EPA/635/R-05/003
August 2005
Peer Consultation Workshop on Research Needs
Related to the IRIS Draft Toxicological Review of Naphthalene
FINAL REPORT
U.S EPA Headquarters Library
Mail code 3404T
1200 Pennsylvania Avenue NW
Washington, DC 20460
202-566-0556
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
'* i.
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NOTICE
This document has been reviewed in accordance with U.S. Environmental Protection
Agency (EPA) policy and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
This report was prepared by the Oak Ridge Institute of Science and Education (ORISE),
Department of Energy, under an Interagency Agreement (No. DW-89939822-01-0, Project 04-
12) with EPA's National Center for Environmental Assessment (NCEA), Office of Research and
Development, as a general record of discussion of the peer consultation meeting. This report
captures the main points of presentations and highlights discussions among the participants. This
report does not contain a verbatim transcript of all issues discussed during the peer consultation,
and it does not embellish, interpret, or enlarge upon matters that were incomplete or unclear.
Except as specifically noted, no statements in this report represent analyses by or positions of
EPA or ORISE.
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TABLE OF CONTENTS
1.0 INTRODUCTION '. 1
1.1 Workshop Purpose 1
1.2 Workshop Participants 1
1.3. Charge Questions :.... 2
2.0 SUMMARY OF OPENING REMARKS AND PRESENTATION 2
2.1 Welcome: Introduction and Charge 2
2.2 Overview of the Genetic Toxicology of Naphthalene and Its Metabolites 2
2.2.1 Bacterial gene mutation assays 3
2.2.2 In vitro eukaryotic gene mutation, cytogenetic, or DNA damage assays .... 3
2.2.3 In vivo eukaryotic gene mutation, cytogenetic, or DNA damage assays 3
2.2.4 Summary of Genetic Toxicity 4
2.3 Discussion of the Genetic Toxicology of Naphthalene and Its Metabolites 4
3.0 * ^SUMMARY OF DISCUSSION OF CHARGE QUESTIONS 5
3.1 Charge Question # 1 5
3.2 Charge Questions # 2 and # 3 '. 13
3.3 Charge Question # 4 15
3.4 Charge Question # 5 22
4.0 SUMMARY OF DISCUSSION OF OTHER RELATED TOPICS 23
4.1 Comparison of Rodent and Non-Human Primate Studies 23
4.2 Relative Timing of Performance of Cytotoxicity and Genotoxicity Studies 24
APPENDIX A LIST OF EXPERT PARTICIPANTS
APPENDIX B LIST OF OBSERVERS
APPENDIX C AGENDA
APPENDIX D PANELISTS' OVERHEADS
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1.0 INTRODUCTION
1.1 Workshop Purpose
Naphthalene has been recently characterized as a likely human carcinogen by the U.S.
Environmental Protection Agency's (EPA) Integrated Risk Information System (IRIS) Program
in a draft Toxicological Review of Naphthalene (U.S. EPA, 2000), based on new information
from a two-year inhalation rat bioassay conducted by the National Toxicology Program (NTP,
2000). In the NTP study, positive trend increases in the incidences of two rare nasal tumors,
olfactory neuroblastomas in males and females and adenomas of the respiratory epithelium in
males, were observed. EPA derived an inhalation unit risk was derived from these findings. The
draft assessment was reviewed by an independent external peer review,panel in July 2004.
Among comments made by the external peer review panel was the desirability of future research
to characterize naphthalene's carcinogenic mode of action. To further discuss this comment,
EPA decided to sponsor a one-day peer consultation workshop, inviting experts in naphthalene
toxicology and chemistry, inhalation toxicology, genetic toxicology, and risk assessment to
discuss the specific types of studies that would improve characterization of the mode of action of
nasal tumor formation and provide estimates of required research time and resources.
The expert opinions and recommendations from this workshop will be considered by
EPA in determining a course of action for the development of a scientifically defensible human
risk assessment for naphthalene inhalation carcinogenicity.
1.2 Workshop Participants
The workshop was organized and conducted by the Oak Ridge Institute of Science and
Education (ORISE), Department of Energy, under an Interagency Agreement with EPA's
National Center for Environmental Assessment (NCEA), Office of Research and Development.
The workshop was held on April 7, 2005, at the Graduate School of Public Health, University of
Pittsburgh, Pittsburgh, Pennsylvania Eight experts (four from the original external peer review
panel, and four additional experts selected by EPA) were invited to present their views and
recommendations. Dr. Bernard Goldstein, Dean of the Graduate School of Public Health at the
University of Pittsburgh, chaired the workshop. The participants were asked to present their
views in response to five charge questions provided by EPA. The final workshop report
represents a summary of the discussion that occurred at the meeting. The report has been
reviewed and accepted by the peer consultation panel as representative of the discussion.
A list of the eight panel participants and their affiliations can be found in Appendix A.
The meeting was attended by approximately 20 observers, who are listed in Appendix B. The
meeting agenda is shown in Appendix C. Overheads presented by panelists are reproduced in
Appendix D.
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1.3 Charge Questions
The panel was asked to focus.the discussion on the following five charge questions:
(1) What specific studies would clarify whether naphthalene induces olfactory epithelial and
respiratory epithelial tumors in rats through a genotoxic mechanism? Discuss specific issues
related to these studies (e.g., if metabolite formation is needed, how would this be accomplished;
if Ames tests are proposed, men what strains and tissue fractions would be best?). .
(2) Which studies would be the most critical for elucidating whether a genotoxic mode of action
is operating?
(3) What resources (level of effort, funds, time) would be required to perform the suggested
studies?
(4) If the critical studies identified above show that genotoxicity is not likely under conditions
that lead to tumors in vivo, what critical studies or evaluations could be used to see if effects on
cell cycling/proliferation (including apoptosis) or cytotoxicity might play a role in tumor
formation?
(5) What resources (level of effort, funds, time) would be required to perform the suggested
studies?
2.0 SUMMARY OF OPENING REMARKS AND PRESENTATIONS
2.1 Welcome: Introduction and Charge
Dr. Goldstein welcomed the panelists and observers, and opened the meeting with brief
introductory remarks on the objectives of the workshop. It was suggested by Dr. Goldstein that
the workshop discussion begin with a focus on areas of concern associated with naphthalene
(NP) inhalation carcinogenicity, identified from the previous comments of the external peer
reviewers. Dr. Goldstein briefly reviewed the comments from the external peer review that were
related to the mode of action of naphthalene carcinogenicity (See Appendix D-l for a copy of the
overheads. See http://ftrww.epa.gov/IRIS/wbatsnew.htni for the complete external peer review
report). The goal was to expand the external peer review discussion within the context of the
charge questions for the present workshop. Dr. Goldstein presented the charge questions and
noted that the key concerns were the roles of genotoxicity versus cytotoxicity in the induction of
naphthalene carcinogenicity, identification of studies that might be conducted to resolve these
issues, and the level of resources needed.
2.2 Overview of the Genetic Toxicology of Naphthalene and Its Metabolites
Following Dr. Goldstein's introductory remarks, Dr. Eastmond provided an overview of
the large body of data on the mutagenicity and genotoxicity of NP and its major metabolites (See
Appendix D-2 for a copy of the overheads).
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2.2.1 Bacterial gene mutation assays
t
In Salmonella typhimurium, all assays were negative except one for 1,2-napthoquinone
(1,2-NQ). SOS response and SOS chromotest were also negative, as was the Pol A or rec assay.
The Mutatox test was positive with S9; however, given the numerous negative studies, Dr.
Eastmond considered this result to be an anomaly.
2.2.2 In vitro eukaryotic gene mutation, cytogenetic, or DNA damage assays
In studies identifying mutations at the HPRT and TK loci, the results were negative at the
TKlocus and equivocal at the HPRT locus for both NP and 1,4-naphthoquinone (1,4-NQ). In
the in vitro micronucleus test, NP was weakly positive (some chromosomal breakage) in MCL5
cells, a cell line transfected with the genes for several cytochrome P450 isoforms and epoxide
hydrolase which likely have bioactivating properties. In the same test, 1,4-NQ was modestly
positive, inducing chromosomal breakage and loss.
NP was strongly positive in the NTP (2000) study with cultured Chinese hamster ovary
(CHO) cells, inducing structural chromosomal aberrations with S9 and significant increases in
sister chromatid exchanges (SCE), with and without metabolic activation. In rat hepatocytes
tested in vitro, NP was negative under conditions of alkaline elution, and NP, 1-naphthol, and 2-
naphthol were also negative for unscheduled DNA synthesis (UDS). NP was also negative in
five cell transformation assays using various cell lines. In contrast, in preimplanted whole
mouse embryo treated in vitro with either NP, 1,2-NQ or 1,4-NQ, structural chromosomal
changes in the presence of S9 were observed.
2.2.3 In vivo eukaryotic gene mutation, cytogenetic, or DNA damage assays
In recombination assays using Drosophila melanogaster, NP induced somatic mutations.
Micronuclei were also induced in the erythrocytes of NP-exposed salamander larvae
(Pleurodeles \valtl). Alkaline elution (measuring single strand breakage) and UDS in rat
hepatocytes treated in vivo were negative. No increases in micronuclei were observed in the
bone marrow of male ICR Swiss mice and male and female CD-I mice. Dr. Eastmond noted
that the focus of these micronucleus assays was on benzene, and these tests were conducted at a
time when guidelines for conducting genotoxicity studies were changing. Therefore, it is
important to evaluate technical aspects of the individual studies when assessing the findings.
Micronuclei were also induced in the erythrocytes of NP-exposed when assessing the findings.
