United Starts • - -J$:
Environmental Prottction tCAO-CIM-OOT
Agency " December, 1991
EPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
POLYCYCLK AROMATIC HYDROCARBONS (PAHS)
Prepared for
OFFICE OF WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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DISCLAIMER
This document has been reviewed 1n accordance with the U.S.
Environmental Protection Agency's peer and administrative review policies
and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Hater Act, as amended In
1986, requires the Administrator of the Environmental Protection Agency to
publish maximum contaminant level goals (MCLGs) and promulgate National
Primary Drinking Water Regulations for each contaminant, which, In the
judgment of the Administrator, may have an adverse effect on public health
and which Is known or anticipated to occur In public water systems. The
MCLG Is nonenforceable and 1s set at a level at which no known or antici-
pated adverse health effects In .humans occur and which allows for an
adequate margin of safety. Factor's considered 1n setting the MCLG Include
health effects data and sources of exposure other than drinking water.
This document provides the health effects basis to be considered 1n
establishing the MCLG. To achieve this objective, data on pharmacok1net1cs,
human exposure, acute and chronic toxkUy to animals and humans, epidemi-
ology and mechanisms of toxldty are evaluated. Specific emphasis 1s placed
on literature data providing dose-response Information. Thus, while the
literature search and evaluation performed In support of this document has
been comprehensive, only the reports considered most pertinent In the deri-
vation of the MCLG are dted 1n the document. The comprehensive literature
data base In support of this document Includes Information published up to
1985; however, more recent data may have been added during the review
process.
When adequate health effects data exist, Health Advisory values for less
than lifetime exposures (1-day, 10-day and longer-term, -10X of an
Individual's lifetime) are Included 1n this document. These values are not
used In setting the MCLG, but serve as Informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
Tudor Davles, Director
Office of Science and Technology
James Elder, Director
Office of Ground Water and Drinking
Water
111
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DOCUMENT DEVELOPMENT
RHa Schoeny, Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Authors
Rita Schoeny
Glenn R1ce
Patricia A. Murphy
Linda S. Erdrelch
Larry Fradkln
Annie Jarabek
Environmental Criteria and
Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Ellen O'Flaherty
Dept. of Environmental Health
University of Cincinnati
Medical Center
Scientific Reviewers
William Bruce Pelrano
Environmental Criteria and
Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Y. Pate!
Office of Drinking Water
Washington, DC
Roy Albert
Department of Environmental Health
University of Cincinnati
Medical Center
3223 Eden Avenue
Cincinnati, OH 45267-0056
Daniel Krewskl
13 Lansfleld Way
Ottawa, Ontario
Canada K2G 3V7
David Warshawsky
University of Cincinnati
3223 Eden Avenue
Cincinnati, OH 45219
Alexander Wood
Hoffman-LaRoche
340 Klngsland Street
Nutley, NO 07110
Edmond LaVole
Rutgers University
College of Pharmacy
Department of Pharmaceutical
Chemistry
Plscataway, NJ 08855
Editorial Reviewers
Judith Olsen
Environmental Criteria and
Assessent Office, Cincinnati
U.S. Environmental Protection Agency
Document Preparation
Technical Support Services Staff:
Office, Cincinnati
Environmental Criteria and Assessment
1v
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TABLE OF CONTENTS
Page
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES II-l
ACENAPHTHYLENE II-7
ANTHRACENE II-7
Production and Use II-7
Occurrence . . . . II-7
BENZ[a]ANTHRACENE II-8
Production and Use II-8
Occurrence II-8
BENZO[a]PYRENE II-8
Production and Use II-8
Occurrence II-8
BENZO[b]FLUORANTHENE II-8
Production and Use II-8
Occurrence . II-9
BENZO[k]FLUORANTHENE. II-9
Production and Use ." II-9
Occurrence II-9
BENZO[g,h,1]PERYLENE. . II-9
Production and Use II-9
Occurrence II-9
CHRYSENE 11-10
Production and Use 11-10
Occurrence . 11-10
DIBENZ[a,h]ANTHRACENE . . . , 11-10
Production and Use 11-10
Occurrence 11-10
FLUORANTHENE 11-10
Production and Use . 11-10
Occurrence 11-10
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TABLE OF CONTENTS (cent.)
Page
FLUORENE 11-11
Production and Use 11-11
Occurrence 11-11
INDENO[l,2,3-cd]PYRENE 11-11
Production and Use 11-11
Occurrence 11-11
NAPHTHALENE 11-12
Production and Use 11-12
Occurrence 11-12
PHENANTHRENE 11-13
Production and Use '. . . . 11-13
Occurrence 11-13
PYRENE 11-13
Production and Use 11-13
Occurrence 11-13
SUMMARY 11-13
III. TOXICOKINETICS III-l
ABSORPTION., III-l
Oral III-l
Inhalation III-6
Dermal 111-13
DISTRIBUTION 111-15
Oral 111-16
Inhalation ..... 111-17
Dermal 111-19
METABOLISM . . . 111-20
Benzo[a]pyrene as a Model of PAH Metabolism. 111-22
Metabolism of Nonalternant PAHs. III-28
Other Toxlflcatlon Pathways 111-28
Comparative Metabolism . . 111-33
v1
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TABLE OF CONTENTS (cent.)
Page
EXCRETION 111-35
Oral 111-38
Inhalation , 111-39
Dermal Ill-41
SUMMARY Ill-42
IV. HUMAN EXPOSURE IV-1
(To be provided by the Office of Drinking Water)
V. HEALTH EFFECTS IN ANIMALS V-l
ACUTE ORAL TOXICITY .V-l
Acenaphthylene V-l
Anthracene . . . . k . . . . V-l
Benz[a]anthracene V-2
Benzo[a]pyrene V-2
Benzo[b]fluoranthene V-2
Benzo[k]fluoranthene V-2
Benzo[g,h,1]perylene . . . . V-2
Chrysene V-2
D1benz[a,h]anthracene V-3
Fluoranthene V-3
Fluorene V-3
Indeno[l,2,3-cd]pyrene V-3
Naphthalene V-3
Phenanthrene V-5
Pyrene V-5
.ACUTE TOXICITY BY OTHER ROUTES V-5
Acenaphthylene V-5
Anthracene . . -. V-6
Benz[a]anthracene. . V-7
Benzo[a]pyrene V-7
Benzo[b]fluoranthene V-8
Benzo[k]f1uoranthene . . . V-8
Benzo[g,h,1]perylene V-8
Chrysene V-8
D1benz[a,h]anthracene V-9
Fluoranthene V-9
Fluorene V-9
Indeno[l,2,3-cd]pyrene V-10
Naphthalene. V-10
Phenanthrene . V-l 3
Pyrene V-14
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TABLE OF CONTENTS (cent.)
Page
SUBCHRONIC AND CHRONIC ORAL TOXICITY V-15
Acenaphthylene V-15
Anthracene V-17
Benz[a]anthracene V-18
Benzo[a]pyrene . V-18
Benzo[b]fluoranthene V-19
Benzo[k]fluoranthene V-19
Benzo[g,h,1]perylene V-19
Chrysene V-20
D1benz[a,h]anthracene V-20
Fluoranthene V-20
Fluorene V-21
Indeno[l,2,3-cd]pyrene V-22
Naphthalene V-23
Phenanthrene.,. V-28
Pyrene % '. . . . V-28
SUBCHRONIC AND CHRONIC TOXICITY BY OTHER ROUTES ....... V-29
Acenaphthylene V-29
Anthracene V-30
Benz[a]anthracene V-30
Benzo[a]pyrene V-30
Benzo[b]fluoranthene V-31
Benzo[k]fluoranthene . V-32
Benzo[g,h,1]perylene V-32
Chrysene V-32
D1benz[a,h]anthracene V-32
Fluoranthene V-33
Fluorene V-34
. Indeno[l,2,3-cd]pyrene . . V-34
Naphthalene V-34
Phenanthrene V-34
Pyrene V-34
TARGET ORGAN TOXICITY . . . . V-35
CARCINOGENICITY, ORAL . V-38
Acenaphthylene V-38
Anthracene V-39
Benz[a]anthracene V-39
Benzo[a]pyrene . . . . V-41
Benzo[b]fluoranthene V-58
Benzo[k]fluoranthene V-58
Benzo[g,h,1]perylene ..... . V-58
Chrysene .• V-58
D1benz[a,h]anthracene V-58
Fluoranthene V-61
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TABLE OF CONTENTS (cont.)
Page
Fluorene V-61
Indeno[l,2,3-cd]pyrene V-63
Naphthalene. V-63
Phenanthrene V-64
Pyrene . V-64
'CARCINOGENICITY, OTHER ROUTES . V-64
Acenaphthylene V-64
Anthracene V-65
Benz[a]anthracene V-65
Benzo[a]pyrene V-69
Benzo[b]fluoranthene and Benzo[k]f1uoranthene V-83
Benzo[g,h,1]perylene V-90
Chrysene V-91
D1benz[a,h]anthracene V-95
Fluoranthene . V-98
Fluorene V-102
Indeno[l,2,3-cd]pyrene V-103
Naphthalene V-107
Phenanthrene V^llO
Pyrene V-lll
REPRODUCTIVE/TERATOGENIC EFFECTS V-113
Anthracene V-113
Benz[a]anthracene V-113
Benzo[a]pyrene V-113
Chrysene V-120
Fluoranthene V-120
Naphthalene V-121
/
MUTAGENICITY/6ENOTOXICITY ... ..... V-123
SYNERGISH AND/OR ANTAGONISM ...... V-123
SUMMARY V-163
VI. HEALTH EFFECTS IN HUMANS VI-1
INTRODUCTION VI-1
CLINICAL CASE STUDIES VI-3
Oral . VI-3
Other Routes VI-5
EPIDEMIOLOGIC STUDIES VI-7
Oral VI-7
Other Routes VI-7
SENSITIVE POPULATIONS VI-14
SUMMARY VI-15
1x
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TABLE OF CONTENTS (cont.)
Page
VII. MECHANISMS OF TOXICITY VII-1
MECHANISMS INVOLVED IN PAH CARCINOGENICITY VII-1
Structure Activity Relationships VII-1
Tissue Specificity of PAH Metabolism VII-9
Genetic Control of PAH Metabolism VII-10
Other Pathways Involved 1n Activation of PAH VII-14
PAH Involvement In Carcinogenic Processes VII-15
MECHANISMS INVOLVED IN NONCARCINOGENIC ENDPOINTS VII-16
Hemolymphatlc System . . . VII-16
Cardiovascular System VII-19
Pulmonary System . VII-20
GastrolntesUnal System VII-21
Gonads VII-21
Endocrine System . . . . VII-22
Integumentary System VII-23
Visual System VII-23
PAH-INDUCED IMMUNOTOXICITY VII-26
SUMMARY VII-28
VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS VIII-1
'INTRODUCTION VIII-1
NONCARCINOGENIC EFFECTS . VIII-6
SHORT-TERM STUDIES IN ANIMALS VIII-7
LONGER-TERM STUDIES IN ANIMALS. VIII-7
Acenaphthylene VIII-8
Anthracene VIII-8
Benz[a]anthracene VIII-9
Benzo[a]pyrene VIII-9
D1benz[a,h]anthracene. VIII-10
Fluoranthene VIII-10
Fluorene VIII-11
Pyrene VIII-11
QUANTIFICATION OF NONCARCINOGENIC EFFECTS VIII-12
Derivation of 1- and 10-Day Health Advisories VIII-12
Derivation of Longer-Term HA VIII-15
Assessment of Lifetime Exposure and Derivation
of DWELs VIII-15
CARCINOGENIC EFFECTS . VIII-21
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TABLE OF CONTENTS (cont.)
Page
CLASSIFICATION OF PAHs AS TO POTENTIAL FOR HUMAN
CARCINOGENICITY . . VIII-23
Acenaphthylene VIII-23
Anthracene , . VIII-23
Benzo[g,h,1]perylene VIII-23
Fluoranthene . . VIII-24
Fluorene ... VIII-24
Naphthalene .'. . VIII-24
Phenanthrene .' VIII-24
Pyrene VIII-25
Benz[a]anthracene. . VIII-25
Benzo[a]pyrene VIII-25
Benzo[b]fluoranthene . ..... VIII-26
Benzo[k]fluoranthene VIII-26 v
Chrysene . .^ VIII-26
D1benz[a,h]anthracene ' VIII-27
Indeno[l,2,3-cd]pyrene VIII-27
QUANTIFICATION OF CARCINOGENIC EFFECTS VIII-27
Benz[a]anthracene VIII-28
Benzo[a]pyrene VIII-28
U.S. EPA (1980d) Approach . . . . VIII-29
Clement Associates (1988) VIII-31
Krewskl and Murdoch (1990) VIII-38
Clement Associates (1990a) VIII-39
U.S. EPA (1991b) VIII-49
Brune et al. (1981 VIII-52
Chouroullnkov et al. (1967) VIII-54
D1benz[a,h]anthracene VIII-62
'SPECIAL CONSIDERATIONS. vin-63
Toxldty Equivalence Factor Approach for PAHs In
Group B2 VIII-63
Interactions with Other Chemicals. . . VIII-77
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS VIII-79
SPECIAL GROUPS AT RISK VIII-80
SUMMARY VIII-81
IX. REFERENCES IX-1
x1
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LIST OF TABLES
No. Title Page
II-l Priority Pollutant Polycycllc Aromatic Hydrocarbons
Found 1n the Environment II-2
II-2 Selected Physical and Chemical Characteristics of
Important PAHs II-3
III-l Examples of Metabolism of PAHs to Biologically Active
Forms by Various Enzyme Systems ..." •. • • • 111-30
V-l Oral Carclnogenlclty Testing of Benz[a]anthracene
Administered by Gavage to Hale B6AF1 Mice . V-40
V-2 Tumor Incidence 1n "Atrium of Stomach" Following Gavage
Administration of Benzo[a]pyrene V-44
V-3 Incidence of Tumors Observed After a Single Gavage Treatment
of Mice with Benzo[a]pyrene 1n Polyethylene Glycol V-45
V-4 Carclnogenlclty of Oral Benzo[a]pyrene In Sprague-
Dawley Rats V-46
V-5 Incidence of Forestomach Paplllomas and Carcinomas 1n
Male and Female CFW Mice Administered Benzo[a]pyrene In
the Diet V-50
V-6 Carclnogenlclty of Benzo[a]pyrene Administered 1n the
Diet to Male and Female Swiss Mice. . V-51
V-7 Induction of Forestomach Tumors 1n Ha/ICR Mice Fed
Dietary Benzo[a]pyrene V-55
V-8 Oral Carclnogenlclty of D1benz[a,h]anthracene In DBA/2
Mice V-60
V-9 Carclnogenlclty Testing of Fluorene Administered 1n the
Diet to Female Buffalo Rats V-62
V-10 Dermal, Injection and Implantation Carclnogenlclty
Assays of Anthracene V-66
V-11 Carclnogenlclty of Benzo[a]pyrene to Male Syrian
Golden Hamsters by Inhalation V-72
V-l2 Carclnogenlclty of Benzo[a]pyrene Administered by
Intratracheal Instillation to Syrian Hamsters V-74
V-l3 Sarcomagenlc Activity of Benzo[a]pyrene 1n Male C3H
Mice Following a Single Subcutaneous Injection V-79
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LIST OF TABLES (cont.)
No. Title Page
V-14 SENCAR Mouse Skin Tumorlgenesls, Benzo[a]pyrene-
Tumor Initiation ................... ... V-81
V-15 Carclnogenlclty of Benzofluoranthenes by Implantation
1n Rat Lungs ........................ V-84
V-16 Cardnogenldty Assay of PAH In Newborn Mice ........ V-85
V-17 Tumor Initiating Activity of Benzofluoranthenes 1n
Crl:CD-l Mice ...... ................. V-87
V-18 Tumor Initiating Activity of Benzofluoranthenes 1n
Crl:CO-l (ICR)BR Mice ............... '. . . . V-89
V-19 Sarcomagenlc Activity of Subcutaneously Injected
D1benzo[a,h]anthracene and Benzo[a]pyrene In Female
NMRI Mice ........................ . V-99
V-20 Incidence of Flbrosarcomas 1n Mice Associated with
Subcutaneous Injections of D1benz[a,h]anthracene ...... V-100
V-21 Cardnogenldty of Indeno[l,2,3-cd]pyrene upon
Implantation In Rat Lungs ................. V-104
V-22 Assay of Pyrene In Newborn Mice ...... ........ V-112
V-23 Embryotoxldty and Malformations 1n Swiss Mice
' Exposed by Intraembryonal Injection of Benzo[a]pyrene
and Derivatives . . ............ ........ V-117
V-24 Results of Short-Term Tests of Polycycllc Aromatic
Hydrocarbons ........................ V-124
V-25 Cocarclnogenlc Activity of Various PAHs with
Benzo[a]pyrene 1n Mouse Skin ................ V-156
V-26 Cocarclnogenlc Activity of Various PAHs with
Benzo[a]pyrene on Mouse Skin ................ V-157
V-27 Co-administration of Pu02 and Benzo[a]pyrene
by Inhalation ....................... V-162
VI-1 IARC Determinations of PAH Cardnogenesls Based on
Human Data ......................... VI-9
VII-1 Reactivity Indices for Polycycllc Hydrocarbons. ...... VII-8
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LIST OF TABLES (cont.)
Title
Immunotoxlclty of Benzo[a]pyrene In Mice after
Short-Term Exposure VIII-13
VIII-2 Comparative Effects of PAH'on Immune Functions In B6C3F1
Mice VIII-14
VIII-3 Incidence of Tumors 1n Mice Treated Intragastrlcally
with Benzo[a]pyrene VIII-30
VIII-4 Forestomach Tumors 1n Mice Fed Benzo[a]pyrene VIII-32
VIII-5 Variable Exposure Data Used to Estimate Parameters
1n the Ingestlon Dose-Response Model for Benzo[a]pyrene . . VIII-35
VIII-6 Forestomach Tumors In Mice Subjected to Variable
Exposures of Benzo[a]pyrene VIII-40
VIII-7 Historical Control Incidence Data for Forestomach Tumors
1n Strains of Swiss (Webster) Bred Mice VIII-42
VIII-8 Maximum Likelihood Parameter Estimates for Two-Stage
Model with Saturation of Growth Rate Function VIII-44
VIII-9 Forestomach Tumors 1n Mice Subjected to Exposures of
Benzo[a]pyrene for at least 70 Days VIII-45
VIII-10 Predicted vs. Observed Tumor Incidence and Data Used to
' Calculate the Dose-Response Model VIII-51
VIII-11 Incidence to Forestomach Only and Total Contact Site
Tumors 1n Sprague-Dawley Rats Exposed to Benzo[a]pyrene
by Gavage or In the Diet VIII-53
VIII-12 Slope Factors for Humans Based on Benzo[a]pyrene
Feeding Studies VIII-56
VIII-13 Cancer Potency Estimates for Excess Risk of 10~5 from
Lifetime Exposure Based on Oral Exposure Data for
D1benz[a,h]anthracene VIII-64
VIII-14 Potency Indices for the Carcinogenic PAH Compounds
Based on Skin Painting Data VIII-65
VIII-15 Ranking of PAHs Based on Estimates of Potency In
Skin Painting Bloassay VIII-67
VIII-16 The Carcinogenic Potency of Various PAHs Measured In Two
Different Animal Bloassay Systems . VIII-68
VIII-17 Summary of Relative Potency Estimates for Indicator PAHs. . VIII-72
x1v
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LIST OF TABLES (cent.)
