risk
0.95
13.6562
9.07745
B-5
-------
Output Bl (con'd): Log-logistic model, male mouse incidence data for pulmonary alveolar proteinosis, with
concurrent control (5% BMR)
Log-Logistic Model with 0.95 Confidence Level
I
c
g
CO
0.6
0.5
0.4
0.3
0.2
0.1
0
Log-Logistic
1
BMDLJ fiiyip
20
15:1408/252003
Output B1 for model fit details.
Benchmark Dose Computation
Specified effect = 0.05
Risk Type = Extra risk
Confidence level = 0.95
HMD = 6.46871
BMDL = 4 .29985
1
40
60
dose
80
100
120
B-6
-------
Output B2: Quantal-linear fit of low-dose male and female mice incidence data for pulmonary alveolar proteinosis,
with concurrent control (10% BMR)
Quantal Linear Model with 0,95 Confidence Level
o
CB
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Quantal Linear
T
BMDL|
10
20
30
40
50
dose
15:2006/132003
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
B-7
-------
Asymptotic Correlation Matrix of Parameter Estimates
Slope
-0.31
1
Parameter Estimates
Variable
Background
Slope
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Est._Prob. Expected Observed
2.06 DF = 1 P-value = 0.1510
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 9.58077
BMDL = 7.26842
-------
Output B2 (con'd): Quantal-linear fit of low-dose male and female mice incidence data for pulmonary alveolar
proteinosis, with concurrent control (5% BMR)
Quantal Linear Model with 0.95 Confidence Level
o
0)
c
o
0.7
0.6
0.5
0.4
'•G 0.3
CO
0.2
0.1
0
Quantal Linear
10
T
^.
T
1
20
30
40
50
dose
15:1908/252003
Benchmark Dose Computation
Specified effect = 0.05
Risk Type = Extra risk
Confidence level = 0.95
HMD = 4.66426
BMDL =
B-9
-------
Output B3: Quantal-linear fit of low-dose male and female mice incidence data for pulmonary alveolar proteinosis,
with historical control (10% BMR)
Quanta! Linear Model with 0.95 Confidence Level
0.7
0.6
CD
| 0.4
•| 0.3
E
^ 0.2
0.1
0
Quantal Linear
10
20
30
40
50
dose
15:2206/132003
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
B-10
-------
Asymptotic Correlation Matrix of Parameter Estimates
Slope
1
Parameter Estimates
Estimate
0
0.0128415
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Log(likelihood) Deviance Test DF P-value
-67.1712
-68.2571 2.17188 2
-271.713 409.083 2
138.514
Goodness of Fit
Scaled
Residual
0
21
Chi-square =
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
HMD = 8.20467
BMDL = 6.49803
B-ll
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