In other rodent studies, NP induced DNA fragmentation in a variety of tissues. For example, in a
study involving normal and p53 heterozygote transgenic mice, NP at moderate doses (^32
mg/kg) induced DNA fragmentation in mouse liver and brain. DNA fragmentation was also
seen in rats administered NP at a higher dose (110 mg/kg) for 120 days. However, all these
studies have some limitations especially those using the higher doses; genotoxic chromosomal
breakage may have occurred as well as breakage associated with cytotoxicity.
Recently, studies using the Comet assay have been conducted assaying DNA damage in
the white blood cells (WBC) of exposed workers. Although these studies focused on multiple
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types of poly cyclic aromatic hydrocarbons (PAH), increases in WBC of PAH-exposed humans
were positively correlated with NP and phenanthrene. The findings were statistically significant;
however, there were numerous confounders in these assays, making the results difficult to
interpret.
2.2.4. Summary of Genetic Toxicity
In in vitro studies, NP appears to be a clastogen, not a mutagen. Results are mixed in in
vivo studies. No genotoxicity studies have been conducted in the target organs of
carcinogenicity. This data gap is particularly important because NP appears to exhibit tissue-
specific metabolism and effects. In target tissues, NP is metabolically activated to reactive
intermediates such as quinones and epoxides. Inhalation exposure also may be critical for NP
toxicity. Dr. Eastmond noted that there are some similarities in the genotoxicity profile of NP
with that of benzene, a compound on which he has worked extensively.
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23 Discussion of the Genetic Toxicology of Naphthalene and Its Metabolites
•j
Considerable discussion followed Dr. Eastmond's presentation. Dr. Penning noted that
many of the mutagenicity studies were flawed and thus, the data were limited for assessment of
mutagenicity. His laboratory has conducted numerous studies with polycyclic aromatic ortho-
quinones using p53 cDNA in in vitro mutagenesis assays. Although 1,2-NQ is an electrophilic
metabolite, it is also redox active. When quinones are allowed to redox-cycle to generate
reactive oxygen species (ROS) they were found to be more mutagenic than the parent quinone.
Therefore, he cautioned that mutagenicity studies should be designed so that mutation in the
presence and absence of redox-cycling could be assessed. NP or its metabolites may not be the
toxic moiety; toxicity may occur via the production of ROS.
A question was raised regarding the certainty with which NP could be classified as a non-
mutagen. Dr. Penning noted that the "jury is still out" on this question, and Dr. Eastmond
concurred, adding that although NP appeared to be a clastogen, a potential for mutagenicity
cannot be ruled out. Oxidative damage or stress may lead to oxidation of DNA leading to 8-oxo-
2-deoxyguanosine, a potentially mutagenic lesion. For example, quinones are highly redox
active molecules and can undergo redox cycling, leading to the formation of numerous ROS.
ROS can cause severe intracellular oxidative stress through the formation of oxidized
macromolecules. Dr. Morris noted that a number of compounds are oxidants but are not
carcinogens; for example, chlorine is a profound oxidant but it is not a nasal carcinogen. Dr.
Center stated that the major limitation of most of the currently available assays is that they are
conducted using standard protocols, with and without liver S9 activation. As some metabolic
enzymes present in NP target tissues are not present in the S9 fraction, mutagenicity associated
with these enzymes cannot be evaluated. Dr. Center suggested that whole-animal in vivo
mutagenesis studies would be useful in elucidating target-specific mechanisms. One can assume
in this case that metabolism and the resulting mutagenicity occurs near the point of contact;
however, one cannot rule out the fact that metabolites generated in the liver may recirculate to
the nasal passages.
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Dr. Van Winkle agreed with these comments. ROS generated by metabolism can cause
DNA damage, and this may be the mechanism of action for NP toxicity and mutagenicity. Dr.
Eastmond noted that both NP metabolites, 1,4-NQ and 1,2-NQ, are potential redox-cyclers. Dr.
Fanucchi added that NP induces two tumor types. With acute NP exposure, frank cytotoxicity is
observed in olfactory epithelial tissue, but no cytotoxicity is evident in respiratory epithelial
cells. Dr. Center stated that these findings occur with a single exposure that induces acute
effects. The effects of repeated exposures are not known. Dr. Penning added that there are
many metabolites that could cause mutagenicity/genotoxicity (e.g., NP-l,2-oxide, 1,2-NQ, 1,4-
NQ). There may also be secondary effects associated with lipid peroxidation. What is needed is
a return to basic biology. Little is known about either the enzymology and kinetics of NP
metabolism and the same is true of its downstream metabolites. He suggested that a basic
approach would be to: (1) list target tissues and gender differences among target tissues; and (2)
assess metabolic profiles of the various metabolites in order to identify putative candidates for
the toxic mode of action evaluation across gender.
»
3.0 SUMMARY OF DISCUSSION ON CHARGE QUESTIONS
3.1 Charge Question #1; What specific studies would clarify whether naphthalene
induces olfactory epithelial and respiratory epithelial tumors in rats through a
genotoxic mechanism?
3.1.1 Initial Studies
What are the first steps to elucidating whether NP-induced tumorigenesis is mediated by
a genotoxic mode of action? Dr. Goldstein asked Dr. Eastmond to begin this discussion.
Dr. Eastmond suggested the performance of a series of specialized genotoxicity studies to
answer this question.
Determine NP genotoxicity in the rat olfactory and respiratory epithelia and in the mouse lung
by measuring covalent binding when NP is administered via inhalation.
The following methods were recommended to measure covalent binding in nasal and
lung tissues: (I) liquid scintillation counting (LSC); (II) 32P-postlabeling; or (III) accelerator
mass spectrometry (AMS). AMS is the most sensitive method for quantitation. This method
detects very low levels of binding but does not necessarily result in the identification of the
binding species.
Examine mutations in target tissues.
(,
Can NP be metabolized into covalent binding metabolites, forming lesions that can be
converted into mutations during the repair process? Dr. Eastmond suggested that one in vivo rat
model that might be used to examine mutations is the Big Blue (BB), a transgenic mouse with
the lad construct as atransgene reporter. The insertion of this construct into the genome allows
for the detection of point mutations or small deletions in any target tissue. Chromosomal
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damage, measured by the formation of micronuclei, and cell proliferation in target tissues can
also be measured. The BB mouse, or Mutamouse, can also measure mutations in target tissues
of the mouse and the BB rat could be used to detect mutations in rat tissues. He noted that the
use of the micronucleus assay is limited if the cells being examined do not contain adequate
amounts of cytoplasm. The sensitivity of the micronucleus assay can be increased by measuring
the incorporation of the 5-bromo-2'-deoxyuridine (BrdU), athymidine analog; this approach can
be used to distinguish replicating from non-replicating cells.
To assess DNA damage in target tissues, Dr. Eastmond recommended the Comet assay,
using single cell electrophoresis that can be modified to detect single strand breakages, double
strand breakages, and oxidative lesions. These types of genotoxic effects can be repaired in
vivo; a limitation of this assay is that it does not give information about whether the observed
lesions are heritable or pre-mutagenic. i
Other secondary assays that might be employed for NP genotoxicity testing include (1)
the Ames test with nasal or lung microsomes; (2) lymphoblastoid cells expressing various CYP
isoforms; and (3) measurement of 8-oxo-2-deoxyguanosine (8-oxo-dG). The 8-oxo-dG test
assesses the formation of oxidative DNA lesions. This test is informative if oxidative damage is
considered to be a critical event in the formation of tumors and might become the primary test
for this end point. Dr. Eastmond noted that establishing whether a chemical compound is not
genotoxic is difficult because it requires a high burden of proof. There are numerous genotoxic
mechanisms that might be occurring in target tissues or via circulating metabolite(s).
Determination of lack of genotoxicity requires a large number of negative tests and a weight-of-
evidence approach.
For investigation of the non-genotoxic mechanisms of action, Dr. Eastmond
recommended modeling toxicity and cell proliferation in the target tissues. This approach would
provide biomarkers that could be useful for identifying the concentrations or doses needed to
induce the carcinogenic effects observed in the NTP (2000) bioassay.
Dr. Center agreed with Dr. Eastmond that the optimal method for determining whether
NP induces olfactory epithelial and respiratory epithelial tumors in rats through a genotoxic
mechanism is to use the BB in vivo mutagenesis assay. An in vivo assay is preferable to one in
vitro because the metabolism of the whole animal is considered, not just that of a single tissue.
She has used the BB assay in a repeated-dose (dietary) study with alachlor, an herbicide with
complex metabolism involving both the liver and olfactory mucosa. An increase in mutant
frequency was seen in the olfactory mucosa (target organ for alachlor carcinogenicity), but no
increases were detected in either respiratory mucosa or liver (neither are target organs). Dr.
Center added that in this study, an increase in thyroid mutant frequency was also not observed.
This is an important finding because the weight-of-evidence for alachlor-induced thyroid tumors
indicates that the mechanism of action is non-genotoxic, and one would not expect to find an
increase in mutant frequency in the thyroid gland. Thus, the BB is a powerful rodent model
system.
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3.1.2 Discussion
In the discussion that followed these test recommendations, Dr. Goldstein observed that
the BB may be a good model for initial testing, given time and cost issues. However, Dr.
Penning disagreed, noting that the most important end point for assessment of genotoxicity
associated with tumor induction is information on the formation of DNA adducts that can be
correlated with tumor incidences. He described a classic experiment in which a linear positive
relationship was observed between the formation of O6-methyl guanine DNA adducts and the
incidence of mouse lung adenomas in AJ mice treated with NNK (a nicotine derived nitrosamine
ketone). The number of tumors increased with increasing adduction. This strongly suggests that
there is a causal relationship between the adduct and the tumor. Dr. Goldstein noted that many
different DNA adducts may be formed due to NP administration. The discussion focused on the
following topics shown in italics.