No. Title Page
VIII-18 Summary of PAHs Relative Potencies VIII-74
VIII-19 Relative Potencies for PAH and PAH-Conta1n1ng Mixtures. . . VIII-76
VIII-20 Comparison of Carcinogenic Potency of PAH Using
Relative Tumor Dose VIII-78
xv
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LIST OF FIGURES
No. Title Page
II-l Chemical Structure of Selected PAHs . II-5
III-l Metabolism Schematic for PAHs 111-23
III-2 Metabolism of Benzo[a]pyrene 111-24
III-3 Mechanisms of Enzymatic Activation of Benzo[a]pyrene
to 7,8-D1ol-9,lO-Epox1des 111-27
III-4 The Positions of Bay-Regions. . 111-29
III-5 Cyclic Scheme of Benzo[a]pyrene D1one/D1ol Involvement
1n Redox Coupling 111-32
xv1
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LIST OF ABBREVIATIONS
AHH Aryl hydrocarbon hydroxylase
B[a]P Benzo[a]pyrene
BUN Blood urea nitrogen
DMSO D1methylsulfox1de
DNA Deoxyrlbonuclelc acid
DWEL Drinking water equivalent level
GI Gastrointestinal
GLC Gas liquid chromatography
GSH Glutathlone
HA Health advisory
HDL High density Upoproteln
HPLC High performance liquid chromatography
l.m. Intramuscular
l.p. Intraperltoneal
1.v. Intravenous
LDL Low density Upoproteln
LOAEL Lowest-observed-adverse-effect level
LOEL Lowest-observed-effect level
_MFO .. Mixed function oxldase
NOAEL No-observed-adverse-effect level
NOEL No-observed-effect level
PAH Polycycllc aromatic hydrocarbon
PEG Polyethylene glycol
q * Potency slope for carcinogenic risk estimate
RfD Reference dose
RNA R1bonucle1c add
s.c. Subcutaneous
SGOT Serum glutamlc oxaloacetlc transamlnase
SGPT Serum glutamlc pyruvlc transamlnase
TPA • 12-o-Tetradecanoyl phorbol-13-acetate
UV Ultraviolet
xv11
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I. SUMMARY
This document Includes Information on 15 polycycllc aromatic hydro-
carbons (PAHs) Included among those compounds Identified as priority (water)
pollutants by the U.S. EPA.
PAHs are a class of diverse compounds that are formed during the
Incomplete combustion of organic material. They are ubiquitous and enter
the environment from many sources. Generally PAHs are not very soluble 1n
water. Solubility decreases greatly with Increasing molecular weight. The
•
log of the octanol/water partition coefficient (P) also Increases rapidly
with Increasing molecular weight. This Increases adsorption to partlculate
matter and exposure of the compounds to mlcroblal degradation. However, 1t
has been found that PAHs with more than four aromatic rings are less amen-
able to mlcroblal action. The larger P values also Increase the probability
of bloaccumulatlon. , Volatilization does not appear to be a significant
route of exit from aquatic systems due to the low vapor pressures of PAHs.
/
As they are highly I1p1d soluble, PAHs readily pass through cellular
membranes. However, the rate of absorption Is Increased when the PAHs are
present 1n an oil carrier vehicle. This 1s particularly true for oral, and
dermal routes of exposure.
The highly I1p1d soluble nature of PAH compounds results 1n their
distribution throughout the body In fatty tissues. The primary sites of
storage In the body have been found to be similar In a variety of mammalian
species. These Include the kidneys, liver and fat with some accumulation
occurring 1n the spleen, adrenals and ovaries.
04380 1-1 03/21/89
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PAH compounds are metabolized by the MFO system of enzymes associated
with cytochrome P-450. The liver has the highest activity for this system
although MFO activity 1s found 1n other organs such as kidneys, lungs and
skin. PAH coumpounds have been found to Induce Increased levels of activity
as well as synthesis of Isozymes of cytochrome P-450-assoc1ated enzymes.
The major routes of excretion for PAH compounds are hepatoblHary and
urinary. Some evidence for mammary gland excretion also exists. Although
PAHs present In the body tend to be present In fatty tissues, available
evidence does not Indicate extensive bloaccumulatlon of PAHs 1n these or
other tissues.
The primary focus of research on biologic effects of PAHs has been on
their carclnogenlclty. There 1s generally a lack of research data on the
noncardnogenlc toxic effects of oral exposures to PAH compounds. What
little research has been conducted has centered on three or four compounds
•
.In this large class. Target organs are diverse, probably due to the wide
distribution of PAH compounds throughout the body. Toxlclty centers
primarily on hematopoletic and lymphold systems. Immunosuppresslon measured
1n various experimental systems has been observed following exposure to a
number of PAHs. Nonoral exposure to PAHs has also been observed to effect
changes 1n lymphold and hematopoletic systems.
There Is an extensive data base on the carclnogenlclty of selected
PAHs. Most studies, however, employed dermal. Inhalation or subcutaneous
rather than oral exposure. Overall there 1s a great deal of variation 1n
carcinogenic potential among this class of compounds. PAHs may produce
04380 1-2 09/24/90
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tumors In the vicinity where they are Introduced; that Is, oral exposure
produces stomach tumors and Inhalation produces lung and upper stomach
tumors. Distant site tumors may form dependent on systemic distribution of
metabolites and/or metabolism at target tissues removed from site of Intro-
duction. Host PAHs have been observed to produce genotoxlc effects 1n one
or more test systems. There are data Indicating that some PAHs can serve as
promoters or cocardnogens.
Reproductive and teratogenlc effects have not been well studied 1n these
compounds. There 1s evidence, however, that Ingestlon of benzo[a]pyrene by
pregnant mice results 1n reproductive deficits 1n the F, generation and
that this compound produces ootoxldty.
While very little Information exists on the effects of specific PAHs on
humans, there are numerous reports Unking exposure to environmental and
occupatlonally generated PAH-conta1n1ng mixtures to human health effects.
.These mixtures Include coal tar, soots, coke oven emissions and cigarette
smoke./
The ability of a PAH .to Induce carcinogenic responses depends on Its
distribution to target organs, the presence of potentially reactive areas 1n
Its structure and Us potential for transformation to reactive electro-
phlles. This last factor 1s species and tissue dependent, and also, to some
extent, a function of 1nduc1b1l1ty of the cytochrome P-450-assoc1ated
enzymes.
04380 1-3 09/24/90
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Target tissues for PAH toxlclty other than cardnogenesls are generally
those engaged 1n active DNA synthesis. Mechanisms Involving PAH-mediated
DNA-damage or suppression of DNA synthesis have been proposed for some
target organs. Naphthalene exposure 1s associated with anemias and cataract
formation; this latter effect 1s not seen with other PAHs.
There were no data suitable for calculation of 1- or 10-day health
advisories for any PAH described 1n this document. For all PAHs In this
document classified as B2, probable human carcinogen, data were Insufficient
for calculation of DWELs. Subchronlc studies (90-day gavage exposure In
CD-I mice) were used as the bases for the following DWELs: anthracene, 10.5
mg/i; f luoranthene, 1.4 mg/i; fluorene, 1.4 mg/l; pyrene, 1.0 mg/l.
Although there Is a 90-day study on acenaphthylene, only frank effects were
reported; data were, thus, not appropriate for derivation of criteria.
Several studies have been evaluated as the basis for a DWEL for naphthalene;
consensus, however, has not been reached as to the critical study.
Evaluation of cardnogenldty data prompted the following classification
of these PAHs: Group D, not classifiable as to human cardnogenldty --
acenaphthylene, anthracene, benzo[g,h,1]perylene, fluoranthene, fluorene,
naphthalene, phenanthrene and pyrene; Group B2, probable human carcinogen —
benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene; benzo[a]-
pyrene, chrysene, d1benz[a,h]anthracene and 1ndeno[l,2,3-cd]pyrene.
Dose-response data for benzo[a]pyrene cardnogenldty were used to
derive an upper bound estimate of the slope of the dose-response curve at
low doses. This evaluation, based on the linearized multistage procedure,
04380 1-4 09/24/90
-------
resulted In an upper-bound slope factor of 11.5 mg/kg/day. This use of the
multistage model was Inadequate because the experimental high-dose data were
excluded from the analysis and a single exposure time was assumed. Other
experimental data and modeling techniques were used. Nine risk estimates
were calculated from three different studies In two species of outbred
rodents. Several different models and data -sets were selected. All nine
*
slope factors spanned less than one order of magnitude; four estimates were
selected from these nine. The data, from which these estimates are derived,
are considered to be less-than-optlmal, but acceptable. These selected
studies have several commonalities, Including mode of administration, tumor
sites, tumor types and the presumed mechanisms of action. The data sets
could not be combined prior to modeling (the preferred approach) because
they employed significantly dissimilar protocols. The range of these four
estimates 1s 4.5-9.0. Each estimate Is based on a low-dose extrapolation
procedure and entails the use of multiple assumptions and default
procedures. The geometric mean from four slope factors, each considered to
be of equal merit, .was used to calculate a single oral slope factor of 5.8
per (mg/kg/day). Using standard assumptions for human body weight and water
consumption, a drinking water unit risk of 1.7E-4 per mg/L was derived.
Concentrations of benzo[a]pyrene corresponding to lifetime risks of 10~s,
10~6 and 10~7 were determined to be 6xlO~2, 6xlO~a and 6xlO~«
, respectively.
An analysis of the relative carcinogenic potency of 12 PAH compounds,
using benzo[a]pyrene as the basis of comparison, was made using the
two-stage model applied to a recent compilation of animal cardnogenesls
data In which each experiment tested both benzo[a]pyrene and one or more of
the other compounds. A peer review panel suggested that different criteria
04380 1-5 10/24/91
-------
be used to select data for a final relative potency determination and that
additional studies be analyzed 1n order to extend the 11st of PAH compounds
for which potencies are evaluated. Until this 1s done, no recommendations
can be made concerning the quantitative cancer risk of the other PAH
compounds.
04380 1-6 10/24/91
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II. PHYSICAL AND CHEMICAL PROPERTIES
Polycycllc aromatic hydrocarbons (PAHs) are a class of diverse compounds
that are formed during the Incomplete combustion of organic material. A
large number of PAHs have been Identified In the environment, most of which
derive from anthropogenic sources '(Santodonato et a!., 1980). As a group,
PAHs are ubiquitous In the atmosphere, water and soil. They enter aquatic
systems from wastewater, urban stormwater runoff, leaching from soil, and
wet and dry deposition from the atmosphere (Kveseth et al., 1982; Andren and
Strand, 1981). Some PAHs have been found 1n surface and grqundwater as well
as finished drinking water. This document focuses on those PAHs Identified
as priority (water) pollutants by the U.S. EPA (U.S. EPA, 1980a-d). Table
II-l lists these PAHs; some of their physical and chemical characteristics
are given 1n Table II-2. The structural formulas are shown schematically 1n
Figures II-l and II-2.
The persistence of PAHs In the aquatic environment 1s a direct function
of their physical and chemical properties. These govern the susceptibility
of the compounds to various degradation processes, Including photolysis,
volatilization, and sedimentation and mlcroblal degradation (Callahan et
\
al., 1979). To begin with, many.of the PAHs have low vapor pressures and,
thus, low volatility. Consequently, volatilization may not be Important 1n
the removal of PAHs from water. Second, the solubility of PAHs In water
tends to be low, which Increases their susceptibility to adsorption onto
aquatic partlculate matter. This. 1n turn, suggests that sedimentation and
ultimately, mlcroblal degradation, constitutes the primary removal process.
04390 II-l 05/22/91
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TABLE 11-2
Selected Physical and Chemical Characteristics of Important PAHsa
Physical and Chemical Characteristics
o
o
i— i
i— <
CO
IXJ
co
Compound
Acenaphthylene
Anthracene
Benz[a]anthracene
22nzo[a]pyrene
Benzo[b]f luoranthene
Benzo[k]f luoranthene
Benzo[g,h,1]perylene
Chrysene
01benz[a,h]anthracene
F luoranthene
Fluorene
Indeno[ 1,2, 3-cd Jpyrene
Naphthalene
CAS
Number
208-96-8
120-12-7
56-55-3
50-32-8
205-99-2
207-08-9
191-24-2
218-01-9
53-70-3
206-44-0
86-73-7
193-39-5
91-20-3
Molecular
Height
152.20
178.23
228.28
252.32
252.32
252.32
276.34
228.28
278.36
202.3
166.21
276.34
128.16
Melting
Point
CC)
92
218
167
178
168
215.7
278
255-256
266.6
111
116-117
162.5-164
80.2
Boiling
Point
CO
265-275
342
435
310-31 2b
480
550
448
375
295
218
Vapor Pressure Solubility
(torr) In H20 at 25*C Density
1xlO~» to 1x10"* 3.93 0.899
1.95xlO"« 0.045, 0.073 1.25
5.0xlO"» 0.014. 0.009 1.274
5.0x10"* 0.0038 1.35
1xlO~" Insoluble
9.59x10"" Insoluble
IxlQ-ioC 0.00026
1x10"" 0.002 1.274
1x10"" 0.0005 1.282
1xlO'»c 0.265 f
1xlO"» 1.68-1.98 1.203d
-1x10""
4.9x10"»« 30-40 1.145
Log Octanol/
Hater Partition
Coefficient
4.07
4.45
5.61
6.04
6.57
6.84
7.23
5.61
5.97
5.20
4.18
7.66
3.37
-------
TABLE II-1
Priority Pollutant Polycycllc Aromatic Hydrocarbons
Found In the Environment*
Acenaphthylene Chrysene
Anthracene D1bent[a»h]anthraeene
Beni[a]anthracene Fluoranthene
Benio[a]pyrene Fluorene
Benio[b]fluoranthene Indeno[l,2,3-
Benio[k]fluoranthene Naphthalene
Benio[g,h,1]perylene Phenanthrene
Pyrene
•Source: U,S, IPA, liSOd
-------
TABLE II-2 (cont.)
CO
lO
o
Physical and Chemical Characteristics
Compound
Phenanthrene
Pyrene
CAS
Number
85-01-8 '
129-00-0
Molecular
Weight
178.23
202.26
Melting
Point
(°C)
100
156
Boiling
Point
CC)
340
385
Vapor Pressure
(torr)
6.8x10-"
6.85x10"'
Solubility
In H20 at 25'C
(rag/i)
1.00. 1.29
0.14. 0.132
Density
0.98f
1.271
Log Octanol/
Water Partition
Coefficient
4.46
5.32
^Source: Smith et al.. 1978; Weast, 1983; Cleland and Klngsbury. 1977; Davis et al., 1942; May et al., 1978; IARC. 1973. 1983; U.S. EPA.
1980c; Hansch and Leo. 1985
°0eterm1ned at 10 mm Hg
^Determined at 25*C
^Determined at 0°C
eDeterm1ned at 19.8°c
'Determined at 4°C
co
-------
Benzlalanthracene
Chrysene
Benzolalpyrene Benzolklfluoranthene
Benzolblfluoranthene Dibenz[a,h]anthracene
Indenol 1,2,3-cd]pyrene
FIGURE II-l
Chemical Structure of Selected PAHs
04390
II-5
07/27/90
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Acenaphthylene
Anthracene
Benzo[g,h,i]perylene
Fluoranthene
Fluorene
Naphthalene
Phenanthrene
Pyrene
FIGURE II-l (conl.)
Chemical Structure of Selected PAHs
04390
II-6
07/27/90
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Those PAHs composed of four or more aromatic rings (therefore, having high
molecular weight) are less amenable to mlcroblal degradation; they are
environmentally more stable compounds. Third, the log octanol/water
partition coefficients of the PAHs also tend to be high, and there 1s a
positive correlation between these coefficients and bloconcentratlon
potential. Therefore, 1f PAH concentrations Increase quickly enough,
toxldty could occur In aquatic life. PAHs, however, do tend to be rapidly
metabolized and excreted. Fourth, the previous characteristics seem to be
most representative of the so-called carcinogenic PAHs (see Chapter VIII),
which Implies that such PAHs are more likely to persist. 1n the aquatic
environment, yielding longer-term exposures In comparison with less potent
PAHs. The production, use and occurrence 1n water for each of the 15 PAHs
described In this document follows.
Acenaphthylene
Pertinent data regarding the production, use and occurrence of acenaph-
•
thylene could not be located In the available literature.
Anthracene
Production and Use. In 1981, U.S. Imports of anthracene totalled
21,000 kg, down sharply from 510,000 kg Imported In 1979. Separate data on
U.S. exports were not available (IARC, 1973). Anthracene has been used as
an Intermediate 1n dye production. It.has also been used In smoke screens,
scintillation counter crystals and organic semiconductor research.
Occurrence. Anthracene occurs ubiquitously as a product_of Incomplete
combustion; U also occurs 1n fossil fuels. It has been Identified 1n
04390 I1-7 05/22/91
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surface water, tap water (1.1-59.7 ng/l), wastewater (1.6-7.0
and dried sediment of lakes (30-650 yg/kg) (IARC, 1973).
BenzFalanthracene
Production and Use. There 1s no commercial production or known use of
•this compound.
Occurrence. Benz[a]anthracene occurs ubiquitously 1n products of
Incomplete combustion; H 1s also found 1n fossil fuels. It has been
Identified 1n surface water, tap water (0'.4-10.7 ng/l), rainfall (3.2-12.3
ng/i), subterranean water (0-1.3 ng/i), wastewater (0.5-4.9 yg/a).
sludge (230-1760 yg/kg) and freeze-drled sewage sludge (0.62-19 mg/kg)
(IARC, 1973).
BenzoFalpyrene
Production and Use. There Is no commercial production or known use of
'this compound.
/
Occurrence. Benzo[a]pyrene occurs ubiquitously 1n products of Incom-
plete combustion; It also occurs In fossil fuels. It has been Identified In
surface water (0.2-13,000 ng/i), tap water (0.2-1000 ng/i), rain water
(2.2-7.3 ng/i), subterranean water (0.4-7 ng/l), wastewater (0.001-6000
yg/l), sludge (3-1330 yg/kg) and freeze-drled sewage sludge
(540-13,300 vg/kg) (IARC, 1973, 1983).
Benzofblfluoranthene
Production and Use. There Is no commercial production or known use of
this compound.
04390 II-8 04/16/91
-------
Occurrence. Benzo[b]fluoranthene occurs ubiquitously 1n products of
Incomplete combustion; 1t also occurs 1n fossil fuels. It has been Identi-
fied In surface water (0.6-1.1 ng/l), tap water (0.4-5.4 ng/l), rain
water (4.4-14.6 ng/l), subterranean water (0.6-9.0 ng/l), wastewater
(0.04-23.7 ng/l) and sludge (510-2160 vg/kg) (IARC, 1973).
BenzoFklfluoranthene
Production and Use. There 1s no commercial production or known use of
this compound.
Occurrence. Benzo[k]fluoranthene occurs ubiquitously as a product of
Incomplete combustion; 1t also occurs 1n fossil fuels. It has been Identi-
fied 1n surface water (0.2-0.8 ng/l), tap water (1-3.4 ng/i), rain water
(1.6-10.1 ng/l), subterranean water (1-3.5 ng/l), effluent discharge
(0.01-8 yg/l) and sludge (150-1270 vg/kg) (IARC, 1973).
6enzo[q.h.1lpery1ene
Production and Use. There 1s no commercial production or known use of
this compound.
Occurrence. Benzo[g,h,1]perylene occurs ubiquitously In products of
incomplete combustion; It also occurs In considerable amounts 1n coal tar
and 1s an Important component of gasoline engine exhaust. It has been
Identified 1n surface water (0.3-28.5 ng/l), tap water (0.8-7.1 ng/l),
rain water (2.3-10.8 ng/l), subterranean water (0.7-6.4 ng/l), waste-
water (0.4-2.8 yg/l), sludge (200-1220 pg/kg), freeze-drled sewage
sludge samples (400-8700 yg/kg) and dried sediments from lakes (1-1930
vQ/kg) (IARC, 1973).
04390 II-9 03/22/89
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Chrysene
Production and Use. There Is no commercial production or known use of
this compound.
Occurrence. Chrysene occurs ubiquitously and In approximately the
same concentration as benzo[a]pyrefie 1n products of Incomplete combustion.
In addition, Chrysene and, preferentially, related structures (methyl-sub-
stituted and partially hydrogenated chrysenes) occur 1n higher concentra-
tions than most of the PAHs 1n fossil fuels such as crude oil and lignite.
Chrysene has been Identified 1n surface water (7.9-62.0 ng/l), wastewater
(0.732-6.44 ng/t), freeze-drled sewage sludge (780-23,700 yg/kg) and
sediments (40-240 yg/kg) (IARC, 1983).
D1benz[a.h]anthracene
Production and Use. There 1s no commercial production or known use of
this compound.
Occurrence. D1benz[a,h]anthracene occurs ubiquitously as a product of
Incomplete combustion; H also occurs 1n fossil fuels. It has been
Identified 1n wastewater (IARC, 1983).
Fluoranthene
Production and Use. There 1s no commercial production or known use of
this compound.
Occurrence. Fluoranthene occurs ubiquitously 1n products of
Incomplete combustion; It also occurs 1n fossil fuels. It has been
04390 11-10 12/31/90
-------
Identified In surface water (4.7-6.5 ng/l), tap water {2.6-132.6 ng/l),
rain water (5.6-1460 ng/l), subterranean water (9.9-100.0 ng/l),
wastewater (0.1-45 yg/l), sludge (580-4090 yg/kg), freeze-drled sewage
sludge (610-5160 yg/kg) and dried sediment of lakes (13-5870 yg/kg)
(IARC, 1983).
*
Fluorene
Production and Use. There 1s no commercial production or known use of
this compound.
Occurrence. Fluorene occurs ubiquitously In products of Incomplete
combustion; It also occurs In fossil fuels. It has been Identified 1n sur-
face water (4.1-102.1 ng/l), tap water (4-16 ng/l) and sewage sludge
(0.61-51.60 mg/kg) (IARC, 1973).
Indenon .2.3-cdlpyrene
Production and Use. There 1s no commercial production or known use of
this compound.
Occurrence. Indeno[l,2,3-cd]pyrene occurs ubiquitously 1n products of
Incomplete combustion; It also occurs In fossil fuels. It has been Identi-
fied 1n surface water (0.2-0.5 ng/l), tap water (0.3-4.8 ng/l), rainfall
(0.2-8.7 ng/l), subterranean water (0.2-5.0 ng/l), wastewater (0.01-15
yg/l), sludge (470-1200 yg/kg), freeze-drled sewage sludge samples
(300-7400 yg/kg) and dried sediment from lakes (1-2070 yg/kg) (IARC,
1973).
04390 11-11 03/22/89
-------
Naphthalene
Production and Use. Domestic production capacity of naphthalene was
estimated to be 660 million pounds annually as of January 1, 1984. Total
domestic consumption of naphthalene for 1985 has been estimated to be 540
million pounds; this amount Includes Imports of 8 million pounds.
Exportation volumes are believed to be 1n the range of 5 million pounds
annually. Major applications of naphthalene Include use as an Intermediate
1n the production of phthallc anhydride (55% of consumption), the
Insecticide carbaryl (20X), B-naphthol (8%), synthetic tanning agents (6%),
surfactants (5%), miscellaneous organic Intermediates (2%), and use as a
moth repellant (2%) (Chemical Economics Handbook, 1981).
Occurrence. Naphthalene Is the most abundant single constituent of
coal tar (Schmeltz et al., 1978). It Is released 1n the environment via
Industrial gaseous and partlculate emissions, aqueous waste streams, and
through consumer uses.
Naphthalene has been detected 1n Industrial effluents (up to 32 mg/i),
municipal wastewater treatment plant effluents (22 vg/l), ambient river
water (2.0 yg/J.), seawater, drinking water (1.4 yg/l), well water,
and groundwater (U.S. EPA, 1980c; Shackelford and Keith, 1976; Eganhouse and
Kaplan, 1982; Oeslderl et al., 1984). Stuermer et al. (1982) detected
naphthalene ' 1n groundwater samples collected near underground coal
gasification sites at concentrations of 380-1800 yg/l 15 months after
gasification activity had ended. Pankow et al. (1984) reported mean
dissolved naphthalene concentrations of 11 and 72 ng/l 1n rainwater
samples collected 1n semlrural and residential locations, respectively, In
Oregon.
04390 11-12 04/16/91
-------
Phenanthrene
Production and Use. There Is no commercial production or known use of
this compound. Its derivative, cyclopentenaphenanthrene, has been used as a
starting material for synthesizing bile adds, cholesterol and other
steroids.
Occurrence. Phenanthrene 1s present 1n products of Incomplete combus-
tion; H also occurs 1n fossil fuels. It has been Identified In surface
water (0-1300 ng/i), tap water (3.1-90 yg/l). wastewater (70 yg/a)
and dried sediment 1n lakes (140-274 yg/kg) (IARC, 1973).
Pyrene
Production and Use. There Is no commercial production or known use of
this compound. Pyrene from coal-tar has been used as the starting material
for the synthesis of benzo(a)pyrene.
Occurrence. Pyrene occurs ubiquitously 1n products of Incomplete
combustion; .H also occurs 1n fossil fuels. It Is found 1n relatively high
quantities 1n coal-tar. Pyrene has been Identified In surface water
(2.0-3.7 ng/i), tap water (1.1 ng/i), rain water (5.8-27.8 ng/i), sub-
terranean water (1.6-2.5 ng/l), wastewater (0.00023-11.8 yg/l), sludge
(900-47,200 yg/kg) and dried sediment from lakes (7-3940 yg/kg) (IARC,
1973).
Summary
PAHs are a class of diverse compounds resulting from Incomplete
combustion. They are ubiquitous pollutants and are found 1n ambient air,
soil and aquatic systems.
04390 11-13 12/31/90
-------
PAHs are characterized by low volatility, very slight water solubility
and a propensity for adsorption to participates 1n aquatic systems. PAHs of
four or "more rings (characteristic of the carcinogenic PAHs) are less
amenable to mlcroblal degradation than are smaller compounds, making them
environmentally stable and, therefore, Increasing the potential for
longer-term exposure. The high log octanol/water partition coefficients of
PAHs Indicate a propensity for bloaccumulatlon. However, animal data
Indicate that PAHs tend to be rapidly metabolized and excreted.
With the exceptions of anthracene and naphthalene there are no reported
commercial productions of any PAH described 1n this document.
04390 .11-14 12/31/90
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III. TOXICOKINETICS
Essentially no human pharmacoklnetic data are available from which to
develop a coherent picture of PAH absorption, distribution and elimination.
The experimental data base consists almost entirely of studies In rodents
(mice, rats and hamsters) with occasional studies In larger mammals. While
there are certainly quantitative differences among these species (1n the
relative Importance of bile and urine as routes of excretion, for example)
the qualitative picture that emerges appears to be much the same across
species. PAH kinetics^, are characterized by rapid, capacity-limited
metabolism to both biologically active and Inactive metabolites. Both
Increased levels of enzymes responsible for the metabolism of PAHs and
Isozymes of these metabolic enzymes are Induclble by chemical Inducers of
the 3-methylcholanthrene class. Excretion of chemically stable metabolites
Into urine and bile 1s rapid. Differences 1n disposition among different
PAHs are associated at least 1n part with differences 1n their
l1pophH1cH1es.
•
Absorption
Oral. Fractional absorption of PAHs from the GI tract 1s dependent on
the specific hydrocarbon Ingested. In general, the more llpophlllc members
of this class must be solublllzed 1n water before they can be absorbed.
PAHs with some Intrinsic hydrophlllc character may be absorbed to some
degree even In the absence of emulslflers such as bile salts. Absorption of
all PAHs, however, 1s expected to be Influenced by the properties of other
chemicals coadmlnUtered or coabsorbed. These Include, of course, the
constituents of the diet (Chang, 1943; Modlca et al., 1983).
04400 III-l 04/24/91
-------
Chang's early (1943) work Illustrates the dependence of fractional
absorption on the compound Ingested. While absorption of naphthalene by the
rat was complete, and that of acenaphthene and phenanthrene nearly so,
anthracene, chrysene, d1benz[a,h]anthracene, benzo[a]pyrene and 3-methyl-
cholanthrene were only 5-50% absorbed either from the diet or from a suspen-
sion In starch solution. Absorption of benzo[a]pyrene was -60% from a stock
diet containing 1% benzo[a]pyrene, and ~40% when the benzo[a]pyrene was
given as the starch suspension. Hodlca et al. (1983) similarly observed
that chrysene was much less efficiently absorbed than either benzanthracene
or trlphenylene by young Tasted rats given the compounds orally as emulsions.
The dependence of PAH absorption on the vehicle 1n which It Is adminis-
tered has been well documented. A group of coordinated studies conducted 1n
the early 1950s (Setala and Ekwall. 1950; Ermala et al., 1951; Ekwall et
al.. 1951; Setala, 1954) examined the effects of single solvents. Benzo[a]-
pyrene was administered to mice and cats by stomach tube, 1n solution or 1n
-a suspension of natural fats (olive oil, arachldls oil); natural fats
emulsified In bile; bile alone; long-chain fatty acids and alcohols (oleyl
alcohol, olelc add); polyethylene glycols; and synthetic emulslflers such
as the Triton ethers, among other vehicles. Animals were fasted for at
least 12 hours prior to benzo[a]pyrene administration and throughout the
experiment, so that the effect of the solvents could be studied 1n the
absence of food. Benzo[a]pyrene was absorbed well 1n the forestomach Irre-
spective of the vehicle. Benzo[a]pyrene In natural fats or other UpophlUc
solvents did not penetrate the stomach wall,-which 1s not surprising since
04400 II1-2 09/26/90
-------
fat Itself Is not absorbed 1n the stomach under normal conditions. Hydro-
phobic solvents, as well as the hydrophoblc PAHs, are unable to penetrate
the protective mucous layer lining the stomach.
Olelc add and oleyl alcohol, predominantly Upophlllc but with hydro-
phlUc groups, effected a slight penetration of benzo[a]pyrene Into the
glandular stomach wall. Greater penetration was associated with administra-
tion of benzo[a]pyrene In vehicles with both hydrophlUc and UpophlUc
properties; penetration was greatest when the hydrophlllc character of the
solvent was dominant (Efcwall et al., 1951). Thus, a solvent capable of
solub1!1z1ng benzo[a]pyrene 1n aqueous solution 1s required for Us
absorption 1n the stomach.
The effective solublUzers studied by this group of Investigators are
not present naturally 1n foods to any significant extent, and H 1s unlikely
that much PAH absorption occurs In the stomach at all under normal condl-
•
-tlons. However, once the PAHs have entered the small Intestine, they are
solublUzed by the bile salts In concert with fatty acid anlons and mono-
glycerldes (Laher and Barrowman, 1983), and are absorbed In both the small
and large Intestines (Ermala et al., 1951).
Working with a group of PAHs of Increasing ring number and correlated
decreasing aqueous solubility, Rahman et al. (1986) showed that the presence
of bile salts 1n the rat Intestine 1s essential for optimum absorption of
anthracene, 7,l2-d1methylbenzanthracene, and benzo[a]pyrene but not for
absorption of 2,6-d1methylnaphthalene or phenanthrene, whose aqueous
solubilities are 2-3 orders of magnitude greater than those of the other
three hydrocarbons. When the bile was diverted from the Intestine by
04400 II1-3 09/26/90
-------
bile duct cannulatlon, benzo[a]pyrene was absorbed only 22% as well as U
was 1n the presence of normal amounts of bile. In addition, fractional
absorption of the 4- and 5-r1nged compounds 7,12-dlmethylbenzanthracene and
benzo[a]pyrene was not as great as that of the smaller hydrocarbons,
particularly In the absence of bile. Thus, coadmlnlstratlon of benzo[a]-
pyrene with food 1s expected to enhance Us absorption as a result of
activation of the natural mechanisms for absorption of dietary llplds.
There 1s no convincing evidence that the absorption of PAHs occurs by
mechanisms other than passive uptake. Rees et al. (1971) proposed the
possibility that uptake might Increase exponentially as the amount of
benzo[a]pyrene In the GI tract Increases. This suggestion was based on
studies of the entry of benzo[a]pyrene Into everted sacs of rat small
Intestine. Transfer of benzo[a]pyrene from sac tissue to the enclosed
medium ^as proportional to the concentration 1n the sac tissue. Uptake by
the sac tissue was not affected by metabolic Inhibitors, Indicating that
•
'uptake was not an energy-requiring process. However, the amount bound to
the sac tissue Increased exponentially with Increasing Incubation medium
concentration >200 yH, suggesting multilayer adsorption to the tissue
surface at high concentrations. That such an adsorption/absorption process
might be relevant to absorption In vivo was suggested by the observation
that the amount of benzo[a]pyrene found 1n retroperltoneal fat and mammary
tissue of young female rats given benzo[a]pyrene Intragastrlcally In sesame
oil 18 hours earlier appeared to be exponentially related to the dose. The
In vitro conditions employed by Rees et al. (1971), however, were grossly
unphyslologlc, and "multilayer adsorption" cannot be a significant component
of benzo[a]pyrene absorption jri vivo. Furthermore, the U» vivo observation
of exponentially Increasing tissue concentrations Is not
04400 II1-4 09/26/90
-------
substantiated by the results of other studies. Bock and Dao (1961) found
that the concentration of 3-methylcholanthrene In mammary tissue and mammary
fat of female rats was proportional to the Intubation dose, while Modlca et
al. (1983) calculated that the area under the tissue concentration curve for
chrysene was less than proportional to the dose 1n blood, liver, brain and
parametMal adipose tissue, and roughly proportional to the dose In mammary
tissue. Thus, there Is no evidence for greater than proportional absorption
with dose.
Uptake of PAHs 1s partly Into thoracic duct lymph but principally Into
•
portal venous blood (Laher et al., 1984). According to Rees et al. (1971),
10-20X of a 10 mg IntragastMc dose of benzo[a]pyrene entered the thoracic
duct lymph of rats. Daniel et al. (1967) found 5% of the radlolabel from an
1ntragastr1c dose of 8.4 yg of 14C-labeled d1benz[a,h]anthracene In
thoracic lymph during the first 24 hours after administration to rats. Reid
(1977) recovered 18% of an absorbed dose of benzo[a]pyrene 1n olive oil from
thoracic lymph of rats given benzo[a]pyrene Intraduodenally.
Gastrointestinal absorption of PAHs 1s rapid, as would be anticipated
for compounds utilizing absorption mechanisms designed for uptake of
nutrients. The concentration of benzo[a]pyrene peaked In the liver ~1 hour
after dosing In female rats Intubated with 22.8 mg of benzo[a]pyrene, while
the blood concentration peaked at 1.5-2 hours, the lag reflecting passage
from liver to blood (Modlca et al., 1983). Excretion of metabolites 1n bile
also follows rapidly, the rate generally peaking by 2 hours Irrespective of
the PAH administered (Rahman et al., 1986). It Is Interesting to note that
1n general the fraction of total metabolites excreted In bile rather than 1n
urine Increases as the PAHs Increase In size and decrease 1n solubility.
04400 II1-5 09/26/90
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Radlolabel from i«C-d1benz[a,h]anthracene given by Intubation continued to
appear In thoracic lymph of two rats -for at least 24 hours, peaking at
around 3-4 hours (Daniel et al., 1967). That some of this radlolabel was
probably associated with metabolite reabsorptlon Is suggested by the
observations that radlolabel did not peak 1n the blood plasma of these two
rats until ~7 hours after dlbenzanthracene administration, and that much of
the radlolabeled material extracted from plasma, urine, and bile was
metabolized d1benz[a,h]anthracene. Rees et al. (1971) reported that
benzo[a]pyrene peaked In the thoracic lymph of rats 3-4 hours after
administration of 10 mg by Intubation, but >the fluorescence-based analytical
technique used by these Investigators would not have distinguished between
benzo[a]pyrene and Us metabolites.
Inhalation
The absorption of PAHs from the lung has been Investigated 1n both
Inhalation studies and 1n experiments 1n which the PAHs were given by Intra-
-tracheal administration. Both pure PAHs and PAHs adsorbed on particles of
various sizes and chemical composition have been used. Adsorption of a PAH
onto ultraflne particles (<1 v,m) from which 1t can be released Into
biologic fluids has been shown to Increase both the lung retention time of
the PAH and Us carclnogenldty. The nature of the adsorbent particle Is
critical: adsorbents that do not readily release adsorbed chemicals are not
as likely to enhance tumor yield and other biologic effects 1n experi-
mental animals as are adsorbents with lower affinity for the carcinogen
(Bevan and Worrell, 1985). In addition to adsorption onto partlculates,
factors such as particle or aerosol size and size distribution, the test
species' airway anatomy, metabolism and defense mechanisms, as well as
04400 III-6 09/26/90
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properties of the PAH Itself, will Influence the penetration, deposition and
retention of an Inhaled PAH and the probability and routes of Us subsequent
absorption. Other chemicals, Including those adsorbed to partlculates, may
also Influence the disposition and biologic activity of PAHs taken Into the
lung. , H
•
Kotln et al. (1959) reported a vehicle effect on Intratracheally
Instilled benzo[a]pyrene. Rad1olabeled-14C-benzo[a]pyrene (25 yg/0.3
ml vehicle) was administered to VMstar male rats either as an aqueous
suspension with distilled water or 1n a solution with tMethylene glycol.