Does one need to test for all putative adducts?
Dr. Penning replied that it was necessary to have a candidate list, of reactive metabolites
which should be followed up by testing to identify the subset that produces the dominant DNA
adducts. Using Liquid Chromatography/Mass Spectrometiy (LC/MS) would be a good approach
as it has high sensitivity. In order to optimize performance, basic information is needed on
adduct chemistry, identification and quantitation. Quantitation would be best achieved using a
stable isotope dilution strategy and would require the synthesis of the isotopically labeled
standards.
Is there enough information on candidate species?
Dr. Penning replied that there are insufficient data and more would be needed. For
example, the glutathione conjugates of 1,2-NQ and 1,4-NQ may be more electrophilic and more
redox active than the unconjugated compounds. However, Dr. Fanucchi noted that nasal tissue
does not appear to contain significantly more glutathione than pulmonary tissue. It was agreed
that more information on possible candidate species for adduct formation was needed.
In order to identify whether or not adducts are produced by putative candidates, assays
must be very sensitive. Dr. Buckpitt stated that he has conducted an experiment with tritium-
labeled NP, looking at the lung. At the time, the nose had not been identified as a target organ of
NP carcinogenicity. Mice were administered 5-10 mCi of radiolabel, and DNA was isolated.
The findings were very clear; no radioactivity was observed in the DNA. If sensitivity can be as
low as 1 adduct/108, one can then make the assumption that adducts are either present or absent.
The limitation of this approach is that it does not detect other candidate species, such as those
which are depurinated and those which result from ROS-derived adducts.
Dr. Buckpitt suggested a tiered approach. First, does binding occur? Second, what
species bind? There are a number of species that do not bind; this is one of the limitations of the
BB model. Where it works, it is a very good system. However, the BB model only detects a
subset of the mutations in the universe of mutations that can cause cancer.
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What is known about extrapolation from rodents to primates?
Dr. Buckpitt noted that species differences, specifically extrapolation from rodents to
primates, is an important issue in mutagenicity and target tissues. Dr. Goldstein stated that
interspecies extrapolation was not part of the charge to the panel. He suggested that species-
specificity of effects and mechanisms of action is a dose issue, not a hazard characterization one.
Other panelists disagreed, noting that human relevance is now being considered as part of hazard
characterization. Dr. Morris stated that if metabolic profiles differ significantly across species
(and there was general agreement that they do), then interspecies extrapolation is an important
concern. He commented that the charge was too "black and white". There are significant
differences between rats and mice; a comprehensive comparison between these two rodent
species would be both interesting and informative. Dr. Penning concurred with Dr. Morris,
noting that charge question #1 was very specific with regard to target tissues and metabolism and
that these issues are species-specific. Dr. Fanucchi noted that if findings in rodents might
possibly occur in humans, then they need to be considered. However, Dr. Buckpitt stated that
the concern is not with the mouse-and-rat world, but with humans.
i
Could a tiered approach to genotoxicity testing be developed?
Discussion reverted to basic issues concerning the continuum from genotoxicity to
cytotoxicity. Dr. Van Winkle noted that protein adducts may not be important in downstream
events leading to carcinogenicity if they are easily and quickly repaired. Dr. Penning stated that
many covalent protein adducts are irreversible, such as those formed with protein kinase C
isoforms. Dr. Eastmond proposed consideration of three endpoints: (1) oxidative damage; (2)
covalent DNA adducts, and (3) interference with enzymes involved in DNA repair and
maintenance, as topoisomerases. Dr. Eastmond mentioned that NQ metabolites had been
reported to inhibit isolated topoisomerase II in isolated systems. Dr. Morris added that tumors
are produced in two separate tissues, an important consideration. He suggested the performance
of in vitro studies, then acute in vivo studies followed by repeated-exposure studies. It was noted
that there are no repeated inhalation exposure studies investigating putative mechanisms of
toxicity/carcinogenicity in the nose.
Dr. Goldstein agreed with Dr. Morris that the tiered approach was a reasonable one, if
genotoxicity was occurring. What types of studies would detect genotoxicity and with what
degree of confidence?
Two sets of studies were suggested by some panelists:
(1) Repeated inhalation exposure with olfactory and nasal epithelia as target tissues, and
measurement of the time course of occurrence of cytotoxicity/toxicity; this type of experiment
would require cell proliferation data (see Charge Question #4); and
(2) Characterization of NP effects in target tissue, followed by detailed investigation of the
causes of these effects; this could be accomplished by mutagenicity assays in target tissues in
conjunction with in vivo mouse micronucleus and BB transgenic studies. This combination of
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studies would detect mutagenic and clastogenic effects including large-scale chromosomal
aberrations, oxidative damage, protein binding, and DNA adduct formation.
With regard to reactive metabolites, Dr. Buckpitt noted that Dr. Penning is identifying
their DNA adducts, and is developing standards for their quantitation using LC/MS, and mat
these data would be very useful for assessment of genotoxicity. Dr. Penning presented his list of
putative candidate metabolites which bind covalently to DNA, to form stable and depurinating
adducts, and those mat induce oxidative damage [See Appendix D-3 for a copy of the overhead].
It was noted mat some of these adducts have large numbers of stereoisomers that would also
have to be measured.
Dr. Goldstein questioned whether studies typically utilized in regulatory testing might be
informative. Dr. Morris replied that the Ames test with nasal S-9 would be'particularry useful.
If this assay yields a positive result, then one can conclude that mutagenicity is occurring and
further testing would be unnecessary. Dr. Fanucchi suggested that the Ames test with
respiratory and olfactory S9 from rats and lung S9 from mice would provide a comprehensive
assessment. Dr. Morris recommended that all 3 tissue-specific microsomal activating fractions
be tested in the Ames assay in both rats and mice. None of these studies have been done.
A simple workup of tiered testing, including cost considerations, followed. It was
agreed by some panel members that the Ames assay should be conducted initially, as it is cost
effective. Inhalation studies should be next, as route of exposure data are crucial to assessment
of in vivo genotoxicity. Dr. Penning noted that all possible candidates would have to be
considered; this is a long list. For example, there are 4 stereoisomers of the NP diol epoxide
which can give rise to 16-stereoisomeric adducts. There are also metabolites that form
depurinating adducts, and several that can induce oxidative damage. Dr. Morris stated, that if all
assays were negative, the weight-of-evidence against genotoxicity would be very strong.
However, the problem with negative data is that one can always say that other genotoxic
candidates may exist.
It was agreed mat a simplified approach would be to use S9 nasal fraction and S9
respiratory fraction in the Ames assays. However, even if these results were to be negative, lack
of genotoxicity could not be assumed. Dr. Penning noted that the inclusion of control mutagens
was important. Therefore, quantitation could be specified down to a level of sensitivity; for
example, a candidate species is not mutagenic down to a level of mutation frequency of 10 "8 to
10 "9nucleotides. However, even under these conditions, it is difficult to conclude that no
genotoxicity is present.
What level of evidence is needed to conclude that NP is not genotoxic?
Dr. Goldstein stated that if one could conclude that NP is reasonably unlikely to be
genotoxic, based on the weight-of-evidence of negative in vitro data, are in vivo studies still
necessary? Dr. Fanucchi replied that circulating metabolites (e.g., from the liver) are unlikely,
but cannot be ruled out in the absence ofin.vivo data. Dr. Penning noted that many metabolites
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are themselves reactive and interpretation of data would be confusing if only in vitro systems
were used.
Dr. Morris stated, that based on what we know about the nasal toxicology of other
compounds, the mechanism of action of NP-induced nasal carcinogenicity has to be genotoxic.
Numerous compounds produce nasal toxicity but not tumors. Therefore, negative genotoxic data
are suspect and difficult to accept. Otherwise, one would have to conclude that NP is "totally
unique".
Dr. Eastmond noted that there are numerous genotoxic mechanisms. The Ames assays
identifies some, but not all, mechanisms. Is mutagenicity the key challenge leading to
downstream events? Is mutagenicity a primary or secondary effect? In order to cover the
spectrum of possibilities, he recommended that the Ames assay be conducted initially. Positive
results would give some indication of possible genotoxicity. However, if NP is compared with
benzene, genotoxicity may not be detected in the Ames assays, because it may be occurring via
clastogenic mechanisms. Therefore, in vivo data are also needed. He commented that the NP
situation is very similar to that of benzene about twenty years ago.
3.1.3 Additional Studies
3.1.3 1 In vitro studies
A discussion of other in vitro tests that might be useful in the first tier of studies
followed.
Use of human or non-human primate S9 fraction from olfactory and respiratory nasal epithelia.
In support of this approach, it was noted that biopsies are not expensive and could be
conducted repeatedly with non-human primates if more tissue is needed. However, Dr. Buckpitt
noted that nonhuman primates will give no information if mutagenesis is S9-activated. The
necessary GYP isoforms are uncommon in non-human primate nasal tissues, occurring in
approximately 1 out of 8 animals. Dr. Morris commented that the ultimate objective of
quantitative risk assessment was to estimate potential human risks. The rat model is usually a
good human model in that it targets the tissues of concern for humans. However, rat nasal
pathways are very different from those of humans and thus may have limited human relevance.
Nonetheless, in order to conduct whole-animal in vivo studies, one needs to know which
metabolites are active. The rat model itself does not provide all the answers; however, it does
reduce uncertainty.