The esophagus was llgated In each to prevent benzo[a]pyrene from entering
the GI tract by mucodllary escalator clearance and swallowing. At 24
hours, 38X of the administered aqueous suspension dose persisted In the
lung, whereas only 17% could be recovered at 4 hours 1n the lungs when
administered In tMethylene glycol. The rapid mobilization was attributed
to the greater speed of solublUzatlon of the fine particles from
trlethylene glycol vs. the slower speed of solublUzatlon of the larger
crystals that form 1n an aqueous suspension.
The role of particles as carriers of PAHs and enhancers of their pulmo-
nary activity has been the subject of a number of Investigations. Coadmln-
Istratlon of PAHs with fine adsorbent particles was shown nearly 30 years
ago to result 1n a higher Incidence of lung tumors In rats than
administration of the PAH alone (Pylev et al., 1969; Safflottl et al., 1965.
1968). Coadmlnlstratlon by Intratracheal Instillation of a suspension of
14C-benzo[a]pyrene with carbon black or asbestos (Pylev et al., 1969) or
by Intratracheal Instillation of particles of ferric oxide, aluminum oxide,
04400 II1-7 09/26/90
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or carbon coated with benzo[a]pyrene (Henry and Kaufman, 1973) resulted 1n
prolongation of radlolabel retention 1n the hamster lung. However, an
Increase In benzo[a]pyrene retention was not necessarily correlated with an
Increase In tumor 1gen1dty. Farrell and Davis (1974) showed that carbon
black was a more effective tumor promoter than ferric oxide 1n the golden
hamster lung; aluminum oxide was only minimally active as a promoter In this
system. In general, the probability of tumor occurrence was shown to
Increase as particle size range decreased when either carbon black or ferric
oxide was used as the adsorbent (Farrell and Davis, 1974; Henry et a!.,
1974). In the absence "of carrier particles, large (77% <42 ym, 0% <10
ym) benzo[a]pyrene particles were cleared less rapidly from the lung and
were more tumorlgenie than small (77X <5.2 ym, 3% <1.3 urn) particles
(Feron et al., 1980). Henry et al. (1975) showed that benzo[a]pyrene had to
be physically adsorbed on the ferric oxide to be most effective as a
promoter.
Henry and Kaufman (1973) suggested that the ability of the hydrocarbon
to be eluted from Us partlculate adsorption sites might be an Important
determinant of Us biologic activity. Creasla et al. (1976) demonstrated
that the rate of elutlon of benzo[a]pyrene from Us adsorption sites on
carbon particles In the mouse lung was greater for small (0.5-1.0 ym)
particles than for large (15-30 ym) particles. Benzo[a]pyrene adsorbed to
the larger particles was cleared with the particles themselves, with a
half-time for elimination of 4-5 days. However, while the smaller particles
themselves were cleared more slowly Ui/o^ days), the benzo[a]pyrene
adsorbed to these particles was cleared more quickly (t,/2=36 hours).
04400 II1-8 09/26/90
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This Indicates that the benzo[a]pyrene was being eluted from the smaller
carbon particles at a rate of 15% per day. Thus, for at least 4 days, 15%
of the Initial dose of benzo[a]pyrene was free to Interact with the respira-
tory tissue. In the absence of carbon particles, 50% of a benzo[a]pyrene
dose was cleared from the lung within 1.5 hours and >95% was cleared within
24 hours. These results established that a combination of prolonged
retention time and biologic availability of the adsorbate are Involved In
the cocardnogenlc effect observed for partlculate and benzo[a]pyrene 1n the
lung.
Based on their studies with model phosphol1p1d bllayer membranes
(Lakowlcz and Hylden, 1978; Lakowlcz et al., 1980) and with mlcrosomal
preparations (Lakowlcz and Bevan, 1979), the authors suggested that cocar-
dnogenlc particles facilitate the uptake of adsorbed chemical carcinogens
by cell membranes. Later work by the same group of Investigators (Bevan et
al., 1981; Bevan and Worrell, 1985) supports this mechanistic hypothesis.
Chang and Hart (1983), who demonstrated that the chrysotlle form of asbestos
enhanced uptake of benzo[a]pyrene Into human dermal Hbroblasts 1_n vitro,
proposed that cocardnogenlclty Is mediated 1n part by enhanced cellular
proliferation. It should be noted that binding to cellular DNA was also
enhanced by the presence of asbestos 1n this study, so that the proposed
mechanisms of enhanced uptake and enhanced cellular proliferation are fully
compatible and not mutually exclusive. Other in vitro studies (Eastman et
al., 1983), using hamster trachea1 epithelial cells Incubated with benzo[a]-
pyrene -and asbestos, showed enhanced benzo[a]pyrene uptake and ONA alkyla-
tlon 4 days post-treatment, which was attributed to the presence of asbestos.
04400 II1-9 09/26/90
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Absorption and distribution of benzo[a]pyrene following Inhalation
exposure of rats to the compound alone or after adsorption onto particles
has been studied by Investigators at the Inhalation Toxicology Research
Institute (Mitchell and Tu, 1979; Mitchell, 1982, 1983; Sun et al., 1983,
1984). Mitchell (1982, 1983) reported the disposition of an aerosol of 500
yg 3H-benzo[a]pyrene/i, 1-2 ym mass median diameter, Inhaled by rats
for <1 hour. Clearance of radlolabel from the respiratory tract was
blphaslc. Fifty percent of the radlolabel was cleared within 2-3 hours;
radlolabel remaining after this time was cleared much more slowly, the
half-life being 25-50 hours, depending on location 1n the lung. The stomach
and small Intestine contained higher concentrations of benzo[a]pyrene 0.5
hour after termination of exposure than any other tissue, and feces con-
tained ~10 times the amount of radlolabel found In urine during the first
day following exposure. The observation that the amount of radlolabel In
the stomach and small Intestine was at Us maximum Immediately following
termination of exposure suggests mucodllary clearance and swallowing of
.Inhaled material. The presence of radioactivity 1n other soft tissues
(e.g.,-kidney and liver) 0.5 hour after exposure Indicates rapid absorption
and distribution of the benzo[a]pyrene from Its Initial site of deposition.
The concentrations of organic-soluble radlolabel, water-soluble radio-
label and covalently-bound radlolabel were determined 1n the lung at 0.5, 6
and 24 hours postexposure (Mitchell, 1982, 1983). The 2-3-hours clearance
half-life appeared to represent loss of organic-soluble radlolabel; after 24
hours,.BOX of total lung radlolabel was covalently bound to macromolecules.
Inviting the speculation that H Is this bound radlolabel that persists 1n
the lung with a half-life of 25-50 hours.
04400 111-10 02/07/91
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Similar Inhalation studies demonstrated that pyrene was also cleared
from the rat lung rapidly, although not 'as rapidly as benzo[a]pyrene
(Mitchell and Tu, 1979). The pyrene aerosol had a mass median diameter of
0.45 ym. The amount of pyrene left In the lung 24 hours after exposure
was 69X of that remaining 0.5 hour after exposure; at 2 days, H was 5%.
Covalent binding was not measured.
Sun et al. (1983) examined the deposition, retention and excretion
patterns of the radlolabel associated with 3H-benzo[a]pyrene coated on
ultraflne (0.1 tint median diameter) particles of gallium oxide, Ga?0_.
The purpose of this study was to determine whether association with ultra-
fine particles affects the disposition of organic air pollutants. Rats were
exposed by Inhalation to the aerosols (3500 ng/i) for 30 minutes, and
tissues and excreta were collected for 16 days after the exposures. Studies
with pure aerosols (600 ng/i) of 3H-benzo[a]pyrene of the same median
diameter were also '.conducted for comparison, with the exposure period
extended to 50 minutes 1n order to Insure that total deposition was similar
to that observed In the particle Inhalation experiments. About 28% of each
aerosol had been deposited 1n the respiratory tract at the end of the
exposure period.
Initial clearance from the lung was rapid, with half-lives of <1 hour
for both aerosols. During the subsequent slow excretion phase, lung levels
of radlolabel resulting from exposure to benzo[a]pyrene-coated particles
were 4-10 times higher than those resulting from exposure to pure benzo[a]-
pyrene aerosols, although the half-lives, had they been calculated, would
probably not have been greatly different. Lung clearance of the pure
04400 III-ll 07/29/90
-------
benzo[a]pyrene aerosol was also very similar to that reported by Mitchell
(1982) for aerosol concentrations 800 times higher. Of particular Interest
1s that levels of radlolabel In the stomach Increased with time after
exposure to coated particles. Thirty-five percent of the benzo[a]pyrene
Initially deposited In the lung was recovered from the stomach 2 hours after
exposure to benzo[a]pyrene-coated particles, while no more than ~0.5X of the
amount of pure aerosolized benzo[a]pyrene (Initially) deposited 1n the lung
was found 1n the stomach at any time following exposure. Thus, exposure to
a benzo[a]pyrene-coated particle rather than to a pure benzo[a]pyrene
aerosol resulted In a very marked shift in the pattern of lung clearance,
from direct absorption Into blood to clearance by mucodllary action
followed by 1ngest1on. Consequently, of the total radlolabel excreted 1n
urine and feces through day 16, -15X was recovered from the urine of animals
exposed to the pure aerosols and only ~8X from the urine of animals exposed
to the coated aerosols.
It should be noted that pure aerosols of 3H-n1tropyrene, as well as
gallium oxide aerosols coated with 3H-n1tropyrene, were studied 1n a
parallel set of experiments, with results that were different from the
benzo[a]pyrene results. Thus, It 1s not advisable to generalize retention
and absorption data for a particular PAH to predict the quantitative
behavior of other PAHs or nHropyrenes under similar conditions.
A possible effect of dlesel exhaust particles on retention of PAHs by
the lung Is currently of some concern (Tyrer et al., 1981). A study
reported by Sun et al. (1984) showed that the retention and excretion
patterns of 3H-benzo[a]pyrene-coated dlesel engine exhaust particles with
04400 II1-12 04/24/91
-------
a mass median diameter of -0.14 pm (4-6 pg/l) were qualitatively
similar to those recorded for the gallium oxide study. However, the amounts
of radlolabel retained 1n the lung during the second, slow phase of
clearance were much higher 1n the dlesel exhaust study than 1n the gallium
oxide study. Host of this radlolabel was present as unchanged compound.
Dermal. PAHs can be absorbed percutaneously. The rate and extent of
absorption are strongly dependent on the size and configuration of the
hydrocarbon molecule, and are also dependent on the concentration of the
hydrocarbon applied to the skin.
Heldelberger and Weiss (1951) showed that 14C-benzo[a]pyrene dissolved
1n benzene was absorbed from a single application site on the shaved back
skin of mice and excreted principally In the feces. Fifty percent of the
radlolabel from a 63.7 yg/cm2 dose of benzo[a]pyrene was lost from the
site of application by 1 hour; only 6X remained after 7 days. D1benz[a,h]-
anthracene was only very slowly lost from the application site and was not
t - •
detected 1n the feces. By contrast. Bock and Burnham (1961) showed that
7,12-dlmethylbenzanthracene and benzo[a]pyrene 1n a 95": 1 mixture of benzene
and mineral oil reached similar concentrations 1n shaved back skin of mice 2
hours after cutaneous application of -400
Sanders et al. (1984) confirmed that benzo[a]pyrene and 7,12-dlmethyl-
benzanthracene are rapidly absorbed across the skin of mice. Six percent of
a dose of 125 yg benzo[a]pyrene/cm2 In acetone had disappeared from the
application site by 1 hour and 40% by 24 hours, with 7% remaining at the
site after 7 days. Eighty percent of a 5.4 yg 7,12-dlmethylbenzanthra-
04400 111-13 11/12/91
-------
cene/cm2 dose had disappeared by 24 hours, and 96% by 1 week. Absorption
was dose-dependent; Increasing the 7,12-dlmethylbenzanthracene dose from 5.4
to 56 and to 515 vg/cm2 reduced absorption during the first 24 hours
from 82% to 71X and to 33X, respectively; decreasing the benzo[a]pyrene dose
from 125 to 12.5 and to 1.25 vg/cm3 Increased absorption during the
first 24 hours from 41% to 83X and to 82X, respectively. Use of muzzles to
prevent grooming of the treated area reduced the fraction of the dose found
1n the stomach and Us contents to one-third of the amount found when
muzzles were not used; this fraction, 0.5-1.5X of the administered dose, was
so small that 1t had no significant Impact on measurement of either
absorption or excretion.
Molecular size (number of rings) and structure affected the
concentrations of 12 different hydrocarbons. Including phenanthrene,
anthracene, benz[a]anthracene and 3-methylcholanthrene, studied In mouse
skin under the conditions used by Bock and Burnham (1961). While this
•
measurement reflects both rate of uptake by the skin and rate of transfer
/
Into the systemic circulation, 1t does Indicate that skin penetration occurs
and that the rate of the overall dermal absorption process 1s dependent on
molecular size and configuration. It was also determined that the nature of
the solvent makes a difference 1n the absorption of PAHs. Small amounts
(<5X) of mineral oil added to benzene resulted 1n greater dermal
absorption. However, the absorption was not as great when a mixture of 50X
mineral oil was used as the solvent.
Fifty percent of a single application of 9.3 yg anthracene/cm2
(dissolved 1n 71 yl of a 1:7 mixture of hexaneracetone) to the shaved
04400 111-14 11/12/91
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back skin of female Sprague-Dawley rats was absorbed 1n 5 days, as measured
by the appearance 1n urine, feces and other tissues (Yang et al., 1986).
Distribution
Elimination of benzo[a]pyrene and of 7,12-dlmethylbenzanthracene from
blood Is blphask, the first phase being exiremely rapid. About 90% of
either chemical was lost In the first 10-30 minutes following administration
of an Intravenous dose to mice or rats (Heldelberger and Weiss, 1951; Kotln
et al., 1959; Lo, 1964; Iqbal et al., 1979). The remainder was lost more
slowly.
Clearance of metabolites accounts for the second, slower phase of
decline of radlolabel 1n the blood. Excretion of metabolites 1n bile and
urine 1s preceded by a period during which the liver and kidney contain
relatively large fractions of the dose. This period 1s brief. By 90
minutes following Intravenous Injection of 14C-benzo[a]pyrene 1n mice, the
liver contained only 1.36% of the radlolabel, and 72% had already entered
the bfle (Heldelberger and Weiss. 1951). D1benz[a,h]anthracene was less
rapidly metabolized; 90 minutes after an Intravenous dose was given to mice,
89% of the radlolabel was still In the liver (Heldelberger and Weiss, 1951).
Selective accumulation does not occur In target tissues of PAH carcino-
genic action. Depending on the I1poph1l1c1ty of the PAH, significant
accumulation 1n body fat 1s likely to occur; early authors attributed the
Induction of mammary tumors by PAHs to the fact that the mammary gland 1s In
Intimate association with fat (Bock and Dao, 1961; Daniel et al., 1967).
Bock and Dao (1961) found Uttle phenanthrene 1n the perlrenal and mammary
04400 111-15 05/13/91
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fat of rats given the hydrocarbon by stomach tube 24 hours earlier. Concen-
trations of 7,12-dlmethylbenzanthracene and 3-methylcholanthrene were some-
what higher, and benzo[a]pyrene was present In the grestest concentration.
These concentrations correlate with the relative I1poph1l1c1ty of phenan-
threne, 7,12-dlmethylbenzanthracene, and benzo[a]pyrene (Rahman et al.,
1986).
Oral. When radlolabeled PAHs are administered Into the GI tract by
gastric Intubation or by feeding, first-pass metabolism destroys the sharp
Initial drop In radlolabel characteristic of Intravenous administration.
Instead, radlolabel peaks after several hours; this radlolabel Is associated
with metabolites. The timing of the peak depends on the particular PAH, and
1s expected to depend on the dose as well. It has been seen to occur at ~7
hours for d1benz[a,h]anthracene and 15 hours for 7,12-dlmethylbenzanthracene
In rats (Daniel et al., 1967).
When a specific analytical technique (cyclohexane extraction of adipose
tissue'followed by gas liquid chromatography) 1s used for the determination
of unchanged PAH, the peak time occurs earlier: at -1.5 hour for benz[a]-
anthracene 1n rats (Hodlca et al., 1983), and at ~1 hour for benz[a]anthra-
cene and chrysene and 2.5-3 hours for trlphenylene 1n rats (Bartosek et al.,
1984). The concentrations of these PAHs In the liver peak shortly before
their peaks 1n the blood (Hodlca et al., 1983; Bartosek et al., 1984). For
this reason, the rate of appearance In the bile also tends to peak early
(1-2 hours) for most PAHs (Kotln et al., 1959; Rahman et al., 1986). As far
as can be determined from published data, distribution from the blood Into
peripheral tissues follows established principles: well-perfused tissues
04400 111-16 05/13/91
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establish a dynamic steady state with the blood early, while adipose tissue
exchanges more slowly. Peak adipose tissue concentrations occurred at 2.5
hours for chrysene, ~4 hours for benz[a]anthracene and 8 hours for
trlphenylene (Bartosek et al., 1984), and at 3 hours for benz[a]anthracene
1n another study (Modlca et al., 1983).
No kinetic evidence for this peripheral distribution 1s found In the
concentrations of PAHs In the blood after a single dose, which can be fit by
a blexponentlal equation with one term representing uptake and the other
loss (Modlca et al., f983; Bartosek et al., 1984). Half-lives In rat
tissues appear to be about the same for benz[a]anthracene and chrysene: 1
hour 1n blood, 0.8 hour 1n liver, 2.5 hours 1n brain, 5 hours In mammary
tissue, and 14 hours In adipose tissue (Modlca et al., 1983; Bartosek et
al., 1984).
Inhalation. As has been discussed earlier (see Absorption Section),
the pattern of distribution following a pulmonary exposure could resemble
the pattern associated with Intravenous administration or the pattern
associated with oral exposure, or could be Intermediate between the two.
Kotln et al. (1959) Instilled 14C-benzo[a]pyrene Intratracheally 1n tr1-
ethylene glycol Into rats whose esophagi had been Ugated to prevent entry
of benzo[a]pyrene Into the GI tract. Except for the high Initial pulmonary
concentration, the pattern of tissue radlolabel was similar to that seen
after subcutaneous or Intravenous administration. One hour after Instilla-
tion. 37% of the radlolabel was 1n the Intestine, 1.3% In the liver, and
43.2% remained 1n the lung.