Use ofp53 in vitro mutagenicity assays
Dr. Penning disagreed with the initial approach suggested in the previous discussion
(Section 3.1.2) to conduct further testing in the Ames assay. In his judgment, sufficient testing
in this system has already been conducted. Further, the Ames assay does not detect mutagenicity
occurring as a result of ROS generation or downstream metabolic events with ease. In his
10
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laboratory, the p53 in vitro mutagenicity assay can distinguish between direct acting mutatgens
and those that require redox-cy cling. He emphasized that the spectrum of metabolites from the
nasal system differs from that of the liver. The p53 in vitro system gives information similar to
that obtained in the Ames assay and also detects ROS derived mutations. Therefore, this model
is preferable. Dr. Eastmond added that the Ames test strains TA102 and 104 were sensitive to
oxidative stress.
It was agreed that the p53 in vitro mutagenicity assay could be considered, using the list
of reactive metabolites to form the DNA adducts being studied by Dr. Penning.
Use of accelerator mass spectrometry (AMS) with in vitro radiolabeling
Although AMS is an excellent system, testing is expensive and there are only a limited
number of such systems worldwide, primarily at Lawrence Livermore National Laboratory.
Limitations of in vitro approaches
Dr. Fanucchi stated that live olfactory epithelial cells were difficult to maintain in
culture; she has never been able to culture them successfully. However, mouse lung tissue can
be maintained in culture. Otherwise, one needs to go into in vivo inhalation studies, which have
different concerns including issues of anatomy and distribution of gas.
3.1.3.2 In vivo Studies
Discussion of in vivo tests that could be.useful to study genotoxicity ensued. Dr. Penning
suggested that an in vivo model could be used to detect adducts, utilizing a sequential series of
standards such as the ones that he is developing. However, he also noted that a cell-based
system could be used to measure these end points.
Use of BE transgenic model(s)
The use of the in vivo BB transgenic model was recommended. Dr. Center noted that
tissues in this system can be dissected and examined separately. A limitation of this model is
that it can identify point mutations and small deletions but will not pick up large deletions or
chromosomal breaks. Dr. Eastmond added that the BB was a very valuable tool but was not
particularly sensitive. If the results were positive, then this model system would be useful.
However, it only measures a subset of the spectrum of possible genotoxic effects, and another
test would be needed to measure chromosomal damage. Further, Dr. Eastmond noted that
although the BB is well accepted as a model, its utility in examining NP genotoxicity raises
several questions. How practical is this model for assessment of genotoxicity in nasal tissues?
What would be the appropriate parameters for an inhalation experiment? What is the
appropriate exposure duration? What is the appropriate expression period?
In response to Dr. Eastmond's questions, Dr. Center recommended an appropriate
exposure duration for the BB model of 1 week if NP is considered to be a direct-acting
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carcinogen, and 3 months if NP is considered to be an indirect-acting carcinogen, such as
alachlor which she is currently testing for mechanisms of action. However she added that little is
currently known about possible effects of repeated inhalation exposure to NP. What are the
critical pre-tumorigenic lesions? What is the level of confidence that these lesions will lead to
tumors?
/•"•*.
Dr. Fanucchi added lhat interspecies differences have to be considered in development of
appropriate parameters for in vivo inhalation testing. For example, the level of metabolic
capacity in rats and mice differs, and this difference would affect time to mutagenesis.
What are relevant end points to be measured in in vivo testing?
Dr. Buckpitt noted that Dr. Penning has generated analytic methods to look at a number
of metabolites; these methods could be used to investigate in vitro adduct formation using
relevant rodent tissues. Identification of metabolites that form adducts would provide
appropriate end points for measurement in in vivo studies.
The panelists agreed that oxidative damage should be considered as an end point. Dr.
Penning concurred with measurement of this end point but noted mat he had concerns about
cross-platform comparisons (interlaboratory differences). He suggested that each DNA adduct
should be studied by a single laboratory to avoid the limitations associated with comparison of
results for the same end point across different laboratories.
Dr. Goldstein expressed concerns about examination of only end points that are
associated with oxidative damage/stress, noting that ozone has been studied since 1967 and has
been repeatedly shown to induce oxidative damage but no tumors. Therefore, the use of only
these end points would not provide a comprehensive and complete assessment. In response, Dr.
Penning replied that evidence for correlation of adducts and tumors would be a necessary
followup step to demonstrate a causal association of adduct formation with tumor development,
as was done with the AJ mice lung adenoma data described previously.
Additional concerns regarding end points associated with oxidative damage were
expressed. Dr. Eastmond noted that measurement of an end point such as an increase in 8-oxo-
2-deoxyguanosine adducts is indicative of the occurrence of oxidative stress but does not tell
whether the oxidative stress is primary (i.e., induced directly by NP administration) or secondary
(i.e., due to other cytotoxic mechanisms or upstream effects). This test generally does not
discriminate between primary or secondary effects. Further, it was agreed that performance of
this assay was difficult to do well, and "easy to do badly". Dr. Eastmond suggested that the
Comet assay as a test with utility for assessment of DNA breakage and oxidative lesions. As this
test does not distinguish between repairable and irreparable lesions in vivo, it would be important
to look at responses in this assay at both early and late time points following in vivo exposure in
order to assess the degree of repair.
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Other assays
Dr. Eastmond noted that none of the above-mentioned assays gives a good indication of
the permanence or heritability of the DNA damage/adduct formation. Dr. Center stated that this
question would be addressed by focusing on the lesioned tissues, i.e, target cells. Dr. Eastmond
agreed and suggested the use of the Comet assay with target cells following in vivo exposure.
3.1.3.3 Preliminary Research Needs Prior to Performance of Extensive Studies
Dr. Penning stated that in his judgment, a significant amount of developmental work was
necessary. If one assumes that covalent adducts are not active in lesion formation, what types of
adducts/metabolites can induce oxidative damage? The most important research need is to
develop analytical methods with a specified level of sensitivity for other potential
adducts/metabolites.' Dr. Penning estimated that this would take approximately 2 years.
Following development of these methods, multiple studies could be performed simultaneously,
with different laboratories working with different adducts to address cross-platform concerns.
He estimated that performance of these studies would take about 1 year. Therefore, a minimum
of 3 years would be needed to conduct this work.
Dr. Morris also noted that there are several research needs with regard to nasal
tumorigenesis. Tumor areas could be dissected and tested in vitro following in vivo inhalation
exposure. However, there was uncertainty with regard to the degree of differentiation of tumor
sites and the localization of tumorigenic regions within the nasal pathways. Dr. Center stated
that lesions appeared to be very regional with regard to location and proposed that selected
regions be identified and that regional counts be done to address these issues.
What level of sensitivity would be needed? How many mg of wet weight tissue would be
needed? If the level of sensitivity was to 10 [ig DNA from 10 mg wet weight tissue, one would
need to pool tissues from mice. In contrast, this level of sensitivity would be easy to attain in a
single rat
It would also be useful to look at urinary levels of DNA adducts in humans; however, it
was not known whether any assays that are currently being developed are also applicable to
human populations.
3.2 Charge Questions #2 and #3; Which studies would be the most critical for
elucidating whether a genotoxic mode of action is operating? What resources (level
of effort, funds, time) would be required to perform the suggested studies?
Two testing strategies, both involving some degree of tiered testing, were put forth to
elucidate whether a genotoxic mode of action induces NP carcinogenicity. Estimated resource
(time and cost) requirements were also presented.
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Test strategy # 1 .
Dr. Penning recommended the following test strategy:
I. Expression profiling of enzyme isoforms to identify metabolic candidates for testing.
Resources: 1.5 months and $200 K.
II. Mutagenicity assay testing, BB rat model testing. Resources: 2 years and $300 K.
III. DNA methods development. Resources: 2 years and $300 K.
He noted that mutagenicity testing could be conducted concurrently with DNA adduct
methods development, and that additional costs, approximating $150 K would be needed for
testing these adducts.
Test strategy # 2
Dr. Eastinond recommended the following test strategy:
I. Mutagenicity testing, using the Ames, crude liquid scintillation counting (LSC), 32P-labeling,
and accelerator mass spectrometry (AMS). assays.
Estimated resources for these tests were 0.5 years and $25 - 40K for crude LSC; $50 - 60K for
32P-labeling; and ^ $75 K for AMS (not including inhalation costs).
He noted that the cost for AMS testing would depend on the complexity of the
experiment including the duration of exposure, the number of samples tested at a given time
point, and the number of different time points sampled.
II. Target tissue analysis using the BB transgenic model. Resources: 1-1.5 years and $150K
(minimal BB assessment) to $400K (whole animal assessment).
III. Comet Assay to assess DNA damage, using single tissue and multiple doses, and
micronucleus test. Resources: 0.5 year and S40-50K for Comet assay and $50 K for
micronucleus test (not including inhalation costs).
Limitations of cost estimates
It was noted that these estimates were for university-based laboratories and that cost
estimates for contract laboratories using GLP might be considerably higher, as much as 4-5 fold
higher, including direct and indirect cost sharing.
Further, these estimates did not reflect costs associated with the conduct of an inhalation
exposure study. Costs estimates were direct, with the assumption that the inhalation exposure
study would be conducted elsewhere. Dr. Eastmond noted that an inhalation study might cost up
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to $200 K for a contract laboratory with GLP procedures. Panelists agreed that all these
estimates may be at the low-end, or underestimates, of actual costs.
3.3 Charge Question #4: If the critical studies identified above show that genotoxicity is
not likely under conditions that lead to tumors in vivo, what critical studies or
evaluations could be used to see if effects on cell cycling/proliferation (including
apoptosis) or cytotoxicity might play a role in tumor formation?
Panelists engaged in broad discussion on a large number of issues regarding testing of
nongenotoxic endpoints. A number of concerns were related to: (1) the need for tissue-
specificity in this type of testing; (2) the paucity of data on the basic biology of the nasal
pathways, and the need for more research in this area; (3) the effects of tissue-specific biological
factors on design of non-genotoxic experiments for NP; and (4) the timing of performance of
non-genotoxicity testing with regard to genotoxicity testing (i.e., should genotoxicity studies be
done first, followed by non-genotoxicity studies or should some or all of these studies be
conducted simultaneously).
3 J.I Issues Associated with Non-genotoxicity Testing of Naphthalene
Development of tolerance in the lung to repeated NP dosing
Dr. Buckpitt noted that the mouse lung is a sensitive tissue, in which proximal through
distal toxicity is observed following acute NP administration. In the rat, no lung lesions occur
following a range of acute NP concentrations given either parenterally or via inhalation
exposure; however, lesions are formed in the rat nasal olfactory mucosal following a single acute
4-hour inhalation exposure of 3.4 ppm.
With repeated dosing in the mouse, exposure tolerance is evident because lung toxicity
disappears. If NP is administered either intraperitoneally or via inhalation at toxic
concentrations (200 mg/kg) for 7 days, mouse lung tissue examined at this time point looks
fairly normal. Further, if a challenge test is done on Day 8, using a high NP dose (300 mg/kg),
there is minimum cytotoxicity on Day 9 that occurs mainly in the Clara cells of the distal lung.
Dr. Van Winkle noted that similar findings have been observed when tolerance is expressed in
vivo (via either acute or repeated dosing) and subsequently challenged in vitro. The
development of tolerance appears to be related to induction of glutathione synthesis via
induction of gamma-glutamyl cysteine synthetase, an essential enzyme required for glutathione
synthesis. Induced glutathione confers protection against target cell injury in the lung. Whether
this occurs in the nose is not known. Further, these findings do not address issues pertaining to
the possible existence of a circulating metabolite that may be acting on lung tissue. It was also
noted that NP administered intermittently may induce greater tissue injury as compared with
continuous exposure because of the development of tolerance under the latter conditions. A
similar pattern of effects has been suggested to occur with benzene.
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Age and gender differences in NP-induced lung injury
Acute exposure studies have been conducted with both adults and neonates, and tissue
injury in the lung assessed. Dr. Fanucchi noted that in neonates, damage was not restricted to the
distal airways but was also seen in the lobar bronchi; observed histopathology included findings
of hyperplastic nodules and mucous cell metaplasia. These findings also showed gender
differences, with the female mouse being more sensitive.
Design of repeated dose studies for assessment ofcytotoxicity
Prior to conducting repeated-dose inhalation studies for the assessment of toxicity, some
basic studies would need to be done to address issues relevant to study design. The most basic
question concerns the time points at which relevant target cells should be harvested. In order to
identify these time points, characterization of the repair system in the nose is required; this
system is reasonably well known for the mouse lung but not for the nasal passages. Repair
systems are complex, and it is uncertain if the repair phase in the rat nose is likely to take more
or less time than that in the mouse lung.' Further, the extent of repair is also not known. It was
noted that with methyl bromide, histological repair occurs with continuous exposure. The target
tissue looks normal; however, metabolic expression is very different if examined
immunologically or histochemically following exposure.
Dr. Morris noted that the dose-response curve for NP toxicity is not known, except at
high doses. Dr. Center stated that data from Dr. Buckpitt's laboratory demonstrates nasal injury
specific to the dorsal meatus at 3.4 ppm following a single acute dose. At higher doses, lesions
occur in the same location, suggesting that the site of the lesion is airflow-driven.
Potential differences in rat strain response
Concerns were expressed with regard to rat strain differences in repair systems and tissue
injury. A number of experiments have been done with Sprague-Dawley rats, whereas the BB
model is F344-based and probably not notably different from the strain used in the NTP (2000)
bioassay. There are very large mouse strain differences and it is likely that rat strains exhibit
similar differences.
Should cytotoxicity studies precede genotoxicity studies?
Dr. Goldstein questioned whether existing data suggest that cytotoxicity studies should
precede genotoxicity studies. Dr. Center noted that it was important to first identify the time
points at which lesions occur, where they occur, and whether they are reversible or irreversible.
It may be possible that cytotoxicity is driving genotoxicity. Some panel members agreed that
cytotoxicity was necessary but not likely to be sufficient for tumor formation, because
cytotoxicity is induced by other compounds that do not produce tumors (e.g., ozone).
Dr. Penning stated that cytotoxicity cannot be separated from genotoxicity. NP may have
both properties, depending on the exposure concentration, target cells examined, and tissue-
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specific modes of oxidative injury repair. There is no vacuum of knowledge with regard to the
dose-response for cytbtoxicity. Formaldehyde exhibits a classic dose-response curve in the form
of the shape of a hockey stick. In contrast, NP shows a flat dose-response cytotoxicity curve,
whereas the dose-response for nasal lesions shows a positive trend. Further, under conditions of
acute exposure, CYP concentrations decrease and glutathione is depleted whether or not
cytotoxicity is observed.
Mapping ofcytotoxic lesions
Dr. Morris stated that it was important to map the distribution of lesions as well as
tumors. For lesions, this can be done with repeated-dose, time-concentration studies that
examine the time-course of occurrence of lesions and their distribution. From these data, a
NOAEL could be determined for lesion formation. Subsequent studies could then be conducted
to assess whether cell proliferation occurs at the NOAEL for lesion formation. Dr. Center noted
that lesions are much more extensive than tumor formation; therefore, studies must be designed
carefully to ensure that the results can be interpreted clearly. Dr. Van Winkle stated that NP
dosing increases cell proliferation in the lung for several days following cessation of exposure
and a similar situation might occur in the nose. Therefore, the timing of cell harvesting is
important and would need to be worked out.
Route of exposure
Route of exposure was also identified as an issue. Intraperitoneal injection is less
relevant than inhalation exposure, and may also induce more NP metabolites. Therefore, the
appropriate route of exposure is via inhalation.
Lack of information on target cells in the nasal passages
It was noted 1hat there was less information available on the nose than on other organ
systems. For example, the identification of the target cells is unclear. Dr. Genter suggested that
stem cells of neuronal origin may be the target cells. Stem cells are undifferentiated, and
olfactory neuroblastomas may be derived directly from the stem cells. The respiratory
epithelium has a basal layer although the histology of this epithelium varies, depending on
location. For example, there is no clear basal cell population in the respiratory epithelium of the
turbinates. Further, the repair system in the respiratory epithelium is not well characterized; cell
regeneration occurs rapidly and there appears to be a high level of plasticity. Stem cells are not
needed for regeneration. Flat basal epithelial cells may divide into daughter cells that mature
and slough off within 30 days. Alternatively, cells may divide into pluripotent cells. The nature
of regeneration is dependent on regional localization.
Summary of Concerns Regarding Studies of Cytotoxicity
Studies linking cytotoxicity (including cell proliferation and apoptosis) to nasal tumor
formation cannot be conducted until some basic information regarding the nasal pathways is
obtained. There is a paucity of data on nasal characteristics, including lack of information on
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regional differences in cell types and contents of the respiratory and olfactory epithelia, and on
repair processes. There may also be large rat strain differences. Further, it is not known how
these variables are affected by NP exposures. Other concerns include time points at which to
harvest cells for cytotoxicity assessment, mapping of nasal lesions and tumors, time-course
determination of lesion formation, reversibility/irreversibility of nasal lesions, and the possibility
of development of tolerance to NP in nasal tissues with repeated dosing, as occurs in the lung.
3.3.2 Human Relevance of Naphthalene Effects in Rat Nasal Pathways
Questions were raised regarding the human relevance of NP effects in the nasal pathways
of rats. Do we need more information on non-human primates and on nasal tissues in humans?
This topic was extensively discussed. It was agreed that human relevance was an important
issue. Large differences in nasal anatomy and air-flow characteristics exist between rodents and
humans; further, nasal tumors have been observed in rats but not mice, indicating significant
species differences among rodents.
Dr. Goldstein noted that there are regulatory guidelines and default assumptions for
interpreting animal data with regard to human relevance, and that existing rodent data should be
interpreted in this context. It was important to get as much information as possible by designing
focused animal experiments to identify mechanisms of nasal tumor formation in rats. Then,
humans or nonhuman primates can be studied and similarities and differences among species
examined. Dr. Goldstein also added that the charge questions were not directed toward
assessment of human relevance and thus this topic was beyond the scope of the workshop
objectives.
3.3.3 Design of Rodent Cytotoxicity Studies
Panelists agreed that a tiered approach was optimal, beginning with acute inhalation
studies.
Acute inhalation study design
This study design would involve testing of single exposure concentrations over a range of
concentrations to determine the time course for induction of nasal histopathology in the F344 rat.
It was noted that the F344 rat was most relevant to assessment of cytotoxicity because NTP
(2000) used this strain for the carcinogenesis bioassay. Dr. Morris suggested that single
exposure concentration-response experiments also be conducted in the mouse; comparisons
between the rat and mouse provide information that is important for assessment of human
relevance.
Further, it was important to establish a range of NP concentrations that induces rat nasal
effects, with the lowest dose being < 1 ppm. There was some concern about whether it was
possible to get a dose as low as < 1 ppm; however, Dr. Buckpitt noted that this could be done
relatively easily by increasing compound dilution. Suggestions were also made to examine a
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subset of blood and/or urinary metabolites in order to identify potential animal biomarkers that
could be correlated with human biomarkers.