04400 111-17 05/13/91
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The study of Kotln et al. (1959) demonstrates that pulmonary absorption
1s rapid. Mitchell (1982) found radlolabel In soft tissues by 0.5 hour
after a 1-hour Inhalation exposure of rats to 3H-benzo[a]pyrene. Radio-
label 1n testls, brain and kidney peaked at ~6 hours and was generally
cleared from these tissues 1n ~1 day. At the 0.5-hour time point, the
radlolabel In liver and kidney wa? largely polar metabolites of benzo[a]-
pyrene (Mitchell, 1983). In every salient point, this distribution pattern
and timing resemble the pattern and timing of distribution of an Intravenous
dose. It can be concluded that an Inhaled aerosol of pure benzo[a]pyrene Is
distributed essentially like a comparable Intravenous dose save for the
Initial high pulmonary concentration. Inhaled pyrene aerosol also behaved
much like an Intravenous dose In rats, although a substantial amount of
pyrene was found In the stomach 0.5 hour after exposure (Mitchell and Tu,
1979).
Clearance from the lung Into the GI tract Is not as rapid a process as
absorption Into blood. Only when PAHs are Inhaled adsorbed to partlculates
/
does their residence time In the lung allow significant transfer to the 61
tract. Sun et al. (1983) showed that during the first 2 hours after an
Inhalation exposure to 3H-benzo[a]pyrene adsorbed onto gallium oxide
particles, radioactivity 1n the stomach Increased. In addition, radlolabel
In the livers and kidneys of these rats was 5-6 times higher at 0.5 and 24
hours than radlolabel 1n the liver and kidneys of the rats Inhaling pure
benzo[a]pyrene aerosol. Although other tissue measurements were not made,
these observations strongly suggest that the distribution pattern of an
Inhaled PAH adsorbed onto partlculate material will have some of the
characteristics of distribution associated with oral exposure. Inhalation
04400 Ill-IB 05/13/91
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studies 1n rats exposed to benzo[a]pyrene adsorbed onto dlesel engine
exhaust particles (Sun et al., 1984) led to the same conclusion.
Tyrer et al. (1981) examined the effect of dlesel exhaust exposure on
the disposition of 1AC-benzo[a]pyrene administered Intratracheally 1n
gelatin solution (20 »il of 0.2X gelatin and 0.5 v9/vl of benzo[a]-
pyrene) to A/J mice. Mice were exposed to raw dlesel exhaust (6 mg/m3 of
particles) 8 hours/day, 7 days/week for 9 months prior to Intratracheal
Instillation of benzo[a]pyrene. Autoradlography sections were processed
from mice killed at 2.24 and 48 hours post-Instillation. Qualitatively
there were no obvious differences between mice exposed to both dlesel fumes
and benzo[a]pyrene or to benzo[a]pyrene only, possibly because of the large
1nter1nd1v1dual variance In expectorate 1ngest1on. Within 2 hours after
placement 1n the lungs, benzo[a]pyrene was found circulating In the blood-
stream and may have entered the esophagus and stomach by swallowing of
material cleared by the mucodllary escalator. 14C-benzo[a]pyrene was.
cleared from the blood by the kidneys and then excreted. By 24 hours,
3H-rad1oact1v1ty accumulated In the stomach, lower GI tract, kidneys,
bladder and slightly 1n the lung.
Dermal. Mitchell and Tu (1979) found trace amounts of pyrene-
equlvalent fluorescence In kidney, liver and trachea of rats 24 hours after
a single application of pyrene to the unshaved back. No pyrene or metabo-
lites were detectable In the lung. The GI tract contained 1.4X of the dose.
Six days after application of anthracene to the shaved back skin of
rats, Yang et al. (1986) recovered a total of 1.3% of the applied dose from
04400 111-19 05/13/91
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15 (unspecified) selected tissues. At this time, 29.1% of the applied dose
had been recovered In the urine and 21.9% In the feces.
To summarize, a number of studies with distribution data Indicate that
1) detectable levels of PAHs can be observed 1n most Internal organs from
minutes to hours after various routes of administration; 2) adipose and
mammary tissues are significant distribution sites where PAHs may be accumu-
lated, stored and slowly released; and 3) after Inhalation exposure, the GI
tract contains relatively high levels of PAH or metabolites as the result of
swallowing unmetabollzed PAH from mucoclHary clearance, or derived from
hepatoblHary excretion of metabolites.
Metabolism
In the past, the relative lack of chemical reactivity for tumorlgenlc
PAHs has been puzzling In light of their dramatic biologic effects. Early
attempts to explain the carc1nogen1c1ty of various PAHs utilized physico-
chemical calculations (Pullman and Pullman, 1955). These early hypotheses
were based on the assumption that those regions of the molecule favoring
substitution or addition reactions would preferentially react with critical
cellular target sites to Initiate a carcinogenic transformation. This
concept, however, did not prove successful for PAHs.
More recently H has been shown that PAHs are metabolized by enzyme-
mediated oxldatlve mechanisms to form reactive electrophlles (reviewed 1n
Conney, 1982; Gelboln, 1980; IARC. 1983; Pelkonen and Nebert, 1982;
Santodonato et a!., 1981; Zedeck, 1980). For many of the PAHs, certain
"bloactlvated" metabolites are formed having the capability for covalent
04400 111-20 05/13/91
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Interaction with cellular constituents {I.e., RNA, DNA. proteins) and
ultimately leading to tumor formation.
The obligatory Involvement of metabolic activation for the expression of
PAH-lnduced carclnogenesls has prompted the Investigation of PAH metabolism
In numerous animal models and human tissues. From these studies has emerged
an understanding of the general mechanisms Involved In PAH blotransformatlon.
It 1s now known that PAHs are metabolized by the cytochrome P-450-dependent
mlcrosomal mixed-function oxldase (HFO) system, often designated aryl hydro-
carbon hydroxylase. The-activity of this enzyme system 1s readily Indudble
by exposure to PAH and 1s found 1n most mammalian tissues, although
predominantly In the liver. The MFO system 1s Involved 1n the metabolism of
endogenous substrates (e.g., steroids) and the detoxification of many
xenoblotlcs (Nebert et al., 1981). Paradoxically, however, the MFO system
also catalyzes the formation of reactive epoxlde metabolites from certain
PAHs, possibly leading to carclnogenesls 1n experimental mammals.
The route by which PAHs and other xenoblotlcs enter the body may deter-
mine their fate and organ specificity. A compound absorbed from the lungs
may bypass the "first pass" effect 1n the liver and reach the peripheral
tissues In high concentrations. The unique enzymes In these extra-rhepatlc
tissues may differ In terms of activity and specificity, from those In the
liver. Thus, extra-hepatic metabolism may be extremely Important In the
b1oava1labH1ty of a chemical to different parts of the body and 1n target
tissue variability. Enzymes capable of metabolizing PAHs are found In the
liver, lung, kidney, adrenals, testes, thyroid, skin, small Intestine and
sebaceous gland 1n a variety of species Including human, baboon, monkey,
04400 II1-21 . 05/13/91
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rat, mouse, hamster, guinea pig, rabbit and dog. Embryonic tissue of rat,
mouse, hamster and chick also possess activity (Zedeck, 1980).
Benzo[a]pyrene as a Model of PAH Metabolism. A discussion of the
metabolism of PAHs 1n mammalian species, Including humans, may be approached
by examining 1n detail the chemical fate of the most representative and
well-studied compound 1n the alternant PAH class, namely benzo[a]pyrene.
The metabolism of benzo[a]pyrene, subject of voluminous research, 1s shown
schematically 1n Figure III-l. Only the most Important pathways will be
presented 1n this discussion that correlates with Figure I.II-2. This 1s a
summary and the reader Is referred to Gelboln (1980), Pelkonen and Nebert
(1982), Yang et al. (1978), Zedeck (1980) and Thakker et al., (1985, 1988)
for primary sources.
A monooxygenase first Introduces an oxygen atom Into any of several
positions of the molecule to produce oxides or primary "simple" epoxldes.
This Initial oxygenatlon 1s catalyzed by one of a number of different forms
/
of P-450 (MFO). The epoxldes then undergo spontaneous rearrangement to
phenols. Another pathway for the epoxldes 1s the reduction back to parent
benzo[a]pyrene. The formation of qulnones through the 6-phenol and 6-oxo
radical 1s less well characterized and 1s discussed briefly 1rv the Other
Tox1f1cat1on Pathways Section. Qulnones can also be produced nonenzymatic-
ally by I1p1d peroxldatlon and aerobic oxidation. The enzyme epoxlde
hydrolase can further metabolize epoxldes to dlhydrodlols. The glutathlone
transferases catalyze conjugation of epoxldes with glutathlone.
04400 II1-22 05/23/91
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(Endoplasmic
Reticulum)
Cytochrome P-450
Mixed-Function Oxidase (MFO)
Glutathione
BaP — SQ -^
petoxification Transferase
Products) (Cytosol)
BaP OXIDES
BaP PHENOLS
Epoxide
Hydrase
(Endoplasmic
Reticulum)
Sulfates
glucuronides
I
BaP Quinones
BaP DIHYDRODIOLS (Proposed Proximate Carcinogens)
MFO
UDP Glucoronosyl Transferase
(Endoplasmic Reticulum)
BaP OIOL EPOXIDES
(Proposed ultimate
Carcinogens)
hU>Soluble Conjugates
(Detoxification Products)
FIGURE ni-i
Metabolism Schematic for PAHs
Source: U.S. EPA, 1980d
04400
111-23
04/24/91
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o
o
CM
[OlQ)
9-OH
®&j
'-<* > *.
3-OH
'o^\\
3.6-0 \\ ,
IG-PHENOXY]
L RADICAL J^-j. CM .1
6-OH
7-OH
T 9, K) epox 9. K)-diol
M
»« iJCJH
n
foTol
LQlOT
4,5-tfiol
9,»fpn
7.8-epox
?;8-diol
-^ [?(8-4ol-9,IO e(J
r • n
[?,§,9,IO-lelfol|
T~
CONJUGATES
—I
BOUND MACROMOLECULES
DNA
RNA
PROTEIN
ro
FIGURE III-2
Metabolism of Benzo[a]pyrene
•
Source: U.S. EPA, 1980d
-------
Epoxldes, dlhydrodlols, phenols and qulnones are generally regarded as
"primary" metabolites of benzo[a]pyrene, which undergo further metabolism.
Dlhydrodlols and phenols can be substrates for the MFO, and another position
of the molecule can become epoxldated (see following discussion of pathway).
Dlhydrodlols, phenols and qulnones can also be conjugated with glucuronlc
add 1n reactions catalyzed by UDP-glucuronosyltransferase or with sulfate
1n reactions catalzyed by sulfotransferase. Dlhydrodlols can be dehydro-
genated to catechols by a soluble dehydrogenase.
The metabolites of benzo[a]pyrene and other PAHs (both alternant and
nonalternant) are often categorized on the basis of solubility 1n various
laboratory extraction protocols (U.S. EPA, 1980d). Those metabolites that
can be extracted from an aqueous Incubation mixture using an organic solvent
Include the phenols, dlhydrodlols, qulnones and hydroxymethyl derivatives of
PAHs having aliphatic side chains. Epoxldes are Included 1n this group
although these are rather labile. The conjugation products, Including those
of glutathlone, glucuronldes and sulfates, remain In the water phase after
extraction. Identification and quantification of metabolites 1n these
fractions provide researchers with Insight on the predominant pathways 1n
the metabolism of a particular PAH.
The pathways leading to the 7,8-d1ol-9,lO-epox1des of benzo[a]pyrene
have been a focus of research since 1t was suggested that these are the
predominant DNA-blndlng species In cell culture (S1ms and Grover, 1974).
For example the major adduct Identified 1n human, hamster, rat and mouse
endometMal DNA, exposed In vitro to benzo(a)pyrene, was antl-benzo(a)-
pyrene-7,8-d1ol-9,lO-epox1de (Kulkarnl et al., 1986). The amount of this
04400 111-25 05/23/91
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adduct formed In hamster and mouse DNA was similar; human levels were 3-4
times higher and rat levels ~5-7 times lower. The quantity of B[a]P bound
In humans was 7 times the amount bound 1n hamsters, while rats and mice were
4 and 2 times higher than hamsters, respectively. This study also showed
that differences exist 1n the proportions and types of other adducts formed
t>y these species.
Although the 4,5-ox1de 1s the most mutagenlc metabolite 1n bacterial
systems relative to the 7,8-and 9,lO-ox1des, other data from assays such as
DNA binding studies, cell transformation assays and cardnogenlcHy
bloassays support the 7,8-d1ol-9,lO-epox1des as ultimate carcinogenic forms
of benzo[a]pyrene (Pelkonen and Nebert, 1982; IARC, 1983; Conney, 1982;
Kulkarnl et al., 1986; Thakker et al., 1988). The metabolic activation
scheme for the formation of the 7,8-d1ol-9,10-epox1de 1s shown 1n Figure
III-3. The 7,8-d1ol formed from benzo[a]pyrene 1s the trans Isomer and
there are four possible stereolsomeHc 9,10-epoxldes derived from this. The
structure and nomenclature of these d1ol-epox1des are also shown 1n Figure
III-3. The two Isomers (antl and syn). also referred to as d1ol-epox1des I
and II, are racemlc and have been synthesized In order to study their
stereochemistry.. D1ol-epox1de II (£y_£-1somer) has been found to be more
chemically reactive than the d1ol-epox1de I (Gelboln, 1980). Both are
unstable In aqueous media and are hydrolyzed nonenzymatlcally to tetrols.
In the presence of NADPH or NADH they are nonenzymatlcally reduced to trlols.
The Idea that 7,8-d1ol-9,lO-epox1de metabolite 1s the most probable
ultimate carcinogen gave rise to the "bay-region" hypothesis (JeMna et al.,
1978) for the metabolic toxlflcatlon of PAHs. The theory was based on the
04400 111-26 05/23/91
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r\>
o
^
v.
r>o
*.
•x.
(O
Metabolic formation of the 7,8-dlol-9,10-epoxldes of benzo(a)pyrene
o«
P-450
H
(*)-BP 7,8-dlol-9,10-epoxld«-2
(*)-BP 7,8-oxlde
(-)-BP 7,8-dlhydrodlol
P-450
H
(-)-BP 7,8-dlol-9,10-«poxlde-1
Bonzo(a)pyr«n«
P-450
P-450
7,8-dlol-9,10-epoxlde-1
(-)-BP 7,8-oxlde
(*)-BP 7,8-dlhydrodlol
/
OH
OH
Potential
Reactions
Trlols
Binding
to
Macromolecules
Tetrols
(-)-BP 7,8-dlol-9,10-epoxlde-2
FIGURE III-3
Mechanisms of Enzymatic Activation of Benzo[a]pyrene to 7,8-D1o1-9,lO-Epox1des
Source: Adapted^pm Yang et al., 1978
-------
assumption that the unusually high chemical reactivity of such d1ol-epox1des
and their high susceptibility to attack by nucleophlles can be attributed to
their electronic properties. The bay-regions of four PAHs, benz[a]anthra-
cene, benzo[a]pyrene, chrysene and phenanthrene, are shown In Figure III-4.
The Implications of the bay-region hypothesis for the mechanisms whereby
*
PAHs may produce cancer are discussed 1n Chapter VII. Table III-l lists
examples of the enzymes Involved In the toxlflcatlon of PAHs from the
production of dlols, phenols and qulnones, and dlol-epoxldes.
Metabolism of Nonalternant PAHs. The cardnogenlcHy -of the alternant
PAHs may be related to the Intrinsic cardnogenlcHy of the bay-region
dlol-epoxldes and the extent to which the parent compounds are metabolic-
ally converted to the bay-region dlol epoxldes (Thakker et al., 1988).
However. H should be noted that there 1s Increasing evidence that the
bay-region theory of activation does not appear to be the principal route of
activation for nonalternate PAHs such as benzo[b]fluoranthene and benzofk]-
fluoranthene and that other mechanisms of activation are Involved for those
/
PAHs that are devoid of a bay-region, such as 1ndeno[l,2,3-cd]pyrene and
benzo[k]fluoranthene (Am1n et al., 1982; Geddle et al., 1982; Hecht et al.,
1980; LaVole et al.. 1980; R1ce et al., 1985a, 1986. 1987a,b).
Other Toxlflcatlon Pathways. In addlton to the HFO mediated formation
of dlol-epoxldes and other oxygenated products, other routes of metabolism
have been shown for PAHs.
One-electron oxidation of PAHs can also form electrophlUc metabolites
that can bind to macromolecules (CavalleH and Rogan, 1983). The 1nter-
04400 II1-28 11/12/91
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BENZ [aJANTHRACENE
BENZO[a]PYRENE
CHRYSENE
PHENANTHRENE
FIGURE III-4
The Positions of Bay-Regions
Source: JeMna et al., 1978
04400
111-29
05/13/91
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TABLE III-l
Examples of Metabolism of PAHs to Biologically Active Forms
by Various Enzyme Systems*
PAH
Toxlflcatlon
Enzymes
Biologically Active
Intermediate-
Benzo[a]pyrene
Benz[a]anthracene
.Chrysene
D1benz[a,h]anthracene
monooxygenase
monooxygenase,
epoxlde hydrolase
monooxygenase
monooxygenase,
epoxlde hydrolase
monooxygenase,
epoxlde hydrolase
monooxygenase,
epoxlde hydrolase
monooxygenase,
epoxlde hydrolase
4,5-oxlde
7,8-d1ol-9,lO-epox1de
6-oxorad1cal
3,4-d1ol-l,2-epox1de
8,9-d1ol-lO,ll-epox1de
!,2-d1ol-3,4-epox1de
3,4-d1ol-l,2-epoxlde
[I0,ll]-d1ol-[l2,l3]-epox1de
*Source: Adapted from Pelkonen and Nebert, 1982
04400
111-30
05/13/91
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mediates In these pathways are radical cations. Benzo[a]pyrene tox1f1cat1on
by free-radical metabolism (see Figure III-2) occurs at position 6. The
6-hydroxybenzo[a]pyrene Is unstable 1n solution and rapidly undergoes
radical formation to the 6-oxobenzo[a]pyrene. This radical may Interact
with macromolecules or be converted to qulnones (Jeftlc and Adams, 1970).
PAH qulnones have been proposed to be biologically active because of
activity 1n oxidation/reduction cycles Involving qulnone, hydroxyqulnone and
molecular oxygen (Lesko et al., 1978). The peroxides and oxygen radicals
formed during these cycles may be responsible for the ultimate cellular
Injury. This oxidation/reduction cycle 1s shown 1n Figure III-5. PAHs,
being photoreactlve, can absorb visible light and become photooxldlzed.
This photooxldatlon can result In the formation of qulnones, dlhydrodlols
and phenols (see Chapter 7).