Repeated inhalation exposure studies in rats
Panelists agreed that the concentration-response data collected from the acute inhalation
exposure studies should then be used to select 3 exposure concentrations for a repeated
inhalation exposure study of 3-6 months duration, with a number of interim sacrifices in order to
examine the time course of histopathologjc effects under conditions of repeated exposure.
What dosing patterns should be employed in this study? There was general agreement
that exposure concentrations should be compatible with existing bioassay data, and some
panelists noted that the highest dose should not be greater than 10 ppm. Discussion ensued
regarding the frequency of interim sacrifices. One suggestion was to conduct daily interim
sacrifices, using the BrdU assay for determination of cell proliferation. An alternate suggestion
was to first obtain time-course data for lesion formation, followed by the BrdU assay at relevant
time points. Dr. Goldstein suggested that mapping be conducted, assuming a standard dose-
response curve is obtained (i.e., increasing response with increasing concentration), and that
blocks of tissue be saved and stored in order to subsequently perform mapping studies. The
objectives of these studies would be to localize the occurrence of lesions and then to correlate
sites of lesion formation with those in which cytotoxicity is occurring.
Dr. Eastmond emphasized that this study was a substantial one as it included repeated
dosing, two sexes, multiple animals per dose group, and numerous interim sacrifices to plot the
time course of cytotoxicity and lesion formation. Further, there were a large number of end
points to be assessed, including multiple metabolites. Dr. Goldstein suggested that at least a
subset of these animals also be tested later for genotoxicity. Retrospective tissue analysis could
be conducted using genotoxicity assays such as the Comet, the rnicronucleus test, and DNA
adduct formation tests.
- Dr. Genter suggested that another end point, DNA methylation, be evaluated. DNA
methylation bridges the gap between genotoxicity and cytotoxicity because this is an effect on
DNA that is traditionally considered to be nongenotoxic. This type of study can also be done
with very small amounts of DNA.
Acute and repeated exposure inhalation studies in mice
Assuming that similar tiered studies will be conducted with mice, what strain of mouse
should be used? One suggestion was to use the CYP2F2 knockout mouse. Cell
cycling/proliferation could be examined with BrdU assays. Apoptosis may be able to be
assessed via histopathologic examination. Specific apoptosis tests were not suggested. The
importance of apoptosis was noted; specifically apoptosis was likely to be involved in
differential selection of cell populations, thus leading to a promoting rather than initiating effect.
Dr. Genter noted that apoptosis would be difficult to quantitate in nasal tissue because it occurs
at a very low rate in the nose. Dr. Penning suggested the use of knockout mouse models for
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PKC, p53, and p21 to examine non-genomic events mediated by NP and its metabolites.
However, he noted that mice only developed lung tumors, not nasal tumors. Dr. Buckpitt stated
that mice did exhibit a weak nasal response in the NTP (2000) bioassay. Had higher
concentrations been used, such as 60 ppm, olfactory lesions may have been observed.
If such studies were to be performed, what level of confidence could be applied to the results?
Dr. Penning noted that this type of study should be testing a hypothesis: specifically,
does NP or its metabolites disrupt cell-cycle check points? It was generally agreed by the
panelists mat affirmation of this hypothesis would indicate a non-genotoxic response, but would
not necessarily be conclusive in terms of non-genotoxicity versus genotoxicity because
cytotoxicity may be only a part of the mode of tumorigenic action. Other mechanisms, including
genotoxic mechanisms, may also be involved.
Other tests: use of a cell-based model to assess effects ofNP on cell-cycling mechanisms
Dr. Penning noted that the hypothesis to be tested is one of basic science, and could also
be tested in a cell-based model using cells from either mouse lung and/or rat nose. Specifically,
can NP and/or its metabolites affect cell-cycling mechanisms mat can be interpreted as indicative
of a promoting capacity, such as interference with protein kinase C activities? Very little
information is known on whether NP has any promoting properties. However, even if this mode
of action were to be observed, one could not conclude that it was relevant to effects occurring in
target tissues. Dr. Morris noted that this type of experiment could be conducted in a single day,
and would provide important non-genotoxic information.
Dr. Buckpitt noted that this type of study could be done in 24 hours, with the use of
single dose and single target zone. If the nasal lesion is present, one can conclude that the lesion
formation is driven by metabolic activity. If the lesion is not present, difficulties arise in the
interpretation of results'. For example, is there delivery of NP to the nasal system? Is NP active
at the selected target site?
With regard to the use of the CYP2F knockout mouse, Dr. Buckpitt stated that NP
inhibits isoforms other than 2F. If findings in this knockout strain were negative, no conclusions
could be reached. If the findings were positive, they would suggest an inhibitory effect on cell
metabolism and could be correlated with results in nonhuman primates. Studies of metabolic
inhibition, including effects on quinone reductase and aldo-keto reductase, could then be
conducted.
Dr. Van Winkle emphasized the importance of characterizing broad-based metabolic
inhibition in nasal tissues. It is not known which CYP might be involved, whether more than
one CYP2F isozyme might be involved, and whether other isoforms and not CYP2F might be
involved.
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It was suggested that CYP isoforms may also be inducible in nasal tissues. However, Dr.
Center disagreed, stating that many people have tried to induce CYP in nasal tissue, with great
difficulty and little success, although induction does occur in the liver.
Could various inducers or hepatectomy be utilized to increase hepatic metabolism or assess
•whether active metabolite(s) are transported from the liver?
Dr. Eastmond questioned whether a hepatectomy would provide useful information to
assess whether an active metabolite was transported from the liver. Dr. Buckpitt stated that this
type of experiment was not informative, because performance of a hepatectomy alters kinetics;
thus, one would not know if the results were due to kinetic changes involving the parent
compound or involving liver metabolites. The question of whether inducers of hepatic
metabolism might be useful was discussed. Dr. Buckpitt noted that mis type of experiment was
very difficult to interpret; for example, an experiment has been conducted in which animals were
given phenobarbital prior to the lung toxicant 4-ipomeanol. Phenobarbital-induced animals
showed decreased lung toxicity because the liver rapidly cleared this compound by conjugation.
Dr. Morris suggested that induction of liver metabolism may also change CYP kinetics in the
nose.
Should cytotoxicity studies focus on the lung rather than on nasal tissues?
The rationale for this approach was presented, mainly that (1) single dose exposures
cause a very predictable time course of tissue injury in the Clara cells of mice, and (2) age and
gender differences have been demonstrated with young and female mice being more susceptible.
Therefore, more data are available on NP-induced toxicity in the mouse lung relative to the rat
nose.
Some panelists noted that it was not clear whether cytotoxicity studies using mouse lung
tissue would yield informative data. If a single NP intraperitoneal injection is administered,
examination of lung cell repair following epithelial sloughing demonstrates the occurrence of
widespread cell proliferation, including proliferation of cells that are not in the target zone. With
repeated NP exposures, the pattern of proliferation is much more confusing; there is no single
spike. Thus, overlapping repair processes co-occur with processes involved in confirmed tissue
injury. There are also a number of data gaps with regard to lung cytotoxicity. For example, do
female mice develop tolerance with repeated exposures? Existing data on tolerance are for
males, not females.
It was noted that the key questions for cytotoxicity involve issues regarding selection of
dose and end points with which to perform metabolic studies. These studies can be conducted
with both lung and nasal tissues; distal airways and nasal tissue are easy to dissect. Ex vivo
experiments are also relatively simple to do. One can manipulate parameters in cell types of
interest in both lung and nasal epithelium.
Dr. Morris agreed that studies should be performed with target tissues in both species. He
emphasized that his interest was in comparing and contrasting responses between mice and rats.
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In rats, there is nasal but not lung toxicity, whereas the reverse occurs in mice. These data
support the conclusion that different mechanistic patterns are occurring between the two species
and thus it was important to study both target tissues.
Additional discussion on the use oftransgenic mouse models
Panelists agreed that transgenic mouse studies should be conducted and that the
following enzymes should be monitored: (1) epoxide hydrolase; (2) GST; and (3) reductases.
Dr. Fanucchi reiterated that there was a paucity of data in target tissues, especially in the nose.
Limited information is available on CYP and glutathione. Changes in enzyme profiles
associated with NP treatment were not well characterized. Expression profiles for some proteins
are available and are more useful than mKNA expression profiles because proteins are
functional. Further, she noted that existing data are for males, and that nothing is known about
females. Dr. Penning agreed that expression profiles for protein were important; however, he
also thought that mRNA expression profiles would be useful information with regard to
genotyping.
Dr. Buckpitt added mat CYP2F in the mouse has an extreme affinity for NP and that
development of this knockout mouse, if none is currently available, would provide information
on whether this CYP isoform is associated with observed changes in target tissues. Dr. Penning
suggested that reductases were also important to assess, particularly dihydrodiol
dehydrogenases, isoforms of the aldo-keto reductase superfamily, and recommended the use of a
transgenic knockout mouse model for their study.
3.4 Charge Question # 5; What resources (level of effort, funds, time) would be
required to perform the suggested (cytotoxicity) studies?
Resources required for acute exposure inhalation studies
A discussion about the general costs of conducting acute inhalation studies ensued
following the recommendations for acute cytotoxicity testing. No specific cost estimates were
offered. Panelists agreed that inhalation exposure is the most expensive testing route; however,
it is also, the most relevant. Histopathologic assessment of nasal pathways is also expensive.
Multiple rats per dose group are needed to reliably determine the time course for occurrence of
nasal histopathology. Nonetheless, these studies are important for determination of the NP
concentration-response curve in the rat.
Resources required for repeated exposure inhalation studies
Assuming the use of GLP for the repeated inhalation exposure studies in the rat and the
mouse, Dr. Goldstein asked for an estimated cost for these studies. None of the panelists offered
cost estimates. Nonetheless, Dr. Goldstein noted that it was important to provide enough
information to design a "Cadillac" study, as if money were no object.