Marnett et al. (1978) and Marnett and Reed (1979) demonstrated oxygena-
tlon of benzo[a]pyrene catalzyed by prostaglandln H synthesis-dependent
oxidation of aractildonlc add metabolism pathway. Benzo[a]pyrene thus
activated has been shown to form covalent bonds with DNA and 1s mutagenlc.
Arachldonlc acid has been shown to be released upon membrane pertubatlon.
This metabolic pathway 1s particularly Intriguing for Instances such as
occur 1n the lung with co-exposure to PAHs and Irritants or partlculates.
These Irritant chemicals may affect membrane physiology such that the
metabolism of the PAH by prostaglandln synthetase could be enhanced locally
1n the extrahepatlc tissue. For example, Uarshawsky et al. (1984) examined
the effects of Fe^O. .on tne rate °f metabolism of benzo[a]pyrene 1n the
lungs of male white New Zealand rabbits. Benzo[a]pyrene was administered
Intratracheally to an Isolated perfused lung preparation with and without
04400 II1-31 05/13/91
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NAD
NADH
BWPHYDROQUINONE .BfrJP QUINONE
CELLS
DNA BREAKAGE
CELL DAMAGE
FIGURE III-5
Cyclic Scheme of Benzo[a]pyrene D1one/D1o1 Involvement 1n Redox Coupling
Source: Lesko et a!., 1978
04400
111-32
04/24/91
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FepOg. After 180 minutes the Fe203 pretreated rabbit lungs showed
an Increase of benzo[a]pyrene metabolism over the benzo[a]pyrene-only
treated rabbit lungs. The benzo[a]pyrene metabolites, namely dlhydrodlols,
were particularly enhanced by pretreatment with Fe.O..
Hydroperoxlde-dependent epoxldatlon of the 7,8-dlol has been
characterized 1n several other systems, such as mlcrosomal llpld
s
peroxldatlon (D1x and Marnett, 1983), hematln catalyzed decomposition of
fatty add hydroepoxldes (Dlx et al., 1985) and Upoxygenase catalyzed
epoxldatlon (Hughes et al., 1989).
The tox1f1cat1on of benzo[a]pyrene by hydroxymethylatlon catalyzed by
"hydroxymethyl synthetase" has also been suggested, but the presence of this
pathway has not been confirmed (Rogan et al., 1980).
Both y-rad1at1on and UV-llght convert benzo[a]pyrene to reactive
•
forms, which bind covalently to macromolecules and are mutagenlc, but the
significance of these mechanisms 1n mammalian species Is not known (Pelkonen
and Nebert, 1982). Metabolites could also disturb DNA structure without
binding covalently by slipping 1n between the planes of the helix
(Intercalation). PAH tetraols have been shown to do this In vitro
(Geadntov et al., 1980).
Comparative Metabolism. One factor that affects the delicate balance
of toxlflcatlon/detoxlflcatlon 1s the tissue site that catalyzes the
formation of chemically reactive metabolites. The degree of Induction or
Inhibition of enzyme systems by exogenous chemicals 1s known to vary for
04400 111-33 05/13/91
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specific organs; for example, liver 1s generally more Indudble than 1s
lung. The stability of reactive metabolites Is also an Important factor.
If the biologic half-life 1s sufficiently long, the metabolite may be
transported to other tissues for action or further metabolism. Several
reactive metabolites 1n one tissue may compete for sites. Differences 1n
site of administration will also affect metabolism.
Freudenthal et al. (1978) examined ttie metabolism of benzo[a]pyrene by
lung mlcrosomes Isolated from humans, rhesus monkeys and Sprague-Dawley
rats. Human samples were normal lung tissues of subjects with pulmonary
tumors. The metabolites of 14C-benzo[a]pyrene were analyzed by HPLC.
Large Individual variation 1n amounts of metabolites produced among monkey
and human samples were seen, probably due to the genetic heterogenldty of
the test subjects. Human subjects also had a wide range of age, were of
both sexes and had dissimilar environmental contacts. Qualitative differ-
ences between the types of metabolites produced by humans as compared with
•
-those produced by the other animal species were less dramatic. This study
demonstrated that the validity of comparison across species may not be
compromised 1f caution Is paid to the considerations of age, gender,
exposure to Inducing agents and other factors (U.S. EPA, 1980d).
That species differences 1n metabolism must be considered In carcino-
genic potency, however. Is demonstrated for chrysene by the work of Weston
et al. (1985). They used short-term cultures of rat skin (In which chrysene
Is not a strong carcinogen), mouse skin (1n which chrysene does cause a
carcinogenic response) and human skin. For each skin type a major pathway
leading to DNA adduct formation Involved formation of chrysene bay-region
04400 II1-34 05/13/91
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d1ol-epox1des (chrysene-1,2-d1ol-3,4-epox1de). Mouse skin, however,
released larger quantHHes of free dlhydrodlols to the medium than either
rats or humans. Moreover, there was greater chrysene adduct formation 1n
mouse skin.
fxcretlon
HepatoblHary excretion and elimination In the feces 1s the principal
route by which metabolites of PAHs are excreted. Early work by Peacock
(1936), Chalmers (1940), and Chalmers and K1rby (1940) established that the
fluorescent material appearing In the bile of rabbits, guinea pigs, rats and
fowl following Intravenous Injection of either colloidal benzp[a]pyrene or
colloidal anthracene was not the administered hydrocarbon but a derivative.
Excretion In the bile was well established within 15 minutes of Injection,
and only a small amount of fluorescence appeared 1n the urine of rabbits and
rats. Almost none of the excreted material was unchanged benzo[a]pyrene.
Chalmers and K1rby (1940) and Berenblum and Schoental (1942) found that only
IX of a subcutaneous dose of 60 mg benzo[a]pyrene was eliminated unchanged
1n the'feces of rats or mice. Kotln et al. (1959) confirmed this fraction,
and noted 1n addition that all elimination of unchanged benzo[a]pyrene
occurred within the first 10-15 minutes following an Intravenous dose.
Metabolism 1s the rate-determining step for excretion Into the bile.
Induction of metabolism with 3-methylcholanthrene enhanced the rate and
extent of excretion of metabolites of benzo[a]pyrene 1n rat bile (Iqbal et
al., 1979). Pre-treatment of rats with either 3-methylcholanthrene or
benzo[a]pyrene Itself also Increased the rate of excretion Into the bile of
metabolites of subsequently administered benzo[a]pyrene. There was,
04400 111-35 05/13/91
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however, no such pretreatment effect on excretion when the metabolites
themselves were given Intravenously, demonstrating that the effect of the
Inducers 1s on metabolite formation and not on metabolite transfer to bile
(Schlede et al., 1970).
Enterohepatlc circulation of metabolites, occurs, and a portion of
<
urinary excretion of metabolites can be attributed to this source. Kotln et
al. (1959) observed that In rats with biliary fistulas, urinary excretion of
benzo[a]pyrene metabolites was reduced from 7-14X to 2-4% of the dose. The
existence of an enterohepatlc circulation suggested by this observation was
confirmed by Chlpman et al. (1982), who collected biliary metabolites of
benzo[a]pyrene and Introduced them Into the duodena of b11e-cannulated rats.
following which the metabolites appeared In both bile and urine.
A very small fraction 1s excreted 1n pancreatic juice. Iqbal et al.
(1979) observed that during the Initial 2-hour period following Intravenous
administration of benzo[a]pyrene to rats, 0.03X of the dose was excreted In
pancreatic juice while 39% was excreted 1n the bile. In view of the low
metabolic potential of pancreatic mlcrosomes In vitro, the authors suggested
that metabolites of benzo[a]pyrene 1n the pancreas may have originated In
the liver and been transferred to the pancreas by the systemic circulation.
Systemlcally absorbed benzo[a]pyrene or Us metabolites (Heldelberger
and Weiss, 1951; Kotln et al.. 1959; Sanders et al., 1984) and systemlcally
absorbed 7,12-dlmethylbenzanthracene or Us metabolites are also not
eliminated 1n expired air (Lo, 1964; Sanders et al., 1984).
04400 111-36 . 05/13/91
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The fractions of 14C-benzo[a]pyrene and 14C-7,12-d1methylbenzanthra-
cene-assodated radlolabel excreted by rats In urine and bile have been
quantHated. Thirty-eight percent of an Intravenous dose of benzo[a]pyrene
of unspecified size was excreted 1n bile by 2 hours; urine was not monitored
1n this study (Iqbal et al., 1979). Kotln et al. (1959) found 39% of
Intravenous doses <134 yg In the "bile at 3 hours, and as much as 96X 1n
the bile by 14 hours. In rats with biliary fistulas, a maximum of only 3-4%
of radioactivity was recovered 1n the urine after a 24-hour period. Intact
rats had a urinary excretion of 7-14% of the administered dose, suggesting
that enterohepatlc circulation serves as a secondary source of radioactivity
that 1s excreted 1n the urine. The amount 1n the bile was not proportional
to dose at doses of >150 yg; only 32% of a 400 yg dose was recovered 1n
the bile at 24 hours. This amount (0.32x400 yg = 128 yg) Is equal to
that found In the bile 14 hours after the 134 yg dose (0.96x134 yg = 129
yg), Illustrating the limited capacity of the rat to metabolize
benzo[a]pyrene.
\ •
Lo' (1964) administered 3 mg doses of l4C-7,l2-d1methylbenzanthracene
Intravenously to three rats, and reported that 10-16% of the radlolabel had
been recovered In the urine and 18-44% 1n the feces by 17 days. Sanders et
al. (1984) found 38% of recovered radlolabel from an Intravenous dose of
1.04 yg 1n rat feces by 7 days, and 26% 1n urine. These observations
Indicate that the urine Is a more Important route of excretion for metabo-
lites of 7,12-dlmethylbenzanthracene than for metabolites of benzo[a]pyrene.
Additional data on relative utilization of urine and feces as routes of
excretion are summarized 1n the following Dermal Section.
04400 II1-37 05/13/91
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Oral. Because most of an absorbed oral dose of a PAH reaches the
liver directly via the portal circulation before reaching the systemic
circulation, hepatic metabolism and biliary excretion may determine the
relationship between administered dose and systemic dose. It 1s to be
expected that at reasonable doses a greater fraction of an oral dose than of
an Intravenous dose would be excreted In the feces. In fact, low doses of
orally administered PAHs could, 1n principle, be fully metabolized and
excreted 1n the bile without reaching the systemic circulation at all.
Twenty-four hours after administration of 50 yg pyrene to rats, 24 yg
was found In the GI tract and none In the liver, kidney, lung or trachea
(Mitchell and Tu, 1979). However, controlled studies of elimination
following oral doses of PAHs have generally utilized large amounts of the
hydrocarbons, so that substantial fractions are not metabolized In the first
pass. Interpretation of biliary excretion data following an oral dose 1s
further complicated by enterohepatlc circulation.
Rahman et al. (1986) administered five radlolabeled PAHs Including
benzo[a]pyrene and 7,12-d1methylbenzanthracene In 1.0 mg doses to rats
Intraduodenally, and cumulative excretion of radlolabel 1n bile and urine
after 24 hours was measured and expressed as a fraction of administered
radlolabel. For benzo[a]pyrene these figures were 6% for urine and 25% for
bile, for 7,l2-d1methylbenzanthracene they were 3% for urine and 25% for
bile. These data for benzo[a]pyrene are consistent with the corresponding
values obtained following Intravenous administration of high doses (see
preceding discussion), suggesting that hepatic capacity to metabolize the
absorbed PAHs on the first pass had been greatly exceeded.
04400 111-38 05/13/91
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Modica et al. (1983) gave emulsions of benz[a]anthracene, chrysene and
trlphenylene to rats by stomach tube. Seventy-two hours later, cumulative
fecal elimination was 6% of the benzanthracene dose of 22.8 mg, 3% of the
trlphenylene dose of 22.8 mg, 38% of the chrysene dose of 22.8 mg, and 41%
of the chrysene dose of 11.4 mg, Indicating that even these relatively
massive doses are absorbed surprisingly well.
Elsele (1985) examined the uptake and distribution of tracer levels of
14C-naphthalene 1n laying pullets, swine and dairy cattle. Each group of
animals received either a single oral dose or dally oral doses of naphtha-
lene for 31 days. The distribution of naphthalene and/or Us metabolites In
various tissues was measured 24 hours after the last dose. Considerable
species differences In the distribution of naphthalene and/or Us metabo-
lites were observed. However, In each of the exposed animals naphthalene
and/or Us metabolites reached the systemic system and distributed to all
tissues examined.
Inhalation. Elimination of an Inhaled dose of a PAH follows Us lung
clearance pattern, that 1s, the fraction that Is systemlcally absorbed from
the lung Is excreted 1n bile and urine, while the fraction that Is cleared
from the lung by mucoclHary action and swallowed Is subject to GI absorp-
tion. In this case, however, the amounts reaching the duodenum may be too
low to approach the capacity of the liver to metabolize them on the first
pass. In support of this supposition, Pylev et al. (1969) observed that
excretion of radlolabel In feces and urine of hamsters given Intratracheal
Instillations of 8H-benzo[a]pyrene alone, or 3H-benzo[a]pyrene-coated
carbon or asbestos particles, followed the same general time course as loss
04400 II1-39 05/13/91
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from the lung, and that for a period of 36 days elimination via urine
remained at ~80X of that 1n feces.
The most detailed and precise studies 1n which excretion was measured
following Inhalation exposure are those from the Inhalation Toxicology
Research Institute, 1n which rats were exposed by Inhalation to pure
3H-benzo[a]pyrene aerosols and to 3H-benzo[a]pyrene adsorbed on gallium
oxide particles, and excretion of radlolabel was followed for time periods
<16 days. Using pure aerosols of 3H-benzo[a]pyrene (500 yg/l) with a
mass median diameter of 1-2 ym, Mitchell (1982) observed that radlolabel
•
was excreted both 1n the feces and 1n the urine, with radioactivity 1n the
feces ~10 times that In the urine during the first 24 hours after exposure.
Subsequently (Sun et al., 1983), this experiment was repeated with a lower
concentration of 3H-benzo[a]pyrene, 600 ng/i, and was extended by
exposures to gallium oxide particles coated with 3H-benzo[a]pyrene. Both
aerosols had the same mass median diameter, -0.1 \an as In the previous
•study. The pure aerosol was, as before, eliminated In urine and feces, with
the total amount of radlolabel eliminated 1n feces through the sixteenth day
after exposure about 6 times the amount eliminated In the urine. Since the
aerosol concentration In this study was 1/800 that used In the. previous
study, H must be concluded that the elimination pattern 1s Independent of
aerosol concentration at least up to ~500 »ig/l. That 1s, these aerosol
concentrations do not stress hepatoblllary metabolism and excretion mecha-
nisms. Based on a comparison with the work of Kotln et al. (1959), this Is
a reasonable conclusion. Kotln et al. (1959) found that oral doses <134
vg/rat did not exceed the maximum metabolic capacity of the animals.
04400 111-40 05/13/91
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When benzo[a]pyrene adsorbed to gallium oxide particles (3500 ng/i)
was Inhaled, the total amount of radlolabel eliminated 1n the feces through
the sixteenth day following exposure was ~12 times the amount eliminated 1n
urine, demonstrating the shift 1n excretion pattern caused by the presence
of the particles.
When benzo[a]pyrene was presented to rats as an aerosol coated on dlesel
exhaust particles (Sun et al., 1984), the amount excreted 1n feces by the
twenty-sixth day following exposure was ~5 times the amount excreted In the
urine. • .
Dermal. Since only an Insignificant amount of radlolabel appears In
the GI tract as a result of grooming by rats whose back skin was painted
with PAHs (Mitchell and Tu, 1979; Sanders et al., 1984), the cumulative
fractions of an absorbed cutaneous dose eliminated 1n urine and 1n feces
should be comparable with the cumulative fractions found after Intravenous
administration. Excretion of radlolabel from 14C-benzo[a]pyrene,
/
l4C-7,!2-d1methylbenzanthracene and 14C-anthracene, absorbed
percutaneously, has been examined. The results are similar to the results
of the Intravenous studies discussed above.
Thirty-five percent of the radlolabel associated with a cutaneous dose
of 12.5 yg 14C-benzo[a]pyrene/cm2 (22.5 yg total dose) was found In
the feces at 24 hours and 80% at 7 days, with -10* 1n the urine at 7 days,
by which time nearly 'all the benzo[a]pyrene had been absorbed (Sanders et
al., 1984). 7,12-dlmethylbenzanthracene metabolites were excreted to a
greater extent 1n the urine than benzo[a]pyrene metabolites. After 7 days,
04400 111-41 05/23/91
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30% of the radlolabel associated with a cutaneous dose of 5.4 yg
l4C-7,l2-d1methylbenzanthracene/cm2 (1.1 yg total dose) was recovered
1n the urine and only 62% 1n the feces (Sanders et al.t 1984). Yang et al.
(1986) found that of the 52% of the absorbed radlolabel from a cutaneous
dose of 9.3 yg a4C-anthracene/cm2, 29% had been excreted 1n the urine
and 21% 1n the feces after 6 days.
Interestingly, these fractions appear to be dose-dependent. Increasing
the benzo[a]pyrene dose from 1.25 to 12.5 to 125 yg/cm3 resulted 1n a
shift In the excretion pattern at 24 hours In favor of the feces. In
contrast. Increasing the 7,l2-d1methylbenzanthracene dose from 5.4 to 56 to
515 yg/cm2 decreased the fraction of the dose excreted 1n both feces and
urine, although fecal excretion was less markedly affected than urinary
excretion (Sanders et al., 1984). These shifts were greater than could be
accounted for by the reduction 1n percentage absorbed at the higher doses,
encouraging the speculation that differential capacity limitations of the
enzyme systems responsible for the formation of different metabolites might
result In a relative decrease 1n production at high doses of metabolites for
which the urine Is the favored route of excretion.
Summary .
The PAHs are a Upophlllc class of xenoblotlcs that are readily absorbed
across cellular membranes. Major routes of environmental exposure to PAHs
are the following: the GI tract by contaminated food or water; the lungs by
Inhalation of aerosols.or by hydrocarbon-adsorbed particles; and the skin by
direct contact. Once absorbed, the PAHs are rapidly and widely
distributed. The differing availability of the PAHs may depend on the
04400 II1-42 05/13/91
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chemical form In which exposure occurs (for example, hydrophlllc or
Upophlllc solution), varying Intestinal absorption, metabolic
transformation or removal rates. PAHs can be observed 1n most Internal
organs from minutes to hours after administration by various routes.
Adipose and mammary tissues are significant distribution sites 1n which PAHs
may be stored and slowly released.
PAH metabolism Is affected by the route of administration. PAHs
administered through the lungs or through 1.p. or 1.v. Injection may avoid
first pass metabolism In the liver. The blood will distribute the PAHs Into
peripheral tissues. This distribution will rapidly result 1n the
establishment of a steady-state concentration between the blood and
well-perfused tissues. Adipose tissues, however, will exchange the
deposited PAHs more slowly and will accumulate and slowly release PAHs. On
the other hand, oral administration of PAHs results In metabolism by the
liver before systematic distribution can occur via the bloodstream;
•
consequently, PAH metabolites will appear In the bloodstream several hours
after administration.
PAHs can be metabolized by enzyme-mediated oxldatlve mechanisms to form
reactive electrophlles. The cytochrome P-450 dependent mlcrosomal mixed
function oxldase (MFO) system (arylhydrocarbon hydrolase) Is Induclble by
PAH exposure. These enzymes are found 1n many mammalian tissues; MFO
enzymes of the liver are the best studied and .have been found to be the most
abundant. Similar types of metabolites are produced by mammalian tissues;
however, the proportions of the phenols, dlols and epoxldes produced may
differ among the various tissues. The mechanism of the dlolepoxlde
04400 111-43 05/13/91
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metabolic pathway for PAH tox1f1cat1on and the bay-region hypothesis are
accepted. Reactive epoxldes, formed by MFO mediated PAH metabolism, have
the ability to Interact with and thereby alter DNA, RNA and proteins. PAH
metabolites are most commonly conjugated with glucuronlc acid, glutathlone
or sulfate. They are excreted by the GI tract after hepatoblHary excretion
or the swallowing of mucus material cleared from the respiratory tract.
Nonenzymatlc mechanisms of biologic PAH alteration also occur.
04400 II1-44 05/23/91
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IV. HUMAN EXPOSURE
Text to be provided by the Office of Drinking Water,
IV-1 08/30/85
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V. HEALTH EFFECTS IN ANIMALS
The general and specific health effects associated with PAH Ingestlon,
and to a lesser degree, Inhalation, peritoneal and dermal exposures, are
examined 1n this chapter. Much of the research on the health effects of
selected PAHs has focused on their potential as skin carcinogens, genotoxlc
agents and on their Inhalation effects (IARC, 1983). Studies of Individual
PAHs administered by the oral route are generally lacking.
Acute Oral ToxIcUv
Acenaphthylene. An English abstract of a paper by Knoblock et al.
(1969) reported that acenaphthylene administration "to the stomach" resulted
1n an LD5Q of 3 g/kg for rats and 2.2 g/kg for mice.
Anthracene. The abstract of a Russian study Indicated that single
oral doses of 1.47 or 2.44 g/kg of commercial grade anthracene or 17 g/kg of
pure anthracene were not lethal to mice (Nagornyl, 1969). Toxic effects
reportedly .Included fatlgabllUy; adynamia; hlstologlc hyperemla In the
kidney, liver, heart and lungs; "Upld dystrophy" In the liver; and leuko-
cytosls with neutrophllla.
Systemlcally administered anthracene (50 mg/mi corn oil by gavage)
followed by UV Irradiation of the skin for 1 hour, 2 hours after dosing,
produced keratUls of the exposed skin In mice (Dayhaw-Barker et al.,
1985).. This effect reportedly was less pronounced 1n mice exposed to UV
light only and was not evident In vehicle controls.
04420 V-l 12/31/90
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Dally Intragastrlc administration to five male Wlstar rats of 100 mg/kg
anthracene 1n olive oil for 4 days produced a nonsignificant (3-fold)
Increase 1n mean liver cytosollc aldehyde dehydrogenase activity_£T6rr6nen
et.al., 1981). Neither treatment-related effects on aldehyde dehydrogenase
activity 1n the liver mlcrosomes or postmltochondrlal fractions of small
Intestinal mucosa nor effects on liver/body weight ratios were observed.
Benz[a1anthracene. Pertinent data regarding the acute oral toxldty
of benz[a]anthracene could not be located 1n the available literature.
BenzoTalpyrene. Pertinent data regarding the acute oral toxldty of
benzo[a]pyrene could not be located 1n the available literature.
Benzofblfluoranthene. Pertinent data regarding the acute oral
toxldty of benzo[b]fluoranthene could not be located In the available
literature.
BenzoFklfluoranthene. Pertinent data regarding the acute oral
toxldty of benzo[k]fluoranthene could not be located In the available
literature.
Benzorg.h.Hperylene. Pertinent data regarding the acute oral
toxldty of benzo[g,h,1]perylene could not be located In the available
literature.
Chrysene. Pertinent data regarding the acute oral toxldty of
chrysene could not be located In the available literature.
04420 V-2 09/21/90
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DlberrzTa.hlanthracene. Pertinent data regarding the acute oral
toxldty of d1benz[a,h]anthracene could not be located 1n the available
literature.
Fluoranthene. the oral LD5Q for male Carworth-Hlstar rats exposed
to fluoranthene 1s -2000 mg/kg (Smyth et al., 1962).
Fluorene. K1zer et al. (1985), 1n an effort to Identify enzyme
changes as early Indicators of hepatocardnogenesls, examined the effect of
several xenoblotlcs, Including fluorene, on both the activity and amount of
hepatic mlcrosomal epoxlde hydrolase. As part of this study, male Holtzman
rats were fed a diet containing 0.06% (10.5 mg/kg/day) fluorene for 3 weeks.
No hepatotoxldty, as measured by SGOT activity, was observed. Furthermore,
fluorene exposure failed to result 1n an appreciable elevation of the
activity of mlcrosomal epoxlde hydrolase. However, a 4-fold Increase over
controls of epoxlde hydrolase antigen was detected by Immunoassay. After
comparing the activity and amount of hepatic mlcrosomal epoxlde hydrolase
Induced by feeding the various xenoblotlcs with their reported carcinogenic
potential, the authors concluded that Induction of mlcrosomal epoxlde
hydrolase 1s not a "key change" leading to malignancy.
IndenoH ,2.3-cdlpyrene. Pertinent data regarding the acute oral
toxUHy of 1ndeno[l,2,3-cd]pyrene could not be located 1n the available
literature.
Naphthalene. The LDcn for oral exposure to naphthalene has been
— ou
determined for several species. The LD5Q values for naphthalene dissolved
04420 V-3 10/08/91
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In peanut oil for male and female Sherman rats were 2200 and 2400 mg/kg,
respectively (Galnes, 1969). In two other studies, the LD5Q values for
rats were 1780 (Toxicology Data Bank, n.d.) and 9430 mg/kg (U.S. EPA,
1980c); the strain and sex of the animals was not specified. For male and
female CD-I mice, the acute oral LD™ values of naphthalene 1n corn oil
were 533 and 710 mg/kg, respectively (Shopp et a!., 1984). In a recent
study (unpublished) conducted by Mallory et al. (1985a), the acute ID.-
values of naphthalene 1n corn oil for male and female Sprague-Dawley rats
were reported to be 2009 and 3310 mg/kg, respectively. An LD5Q of 353
mg/kg was determined for CD-I mice In a study In which Plasterer et al.
(1985) treated mice at doses ranging from 125-2000 mg/kg/day for 8 days.
Although cataract formation 1n rats following oral administration of
naphthalene has been known for many years (FHzhugh and Buschke, 1949),
recent studies have shown that ocular changes can result from a single dose
of naphthalene, van Heynlngen and Plrle (1967) found that lens changes
developed In the eyes of rabbits after a single gavage dose of 1000 mg/kg.
In CD-I mice, oral doses of >400 mg/kg for males and >600 mg/kg for females
resulted 1n ptosls with clear, red secretions around the eyes within 1 hour
of dosing (Shopp et al., 1984).
Ikemoto and Iwata (1978) reported that oral administration of naphtha-
lene (1 g/kg) to male and female albino rabbits for 2 consecutive days
resulted In cataract formation. Occurrence of cataracts was accompanied by
a decrease 1n sufhydryl content In both soluble and Insoluble lens protein.
04420 V-4 05/14/91
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Rao and Pandya (1981) orally administered male BHD and AR albino rats
1000 mg/kg naphthalene each day for 10 days. Significant Increases In the
relative liver weight (p<0.01), and 1n liver aniline hydroxylase activity
and I1p1d peroxldatlon (p<0.001) were observed 1n the treated animals. A
moderate (nonsignificant) Increase 1n llpld peroxldatlon was also observed
In the eyes. No treatment-related changes In sulfhydryl content or alkaline
phosphatase activity were observed In the liver, kidneys or eyes.
Yamauchl et al. (1986) reported that a single oral dose of napthalene
(1000 mg/kg) to male Wlstar rats resulted In a significant Increase 1n serum
I1p1d peroxide levels (p<0.05) beginning on the 4th day after administration
and continuing through the 20-day observation period. This Increase was
paralleled by a significant (p<0.05) decrease In GSH content In the lens of
the exposed animals. Zuelzer and Apt (1949) reported a hemolytlc effect
when naphthalene was administered to dogs 1n their diets.
•
Phenanthrene. 'Pertinent data regarding the acute oral toxlclty of
phenant'hrene could not be located 1n the available literature.
Pyrene. Pertinent data regarding the acute oral toxlclty of pyrene
could not be located In the available literature.
Acute Toxlclty By Other Routes
Acenaphthylene. In a study published 1n Russian (Rotenberg and
Mashblts, 1965), acenaphthylene was administered to white rats Intratra-
cheally 1n a sunflower oil solution or by blowing acenaphthylene powder Into
the trachea. The dosing schedule used was not provided. The pulmonary
tracts of animals sacrificed 1 month after the experiment began showed signs
04420 V-5 05/14/91
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of tracheobronchHIs and hyperemla, edema and necrosis of the epithelium 1n
the trachea and bronchi with the formation of ulcers. No further details of
this study were available.
In a corn oil gavage study acenaphthylene was administered dally to 10
CD-I mice/sex/group at 0, 125, 250, 500, 750 and 1000 mg/kg/day. In the
1000 mg/kg/day group the mean body weights were significantly reduced 1n
both sexes at the end of the first week. Dose-related clinical symptomology
was noted 1n groups receiving 500, 750 and 1000 mg/kg/day; these symptoms
•*
Included languid behavior, prostration, decrease 1n body temperature (cold
to touch) and toxic effects to the eyes. These eye effects Included
enlargement, drying out and eventual crusting over. In the 1000 mg/kg/day
group eyes were adversely affected 1n 40% of the males and 50% of the
females. The survival rate 1n the 750 mg/kg/day group was 90% among the
males and 80% among the females; 1n the 1000 mg/kg/day group survival was
significantly reduced In both sexes at 40%. Gross pathology from the 750
and 1000 mg/kg/day groups Indicated that the liver, stomach, eyes and
/
subcutaneous tissues were affected by the treatment (Hazelton Laboratories
America Inc., 1989a).
Anthracene. Mice treated 1.p. with anthracene were found to have an
LD50 of >430 mg/kg bw (Salamone, 1981). In a study by Gerarde (1960),
mice were given 1.p. Injections of 500 mg/kg bw/day for 7 days. Of the 10
mice treated, 9 survived. The ID,.- (skin Irritant dose) for the mouse was
found to be 6.6xlO~4 mmol/ear (Brune et al., 1978).
In a corn oil gavage study anthracene was administered dally for 14 days
to 10 CD-I mice/sex/group at 0, 125, 250, 500, 750 and 1000 mg/kg/day. No
04420 V-6 11/12/91
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treatment-related differences were observed between the groups 1n body
weight gain, mortality and gros.s pathologic examination (Hazelton
Laboratories America, Inc., 1989b).
-\
Benzfalanthracene. Pertinent data regarding the acute nonoral toxlc-
1ty of benz[a]anthracene could not be located \n the available literature.
BenzoTalpyrene. The mouse L05Q (1.p.) for benzo[a]pyrene 1s ~250
mg/kg (Salamone, 1981). The ID™ for skin Irritation In mice 1s
5.6xlO"5 mmol/ear (Brune et al., 1978).
A single 1.p. dose of 10 mg benzo[a]pyrene produced a reduction In the
growth rate of Immature rats (Haddow et al., 1937). He 11 man et al. (1984)
studied the acute toxldty of Injections of benzo[a]pyrene In C57B1 male
mice. Groups of 7-10 animals were Injected 1.p. with 1.12x10~4 mol/kg
benzo[a]pyrene 1n corn oil. Two hours before sacrifice animals were Inject-
ed with 8H-thym1d1ne to determine the rate of DNA turnover. A significant
/
decrease (40X) In 8H-thym1d1ne Incorporation Into the thymus, spleen,
small Intestine and testls occurred 48 hours after benzo[a]pyrene treatment,
which was Indicative of decreased DNA synthesis 1n those organs. There was
a stimulatory effect on thymldlne Incorporation In the liver 48 hours after
the Injection of benzo[a]pyrene.
Robinson et al. (1975) showed that "responsive" mice (those capable of
producing Increased levels of cytochrome P-450 mediated enzymes as a conse-
quence of PAH exposure) had reduced survival time following a single 1.p.
dose of 500 mg/kg bw benzo[a]pyrene.
04420 V-7 11/12/91
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Wojdanl et al. (1984) reported that target cell killing by lymphocytes
from two mouse strains was decreased following a single 1.p. Injection of 5
or 50 mg benzo[a]pyrene/kg. This study Is described 1n the Target Organ
Toxldty Section.
Subcutaneous Injections of benzo[a]pyrene In corn oil at doses of 5, 20
or 40 mg/kg 1n female B6C3F1 mice produced a dose-related suppression of
antibody production to both T-cell Independent and T-cell dependent antigens
(White and Holsapple, 1984).
•
Benzo[b]fluoranthene. Pertinent data regarding the acute nonoral
toxldty of benzo[b]fluoranthene could not be located 1n the available
literature.
BenzoTklfluoranthene. Pertinent data regarding the acute nonoral
toxldty of benzo[k]fluoranthene could not be located 1n the available
•
literature.
Benzofq.h.Uperylene. Pertinent data regarding the acute nonoral
toxldty of benzo[g,h,1]perylene could not be located 1n the available
literature.
Chrysene. The LD50 (1.p.) for chrysene was found to be >320 mg/kg
bw 1n mice (Simmon et al., 1979). A single l.p. Injection of 7.5 mg
chrysene 1n sesame oil produced no toxic effects 1n AKR/J or C57B1/60 mice
during a 20-day observation period or upon necropsy.
04420 V-8 03/20/91
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D1benz[a.h]anthracene. One or two Intraperltoneal administrations of
3-90 mg/kg bw d1benz[a,h]anthracene 1n sesame oil produced a reduction 1n
the growth rate of young Lister strain hooded rats. This persisted for at
least 15 weeks (Haddow et al.. 1937).
Fluoranthene. The 24-hour CDC_ value after dermal exposure to
5U
fluoranthene was found to be 3180 mg/kg In rabbits (Smyth et al., 1962). No
Information was reported concerning target organs or specific cause of
death. As part of the same study, Smyth et al. (1962) observed no mortality
1n six male and six female albino rats exposed to concentrated vapors of
fluoranthene for 8 hours.
Haddow et al. (1937) examined the effect of various PAHs. Including
fluoranthene, on body growth In hooded rats of the Lister strain. A single
1.p. Injection of 10 mg/kg fluoranthene dissolved In sesame oil had no
adverse effect on body weight gain over a 24-day observation period.
Aso'kan et al. (1986) examined the Induction of cutaneous and hepatic
monoxygenase activities by fluoranthene. Twenty-four hours after a single
topical application of 10 mg/kg fluoranthene to the backs of Sprague-Dawley
rats, significant Increases 1n aryl hydrocarbon hydroxylase, 7-ethoxyreso-
rufln 0-deethylase and 7-ethoxycoumarln 0-deethylase activities were
observed 1n the skin and liver.
Fluorene. Pertinent data regarding the acute nonoral toxldty of
fluorene could not be located 1n the available literature.
04420 V-9 03/20/91
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IndenoH,2.3-cdlpyrene. Pertinent data regarding the acute nonoral
toxlclty of 1ndeno[l,2,3-cd]pyrene could not be located In the available
literature.
Naphthalene. Ir1e et al. (1973) studied the effects of naphthalene 1n
mice (strain not specified) following s1ng>e subcutaneous Injections at
doses of 650-1348 mg/kg. Vigorous tremors were noted In the mice for 3-4
days following dosing. The LD5Q value was calculated to be 969 (891-1053)
mg/kg.
»
The 24-hour LD5Q value of naphthalene In Swiss-Webster .mice was 380
(350-413) mg/kg following 1.p. Injection (Warren et al., 1982; Shank et al..
1980). Those mice that died did so within 24 hours; survivors were observed
for an additional 6 days. The target organ was Identified by Warren et al.
(1982) as the lungs, 1n which naphthalene caused a dose-dependent Increase
In bronchlolar epithelial necrosis at doses >200 mg/kg. The pulmonary
damage and lethality resulting from naphthalene administration were markedly
inhibited by prior treatment with plperonyl butoxlde and enhanced by prior
treatment with dlethyl maleate. This supports the view that P-450-dependent
metabolism of naphthalene 1s responsible for the observed toxldty and that
glutathlone plays an Important role In the detoxification of the lung-
damaging metabollte(s).
Reid et -al. (1973) gave napthalene dissolved 1n sesame oil to C57B1/6J
mice by the 1ntraper1toneal route and found coagulatlve necrosis of the
bronchlolar and bronchial epithelium at a dose of 600 mg/kg. Controls
received sesame oil alone, and no adverse effects were reported for this
group. The size of the treatment groups was not stated.
04420 V-10 10/24/91
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Mahvl et al. (1977) administered naphthalene In corn oil Intraperlto-
neally to C57B1/6J mice. Two groups of 63 mice received corn oil alone or
remained untreated. Groups of 21 mice each were given 67.4, 128 or 256
mg/kg. Three animals from each dosage group were sacrificed at 10 minutes,
1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and 7 days following
treatment. Lung tissue was rapidly fixed and examined by light, scanning
electron, and transmission electron microscopy. No changes were noted 1n
either control group. Minor bronchlolar epithelial changes were noted 1n
the group receiving 67.4 mg/kg. Mice In the higher dose groups developed
necrosis of secretory nonclHated bronchlolar cells and the adjacent
dilated cells. Epithelial structure returned to normal within 7 days 1n
all cases.
Tong et al. (1982) found hlstologlc changes In the lungs of C57B1/6J
mice treated 1.p. with 225 mg/kg naphthalene. One day after dosing, the
Clara cells In the terminal bronchioles were pyknotlc, and hypereoslnophlUc
'nuclei appeared to be detaching from the bronchlolar wall. Three days after
dosing, some surfaces appeared to be completely denuded of Clara cells,
whereas other surfaces appeared to have Immature Clara cells scattered
drcumferentlally. Five days after treatment, there was still evidence of
Incomplete recovery; by 8 days, most of the terminal bronchioles were
reeplthellaHzed; and by 15 days, mature Clara cells were common, but
recovery was evidently not complete.