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Panelists agreed that these types of studies would be very expensive, approaching the
standard two-year animal bioassays conducted by NTP (2000) which cost approximately $6
million. Addition of transgenic studies would accelerate the costs, possibly exponentially.
4.0 SUMMARY OF DISCUSSION ON OTHER RELATED TOPICS
4.1 Comparison of Rodent and Non-Human Primate Studies
Dr. Van Winkle noted that Dr. Buckpitt had done some work with NP in monkeys. In
monkeys, NP metabolism is a lot slower than in either rodent species. However, metabolism to
reactive metabolites which become bound covalently to protein appears to be similar to that of
t the rat. It is not known whether these effects are associated with cytotoxicity or genotoxicity.
Dr. Buckpitt thought that this question could be answered by developing biomarkers not of
exposure but of events driving the cytotoxic (or carcinogenic) response. Correlation of a specific
protein adduct (or adducts) with cytotoxicity would allow investigators the ability to measure
these across species and in exposed humans to determine relative sensitivity. Similarly, if
naphthalene metabolites generate DNA adducts, these may be able to be measured in biofluids of
exposed populations. In this case animal correlations would be essential. A high rate of
adduction occurs, in the order of 0.7 |ig adducts/nanomole. This suggests that there are multiple
mechanisms of adduct formation and that a number of proteins are adducted, as indicated by
protein folding.
Would a short-term inhalation toxicity study in primates provide useful information?
Dr. Buckpitt stated mat he thought that this type of study would be very useful. If NP
exposure concentrations up to 60 ppm are administered to monkeys and no effects are observed,
strong support would be provided for the conclusion that marked species differences occur, most
likely through differences in the rate of naphthalene bioactivation. Glutathione depletion could
potentially be used to monitor this. He added that this type of study could not be done following
intraperitoneal injection of NP and would have to be conducted via inhalation exposure.
Dr. Penning noted that 1,2-NQ and 1,4-NQ are redox active compounds. Could
mechanisms be discerned by measurement of these compounds alone in experimental studies?
These metabolites are bi-functional: they are electrophilic and will generate ROS. It would be
useful to identify biomarkers that measure the consequence of electrophile or ROS formation
following NP administration in non-human primates. Dr. Morris agreed with this approach.
However, Dr. Goldstein disagreed, noting that primate studies should not be conducted without
more information on relevant end points and metabolism in rodents. He suggested that the
approach used with butadiene might be suitable for NP. However, given the paucity of data on
metabolic pathways and end points, this approach does not appear to be useful at this time. Dr.
Goldstein added that it might be worthwhile to conduct non-human primate studies to inform the
issue of interspecies extrapolation. However, again he did not think that sufficient information
on metabolic pathways and end points were currently available to obtain useful information in
non-human primates.
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Dr. Penning noted that quinones appear to cause oxidati ve stress and if this can be
confiimed, it would be a good end point to measure in non-human primates. A short-term
primate study could demonstrate that in the absence of quinone generation, downstream
metabolites do not form. Does quinone formation, which occurs in rodents, also occur in
primates? Dr. Buckpitt noted that in one of his primate studies, quinones were not generated
following NP administration. However, this end point was not the focus of the study; therefore,
the reliability of the finding is uncertain. It was agreed that the first metabolic step is essential in
rodents for the cascade of downstream events induced by NP exposures. If it does not occur in
primates, then this finding would be indicative of marked species differences. Dr. Eastmond
expressed concerns that quinone generation in non-human primates might occur at very low
levels or below the limit of detection. Dr. Buckpitt replied that quinone generation can be
measured at very low levels and via the use of radiolabeled material. .Dr. Eastmond noted that
there were regulatory guidelines for interpretation of findings with regard to determination of a
threshold versus non-threshold (low-dose linear) response, and mat it would be important to
consider these guidelines in designing a primate study. '
Are there any data on naphthalene cytotoxicity in humans?
Dr. Goldstein asked if there were any human studies available following NP exposure.
Dr. Fanucchi noted that there were no human studies of which she was aware. Dr. Genter added
that a human biopsy would be difficult to obtain in the relevant region of the olfactory pathways
because this region is too close to the brain, unless there was a medical condition necessitating
removal of the whole turbinates. It would be difficult, but possible, to obtain human respiratory
epithelium. Dr. Eastmond stated that human biomonitoring was neither useful nor heuristic until
there were better precursor or biomarker data. He suggested epoxide or quinone adducts of
proteins as potential biomarkers and noted that one investigator, Stephen Rappaport, has looked
at these biomarkers as end points for benzene toxicity in humans.
4.2 Relative Timing of Performance of Cytotoxicity and Genotoxicity Studies
Among cytotoxicity experiments previously suggested, which ones.are really needed
prior to performance of genotoxicity studies? Which of these studies could be done
simultaneously with genotoxicity studies and which could be done following genotoxicity
studies?
Dr. Goldstein stated that at this time, none of the suggested studies would conclusively
differentiate between cytotoxicity and genotoxicity. How might this fact affect the ordering of
data collection? Dr. Goldstein added that NP may not become fully genotoxic unless a high
level of cell proliferation occurs. Therefore, defining the conditions under which cell
proliferation occurs and the extent of cell proliferation in target tissues are important data to
generate prior to assessment of genotoxicity. This type of information has been determined for
formaldehyde and acetaldehyde in in vivo studies. Dr. Eastmond suggested that cell
proliferation may occur prior to or concurrent with genotoxicity; an adduct or other pre-
mutagenic lesion would become fixed into a mutation during cell replication. For heritable
chromosomal damage to occur, the cell would ordinarily need to go through a mitosis.
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Therefore, the observed genotoxic effects could be due to a combination of cytotoxicity and
direct DNA damage. Alternatively, cytotoxicity may be the key event in the absence of
genotoxicity. Although DNA adducts may occur in target tissues, if there is no cell replication,
fixation of damage is less likely to occur. Therefore, performance of cytotoxicity studies prior to
genotoxicity studies would be beneficial.
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APPENDIX A
List of Expert Participants
Bernard D.Goldstein, M.D.
Graduate School of Public Health
University of Pittsburgh
Pittsburgh, PA
Mary Beth Center, Ph.D.
Department of Environmental Health
University of Cincinnati
Cincinnati, OH
John Morris, Ph.D.
Department of Pharmaceutical Sciences
University of Connecticut
Starrs, CT
Laura Van Winkle, Ph.D. DABT
School of Veterinary Medicine
Department of Anatomy, Physiology and Cell Biology
University of California-Davis
Davis, CA
Michelle Fanucchi, Ph.D.
School of Veterinary Medicine
Department of Anatomy, Physiology and Cell Biology
University of California-Davis
Davis, CA
Alan Buckpitt, Ph.D.
School of Veterinary Medicine
Department of Molecular Biosciences
University of California-Davis
Davis, CA
David Eastmond, Ph.D.
Environmental Toxicology Graduate Program
University of California-Riverside
Riverside, CA,
Trevor Penning, Ph.D
School of Medicine
Department of Pharmacology
University of Pennsylvania
Philadelphia, PA
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APPENDIX B
List of Observers
Lauren A. Wally
Graduate School of Public Health
University of Pittsburgh
Leslie Shapard
ORISE ->
Brian Herndon
ORISE
Anne LeHurray
American Chemistry Council
Cynthia Davin
EMBSI
John Gibbs
Kerr-McGee Corporation
Jane Patarcity
Beazer East, Inc.
Thomas Zordan
Zordan Associates, Inc.
Jonathan Gledhill
Policy Navigation Group
George Cruzan
Tox Works
Michael Juba
Koppers, Inc.
Vincent Piccirillo
VJP Consulting, Inc.
Peter Preuss
US Environmental Protection Agency
Lynn Flowers
US Environmental Protection Agency
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Bonnie Stern
BR Stern and Associates
Donald McGraw
Corporate Environmental Solutions
Channa Keshava
US Environmental Protection Agency
Ken Fontaine
Morgan Research Corporation
Linda Wennerberg
US Department of Defense
William Luttrell
Navy Environmental Health Center
Nancy Beck
US Office of Management and Budget
Maxine Wright-Walters
University of Pittsburgh
Mike Rick
University of Pittsburgh
David C. Ailor
ACCCI
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APPENDIX C
Peer Consultation Workshop on Research Needs Related to the IRIS Draft Toxicological
Review of Naphthalene
University of Pittsburgh
Pittsburgh, PA
April 7,2005
MEETING AGENDA
Location: Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA
8:3 0 am Registration
9:00
Orientation, Instructions and Conflict of Interest Discussion
(Brian Herndon, ORISE)
9:15
9:30
9:45
11:00
Introduction to the Workshop (Dr. Goldstein, Chair)
Panel Chair's Intreduction and Overview of Charge
Charge Question #1 (Panel)
Charge Question #2 (Panel)
12:00 noon Lunch
1:00 pm Charge Question #3 (Panel)
2:00 Charge Question #4 (Panel)
3:30 Charge Question #5 (Panel)
4:30 Conclude Meeting (Dr. Goldstein, Chair)
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APPENDIX D
Panelists' Overheads
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APPENDIX D-l
Dr. Bernard Goldstein
Comments from the external peer review (July 2004) that were related to the mode of
action of naphthalene carcinogenicily:
Charge Question:
4. Inhalation Carcinogenicity of Naphthalene
4. a An assumption has been made that the nasal tumors in rats and lung tumors in
mice are relevant to human carcinogenesis. Has this assumption been transparently
and objectively described?
Laura Van Winkle:
...However, it would be nice to have some human data that shows an increase in tumors
after NA exposure, or barring that, data in nonhuman primates on NA metabolism and
cytotoxicity.
Charge Question:
4. b Naphthalene is described as likely to be carcinogenic to humans via the inhalation
route of exposure based on the U,S, EPA 1999 Draft Revised Cancer Guidelines
(www. epa.goy/ncea). Do the available data support this statement?
James Chen:
...I suggest adding a statement that the overall evidence for naphthalene in the group of
agents designed as likely human carcinogens is at the low end.
Michael Dourson:
I am comfortable with this statement and agree with several panel members that the
supporting data fall at the animal end of the range of data in this definition. I may or may
not agree with adding a statement that "naphthalene is likely to be carcinogenic to
humans at doses that exceed a threshold for cytotoxicity", depending on further
suggested work as shown in the answer to question 3.
John Morris:
...In my view, the absence of in vitro genotoxicity tests that include a target organ (e.g.
nasal S9) activating system represents a critical data gap that might be highlighted.
Laura Van Winkle:
...The current data appear to be at the minimal end of fulfilling the requirement for NA to
be considered as likely to be carcinogenic to humans because there is no convincing
human data. Further information, not available at this time, regarding NA toxicity or
carcinogenicity in humans and nonhuman primates might well justify reclassification of
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NA to "suggestive", particularly if it is shown that the rodent carcinogenicity and MOA
are not relevant to humans and/or nonhuman primates. In the absence of this information
we must assume that the animal carcinogenicity and mode of action are relevant to
humans, but this is not known.
Charge Question:
4,c An inhalation unit risk has been derived utilizing benchmark dose modeling to
define the point of departure of 10% extra risk followed by linear low-dose
extrapolation below the point of departure.
4,c.l The inhalation dosimetry equations used in the calculation of the human
equivalent concentrations are for a category 1 gas (U.S. EPA, 1994). Is the explanation
for the dosimetry choice in the derivation of the inhalation unit risk scientifically
justified and transparently described?
Michael Dourson:
I do not agree with EPA's use of a category 1 RGDR, nor with observer comments on the
use of a category 3 RGDR. Naphthalene is a category 2 gas. EPA needs to do its
homework here and parse out the proportion of dose expected to arrive at the target
tissues by way of systemic circulation and direct absorption. If the data are truly not
helpful in making a reasonable guess, then I would be comfortable with a 50/50 split...
Mary Beth Center:
Based on the discussions and definitions of Category 1,2, and 3 gases, naphthalene best
fits the description of a Category 2 gas, not Category 1.
John Morris:
...A careful application of the RfC methodology would suggest that naphthalene is in fact
best modeled as a category 2 gas.
...As is documented for other compounds (e.g. acetaldehyde) the relative quantitative
importance of metabolism may differ dramatically at high compared to low exposure
concentrations. This represents another factor that should be considered relative to the
inhalation dosimetry.
Charge Question:
4.C.2 Has support for the use of linear low dose extrapolation been objectively and
transparently presented? Are there other modeling approaches that should have been
considered instead of or in addition to the low dose linear extrapolation approach?
Michael Dourson:
I am not comfortable with the support for the linear case being made by EPA, especially
in light of the public comments, which show little support for naphthalene's supposed
mutagenicity. Even EPA acknowledges this. EPA needs to define what data would
cause it to move from this linear default, and not just state that an understanding of
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naphthalene's MO A is not sufficient. Although modeling anoncancer cytotoxicity
precursor is unlikely to yield credible results, since the incidence of these noncancer
lesions is so high at low dose and the dose response behavior is so fiat, it would be
enlightening to model both a linear and nonlinear curve at the same time—like a hockey
stick, but with no threshold. Consistent with this duel approach is the fact that tumors are
only found with extensive noncancer toxicity, and that no genotoxicity is found except
with some metabolites that make up an unknown, but suspected to be small, fraction of
the overall metabolites.
Michelle Fanucchi:
The support is clearly presented. This appears to be the appropriate modeling approach.
David Gaylor:
Low dose linear extrapolation is justified, but likely to be overly conservative...
John Morris:
The rationale for the linear low dose extrapolation was clearly and objectively described
in my view. It is my belief that a significant advance would be made by reliance on a
dosimetrically-based PBPK model for low dose extrapolation. The state-of-the-art is
sufficiently advanced to allow inclusion of inhalation pharmacokinetic considerations in
the risk assessment process. Greater clarity and objectivity would be provided by text
that lays out the alternative risk assessment approaches (cytotoxicity driven non-linear, or
mixed mode of action), and also explicitly indicates the basis for selection of the
approach that was utilized.
Charge Question:
4.C.3 The inhalation unit risk is based upon the summed risks of developing olfactory
neuroblastomas and respiratory epithelial adenomas in male rats derived from a time-
to-tumor analysis. Is this approach scientifically justified? Are there other modeling
approaches that should have been considered instead of or in addition to the approach
taken? Has the best data set been chosen for derivation of the inhalation unit risk?
Has the modeling been accurately and transparently described?
Michael Dourson:
...After I think that I understood it, my first question was why not add up all the
nonsignificant tumors in males? Why just stop with neuroblastomas for males and
epithelial adenomas for females? Why not take the approach adding both the male and
female responses for individual tumor types?...
It appears mat the answers to these questions might be that EPA is trying to maximize the
cancer potency factor. I have no conceptual problem with this, but if this is the case,
EPA needs to clearly state it.
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Michelle Fanucchi:
This approach is very conservative, but given the lack of complete information, it appears
to be justified...
David Gaylor:
...The summed risks correctly estimate the risk of an olfactory neuroblastoma or a
respiratory epithelial adenoma. This is the most conservative approach. However, if it is
desired to estimate the risk that an individual develops either type of tumor (the more
usual calculation), then the risk of an individual producing both types of tumors must be
subtracted from the sum to avoid double counting of individuals with both types. For
rare tumors, as is the case here, this is a minor adjustment...
Mary Beth Genter:
I believe that the approach is valid, although this tumor type is very unusual...
Laura Van Winkle:
The inhalation unit risk is based upon the summed risks of developing olfactory
neuroblastomas and respiratory epithelial adenomas in male rats derived from a time-to-
tumor analysis. Is this approach scientifically justified? Yes from a "choice of models"
point of view. It is a strength of this modeling approach that it allows different tumor
types to contribute to the estimate of risk...
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APPENDIX D-2
Dr. David Eastmond
Research Recommendations to determine whether naphthalene is genotoxic in the rat
olfactory epithelial and respiratory epithelial tissues. Similar studies could/should be
done for the female mouse hong. Exposures should be by inhalation
Covalent binding to nasal (and/or lung) tissues
LSC
32P
AMS
Mutation in target tissues (or elsewhere)
Rat-Big Blue
Mouse-Big Blue or Mutamouse
Chromosomal damage and cell proliferation in the target
Micronucleus assay
BrdU incorporation
DNA damage in target tissue
Comet assay-DS vs SS breaks, oxidative lesions
Secondary assays-
Ames with nasal or lung microsomes
Cell assays with lymphoblastoid cells expressing specific Cyp isoforms
8-hydroxy dG studies
To convincingly establish that something is non genotoxic requires a fairly high standard
of proof, in part because there are a number of genotoxic mechanisms and because one is
trying to prove a negative
For non genotoxic mechanisms of action, I would recommend modeling toxicity and cell
proliferation in the target tissues. These could be used as biomarkers to provide insights
into the concentrations/doses where effects are likely to be seen in the cancer bioassay.
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APPENDIX D-3
Dr. Trevor Penning
Potential DNA-Adducts that Arise from Naphthalene Metabolism
Covalent-Bulkv Stable Adducts
NP-l,2-oxide-N2-dGuo (2 trans-opened and 2 cis-opened adducts)-4 total
NP-l,2-oxide-N6-dAdo (2 trans-opened and 2 cis-opened adducts)-4 total
NP-l,2-dione-N2-dGuo (1,4- and 1,6-addition possible) 2-total
NP-l,2-dione-N6-dAdo (1,4- and 1,6-addition possible) 2-total
(+)-trans-anti-NPDE (diol epoxide)-N2-dGuo .
(-)-trans-anti-NPDE (diol epoxide)-N2-dGuo
(+)-cis-anti-NPDE (diol epoxide)-N2-dGuo
(-)-cis-anti-NPDE (diol epoxide)-N2-dGuo
(+)-trans-anti-NPDE (diol epoxide)-N6-dAdo
(-)-trans-anti-NPDE (diol epoxide)-N6-dAdo
(+)-cis-anti-NPDE (diol epoxide)-N6-dAdo
(-)-cis-anti-NPDE (diol epoxide)-N6-dAdo
(8-total diol-epoxide adducts which become 16 if the syn diol epoxide is formed instead of the
anti diol epoxide)
Covalent-Depurinating Adducts
NP-l,2-dione-N7-Gua (1,4- and 1,6-addition possible) 2rtotal
NP-l,2-dione-N7-Ade (1,4- and 1,6-addition possible) 2-total
NP-l,4-dione-N7-Gua (1,4- and 1,6-addition possible) 2-total
NP-l,4-dione-N7-Ade (1,4- and 1,6-addition possible) 2-total
Oxidative DNA Adducts
8-oxo-dGuo
Ml-dG (malondialdehyde)
4-hydroxy-2-nonenal propano-dGuo
4-oxo-2-nonenal etheno-dGuo, etheno-dAdo and etheno-dCyd
[dGuo, 2'-deoxyguanosine; dAdo, 2'-deoxyadenosine; dCyd, 2'-deoxycytidine; Gua, guanine;
and Ade, adenine]
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