O'Brien et al. (1985) Investigated differences 1n naphthalene-Induced
toxldty subsequent to 1.p. treatment of male Swiss T.O. mice and male
Wlstar-derlved rats. In mice doses >200 mg/kg resulted In damage to the
04420 V-ll 03/20/91
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nondllated bronchlolar epithelial cells, and doses >400 mg/kg resulted In
damage to cells 1n the proximal tubules of the kidney. Rats were more
resistant to the cytotoxlc effects of naphthalene, as doses of naphthalene
as high as 1600 mg/kg caused no detectable pulmonary or renal damage. This
species difference In toxldty was reflected by a larger depletion of
*
nonproteln sulfhydryls 1n the lung and kidney of the mouse than 1n those
organs 1n the rat.
It previously had been reported that the 8-hour Inhalation LC5Q value
for naphthalene was 100 ppm (Union Carbide, 1968). However, Buckpltt (1985)
suggested that this value may be too low. He estimated that 1n 8 hours the
body burden would be <30 mg/rat, or ~150-200 mg/kg. This concentration 1s
less than the oral or 1.p. ID™ values for rats. Fait and Nachrelner
(1985) reported that exposure of male and female VMstar rats to 78 ppm
naphthalene for 4 hours resulted In no mortalities, nor any lung, liver,
kidney, or nasal passage abnormalities. . In an unpublished Inhalation study
with male Swlss-Wesbster mice, no deaths were noted following nose-only
exposures to 90 ppm for 4 hours. Lung lesions however, were reported
(BuckpHt, 1985).
van Heynlngen and P1r1e (1967) treated one rabbit Intravenously with 300
mg of a dlhydrodlol metabolite of naphthalene 1n divided doses over 3 days
and noted retinal lesions. They also noted lens changes In four rabbits
dosed externally with eye drops of the same compound (IX dissolved In water)
over a period of 2-5 days for a total of 40-70 mg per rabbit.
Acute ocular Irritation was noted 1n 2/6 New Zealand white rabbits
receiving no postdose eye rinse after 24 and 48 hours of exposure to 0.1 mg
04420 V-12 11/12/91
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naphthalene (Mallory et al., 1985c). This response Included slight 1r1t1s,
moderate redness and slight swelling and discharge. All animals appeared
normal by 72 hours postdoslng. No positive response was noted In rabbits
(three) receiving a postdose rinse.
No deaths occurred when 2500 mg/kg of naphthalene was applied to the
skin of male and female Sherman rats (Galnes, 1969). The application of
2000 mg naphthalene/kg (dissolved 1n acetone) to the skin of New Zealand
white rabbits did not cause mortality; the LD ' was, thus, >2000 mg/kg
(Mallory et al., 1985b). These studies suggest that naphthalene may not be
as readily absorbed through the skin as It 1s through the Intestinal mucosa.
Naphthalene (moistened with 2 mi of acetone) was found to be slightly
to moderately Irritating to the skin of male and female New Zealand white
rabbits 30-60 minutes postdoslng (Mallory et al., 1985d). Dermal Irritation
was still evident up to 5 days after test material application. Flssurlng
'of the skin was also noted.
/
Naphthalene (100X) did not cause delayed hypersensUlvUy In Hartley
guinea pigs (Mallory et al., 1985e).
Phenanthrene. The LD™ for mice (1,p.) 1s 700 mg/kg bw (Simmon et
al., 1979).
Yoshlkawa et al. (1985) Investigated effects of 1.p. exposure to phenan-
threne, pyrene and some of their oxidized products 1n Sprague-Dawley rats.
As part of this experiment, groups of three males each were Injected with
04420 V-13 03/20/91
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either 3.0 ml/kg saline, 3.0 ml/kg DMSO or 150 mg/kg phenanthrene 1n
DMSO. An additional set of animals was similarly treated on a second
occasion. Blood was obtained at 24 or 72 hours post-treatment by cardiac
puncture and the following measurements taken: aspartate amlnotransferase
(AST), alanlne amlnotransferase, -r-glutamyl transpeptldase (G6TP), lactic
dehydrogenase, glucose, b1!1rub1n, BUN and creatlnlne. Gross observation
was made of organs. Phenanthrene treatment resulted In a significant
elevation of serum AST and GGTP by comparison with both controls. GGTP
returned to control levels by 72 hours. Livers of animals killed at either
24 or 72 hours were described as congested with a distinct'lobular pattern.
Kidneys were reported to be somewhat smaller In size and congested.
Pyrene. The Intraperltoneal LD5om (dose lethal to half the
animals 1n 7 days) for B6C3F1 mice was found to be 514 mg/kg pyrene and the
Intraperltoneal LD5Q... (dose lethal to half the animals 1n 4 days) was
678 mg/kg bw (Salamone. 1981). The growth rate of young (46-48 days old)
Lister rats was not affected by Intraperltoneal administration of 10 mg of
pyrene 1n sesame oil (Haddow et al., 1937).
Yoshlkawa et al. (1985) administered 150 mg/kg pyrene to male Sprague-
Dawley rats as described In the preceding section. This resulted 1n a small
but significant elevation In both serum AST and blllrubln. At both 24 and
72 hours sacrificed rats were observed to have minimal congestion and swell-
Ing of livers.
Yoshlkawa, T., W. Flory, L.P. Ruhr et al. (1987) treated male
Sprague-Dawley rats with a single 1.p. Injection of 150 mg/kg pyrene In DMSO
04420 V-14 11/12/91
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and evaluated serum chemistry at 24 and 72 hours for Indications of
hepatotoxldty. No statistically significant differences were found for
AST, alanlne amlnotransferase, sorbHol dehydrogenase, GGTP, lactate
dehydrogenase, glucose, BUN or creatlne between control rats receiving DMSO
and rats administered pyrene. Furthermore, no hlstopathologlc alterations
were noted 1n animals necropsled 72 hours after treatment with pyrene.
Subchronlc and Chronic Oral ToxIcUy
Bloassays for carc1nogen1c1ty are reported 1n this section only when
they refer to health endpolnts other than tumor Incidence. '
Acenaphthylene. Knobloch et al. (1969) reported on the effects asso-
ciated with Ingestlon of both acenaphthylene and acenaphthene. One group of
seven rats was given an oral dose of 0.6 g/kg bw of acenaphthylene 1n olive
oil for 40 days. A second group of seven rats was given an oral dose of 2.0
g/kg bw of acenaphthene 1n olive oil for 32 days. Both PAHs yielded the
following physiologic effects: "considerable" body weight loss, changes 1n
/
the peripheral blood pattern, changes 1n renal function, and Increased serum
amlnotransferase activities. Additional effects associated with acenaph-
thene only Included mild morphologic damage to the liver and kidney, lung
changes consisting of mild bronchitis, and localized Inflammation of perl-
bronchial tissue. Since the source of this Information 1s an abstract, more
precise quantification of the health effects 1s not possible.
In a Russian study (Rotenberg and Mashblts, 1965), acenaphthylene In oil
was administered orally to white mice at a dose 1/10 the ID,.. [LD5Q=1760
(range of 1100-2800) mg/kg] every other day for 2 months. Treated mice
04420 V-15 11/12/91
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showed a significant lag 1n weight gain as.compared with controls. H1sto-
pathologlc examination of organs showed signs of stasis In the parenchyma-
tous organs and albuminoid degeneration of the liver. The most severe
changes were observed 1n the lungs, which showed hemorrhage with destruction
of the Interalveolar septa and focal bronchial pneumonia. Purulent fod
were observed 1n Isolated cases, and bronchogenlc lung cancer was diagnosed
1n one mouse. Further details of this study were not provided.
In a study conducted by Hazelton Laboratories America, Inc. (1989C) for
U.S. EPA acenaphthylene was administered to CD-I mice (20/sex/group) by
gavage at dosage levels of 0, 100, 200, or 400 mg/kg/day for at least 90
days. Effects examined Included mortality, clinical signs, body weights,
food consumption, opthalmology, hematology, clinical chemistry, organ
weights, and gross and hlstopathology. There was no Increase 1n mortality
among males tested; however, the Incidences of treatment-releated deaths 1n
females were 15, 25 and 40X for low-, mid- and high-dose groups, respec-
tively, compared with no deaths 1n female controls. No significant changes
In mean body weights, body weight gains, or food consumption were found.
Statistically significant (p<0.05) treatment-related hematologlc effects
Included decreased erythrocyte count 1n all male dose groups and high-dose
females; decreased hemoglobin and hematocrlt In mid- and high-dose males and
high-dose females; Increased platelet counts 1n mid- and high-dose males;
and Increased leucocyte and segmented neutrophll counts In high-dose
females. Treatment-related clinical findings Included significantly
Increased cholesterol* and albumin In high-dose males and all groups of
treated females, and Increased total protein In mid- and high-dose males and
all three female treatment groups. Nean absolute and relative liver weights
04420 V-16 11/12/91
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were significantly Increased 1n all treatment groups of both sexes, accom-
panied by a dose-related Increase In gross pathologic findings, Including
enlarged, dark, mottled, prominent retlcular pattern or pale areas.
An Increase 1n the Incidence and severity of centMlobular hepatocellu-
lar hypertrophy was seen 1n all female treatment groups and 1n high-dose
males. Individual cell necrosis was observed In high-dose females. No
significant changes In mean absolute or relative kidney weights were found,
but gross examination revealed a treatment-related Increase In the occur-
rence of granular, pitted, rough, mottled, or small appearance. Nephro-
pathy, Increased Incidence and severity of renal tubular dilatation, epithe-
lial hyperplasla of the collecting ducts, slight hyperplasla of the transi-
tional epithelium In the renal pelvis, and renal tubule mlcroconcretlons
were observed In the kidneys of all treated females. An Increase In the
Incidence of renal tubule regeneration was seen 1n high-dose males. Mean
absolute and relative ovary weight decreases 1n mid- and high-dose females
'were accompanied by a slight Increase In Incidence and degree of Inactivity,
and fewer and smaller corpora lutea 1n the high-dose group. A small
Increase In the number of grossly observed ocular opacities was observed,
but opthalmoscoplc examination revealed no treatment-related ocular lesions.
Based on liver and kidney changes and deaths In females the LOAEL was deter-
mined to be 100 mg/kg/day; no NOAEL could be determined since 100 mg/kg/day
was the lowest dose given.
Anthracene. In a chronic bloassay for carcinogenic effects (Schmahl,
1955), a group of 28 BDI and BDIII rats received anthracene In the diet,
starting when the rats were ~100 days old. The dally dosage was 5-15
04420 V-17 03/20/91
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mg/rat, and the experiment was terminated when a total dose of 4.5 g/rat was
achieved on the 550th experimental day. The rats were observed until they
died, with some living more than 1000 days. No treatment-related effects on
Hfespan or gross and histologic appearance of tissues were observed. Body
weights were not mentioned, and hematologlc parameters were not measured.
No chronic LOAEL could be determined from this study.
In a 90-day subchronlc toxldty study, the U.S. EPA (1989a) administered
anthracene to groups of 20 male and female CD-I (ICR)BR mice by gavage.
Dose levels were 0, 250, 500 and 1000 mg/kg/day. Criteria evaluated for
»
compound-related effects were mortality, clinical signs, body weights, food
consumption, opthalmology, hematology, clinical chemistry, organ weights,
organ-to-body weight ratios, gross pathology and hlstopathology. No
treatment-related effects were noted; therefore, the NOAEL determined from
this study Is 1000 mg/kg/day.
•
BenzTalanthracene. Pertinent data regarding the subchronlc and
chronic oral toxldty of benz[a]anthracene could not be located In the
available literature.
Benzofalpyrene. Aplastlc anemia, and ultimately death, have- been
linked to subchronlc oral exposures to benzo[a]pyrene (Robinson et al.,
1975). Strains of mice used In the experiment had been classified as either
"responsive" or "nonresponslve", based on the strain's susceptibility to
Induction of cytochrome P-450 and associated -enzymes by PAHs. Treatment
groups, consisting of 30 animals/strain, were fed a laboratory diet ad
libitum that had been soaked 1n corn oil containing benzo[a]pyrene; the
04420 V-18 . 03/20/91
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estimated oral dose was -120 mg/kg/day. Responsive and nonresponslve
control groups, each consisting of 30 animals, were fed the same diet that
had been soaked In unadulterated corn oil. In three responsive strains
(C57B1/6, C3H/HeN, BALB/cAnN) fed benzo[ajpyrene, the following numbers of
mice/group died over a 180-day period: 2/30, 3/30 and 1/30, respectively.
This was by comparison with one iflouse 1n the responsive strain (C57B1/6)
control group. Among the nonresponslve strains (AKR/N, DBA/2), all of the
mice In the treatment groups died with at least half the deaths occurring
within 15 days. Only two mice died In the nonresponslve (DBA/2) control
group over the same period of time. The high mortality among the treated
nonresponslve mice was attributed to pancytopenla, which led to death from
hemorrhaglng or overwhelming Infection. The nonresponslve benzo[a]pyrene-
treated mice also experienced a significant Increase In relative Hver-to-
body weight ratios.
Benzofblfluoranthene. Pertinent data regarding the subchronlc and
chronic oral toxldty of benzo[b]fluoranthene could not be located 1n the
available literature.
Benzo[k]f1uoranthene. Pertinent data regarding the subchronlc and
chronic oral toxldty of benzo[k]fluoranthene could not be located 1n the
available literature.
Benzorg.h.Hperylene. Pertinent data regarding the subchronlc and
chronic oral toxldty of benzo[g,h,1]perylene could not be located In the
available literature.
04420 V-19 03/20/91
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Chrysene. Pertinent data regarding the subchronlc and chronic oral
toxldty of chrysene could not be located 1n the available literature.
D1benz[a.h]anthracene. Pertinent data regarding the subchronlc and
chronic oral toxldty of d1benz[a,h]anthracene could not be located In the
available literature.
Fluoranthene. The U.S. EPA (1988) evaluated the oral toxldty of
fluoranthene 1n a subchronlc bloassay. For 13 weeks male and female CD-I
mice (20/sex/group) received either 0, 125, 250 or 500 mg/kg/day of fluoran-
thene dissolved 1n corn oil. Baseline blood evaluations were determined
during the first week of study from an additional 30 animals/sex/group.
Body weights, food consumption and clinical signs of toxldty were monitored
at regular Intervals during the experimental period. At the end of the
study period the animals were sacrificed, submitted for autopsy, and
hematologlc and serum chemistry evaluations were performed.
All treatment groups exhibited Increased salivation (never exceeding 10%
of the population of any treatment group). There were dose-dependent
Increases 1n pigment accumulation In the liver and mild nephropathy charac-
terized by the presence of multiple fod of tubular regeneration. A small.
but statistically significant (p<0.01). Increase In relative liver weight
was observed In mice receiving 125 mg/kg/day of fluoranthene. Statistically
significant (p<0.05) changes 1n mice receiving 250 mg/kg/day Included
Increases 1n SGPT and absolute and relative liver weights, as well as
decreases 1n packed cell volume and red blood cell numbers (females only)
and albumin/globulin ratios. Statistically significant (p<0.05) observa-
04420 V-20 03/20/91
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tlons In mice receiving 500 mg/kg/day Included Increases 1n SGPT, serum
globulin and absolute and relative liver weights, as well as decreases 1n
packed cell volume (females only) and albumin/globulin ratios. Since there
was no dose-related Increase 1n clinical signs (I.e., salivation) and the
changes In kidney and liver hlstopathology observed at 125 mg/kg/day were
not considered adverse by the U.S. EPA (1991a), this dose level 1s
considered the NOAEL. Based on hematologlc alterations, Increased SGPT
levels, and changes In kidney and liver hlstopathology 1n animals receiving
250 mg/kg/day, the U.S. EPA (1991a) considered this dose level as the LOAEL.
Fluorene. Wilson et al. (1947) provided anecdotal reports on gross
and hlstologlc appearance of organs of rats exposed to fluorene 1n the diet
as part of an oncology study. They observed significant decreases 1n the
rate of growth among albino rats consuming 0.5 and 1.054 fluorene In the diet
for 105 days. Furthermore, liver weights were Increased In rats receiving
>0.25X fluorene, spleen weights were decreased In all treated animals and
testes weights were decreased 1n the high-dose rats. Neither numbers of
/
animals nor any organ weights were published.
The U.S. EPA (1989a) conducted a subchronlc toxlclty study 1n which CD-I
mice (25/sex/group) were exposed for 13 weeks via gavage to 0, 125, 250 or
500 mg/kg/day fluorene suspended 1n corn oil. Parameters used to assess
toxlclty Included food Intake, body weight, clinical observations, hemato-
logy and serum chemistry and gross and hlstopathologlc examinations.
Increased salivation, hypoact1v1ty and urine-wet abdomens In males were
observed In all treated animals. The percentage of mice exhibiting hypo-
activity was dose-related. In mice exposed at 500 mg/kg/day, labored
04420 V-21 11/12/91
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respiration, ptosls (drooping eyelids) and unkempt appearance were also
observed. A significant decrease 1n erythrocyte count and hematocrlt was
observed 1n females treated with 250 mg/kg/day fluorene and 1n males and
females exposed to 500 mg/kg/day. Decreased hemoglobin concentration and
Increased total serum blUrubln levels were also observed 1n the 500
mg/kg/day group. Decreases In ery'throcyte count, hematocrH and hemoglobin
concentration were all observed at 125 mg/kg. These effects, although
apparently dose dependent, were not statistically significant at 125 mg/kg
by comparison with controls. A significant decreasing trend 1n BUN and a
significant Increasing trend 1n total serum blUrubln were observed for
high-dosed males and females. A dose-related Increase 1n relative liver
weight was observed 1n treated mice; a significant Increase In absolute
liver weight was also observed In the mice treated with >250 mg/kg/day
fluorene. A significant Increase 1n absolute and relative spleen and kidney
weight was observed 1n males and females exposed to 500 mg/kg/day and males
at 500 mg/kg/day, respectively. Increases 1n the absolute and relative
liver and spleen weights 1n the high-dose males and females were accompanied
by Increased amounts of hemoslderln In the spleen and Increased numbers of
Kupffer cells of the liver. No other hlstopathologlc lesions were
observed. Using the data from U.S. EPA (1989a), the U.S. EPA (1991a)
Identified a LOAEL of 250 mg/kg/day for hematologic effects; the
corresponding NOAEL 1s 125 mg/kg/day.
IndenoM,2.3-cdlpyrene. Pertinent data regarding the subchronlc and
chronic oral toxIcHy of 1ndeno[l,2,3-cd]pyrene could not be located 1n the
available literature.
04420 V-22 10/24/91
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Naphthalene. Shopp et al. (1984) conducted a 14-day and a 90-day
study on groups of male and female CD-I mice administered naphthalene 1n
corn oil by gavage. In the 14-day study, six groups of male and female mice
(40-112/group) were given doses of 0, 27, 53 or 267 mg/kg/day; the highest
dose was one-half the LD5Q for male mice. Male mice demonstrated lower
survival rates than females, apparently due to the aggressive behavior of
group-housed male mice; however, the mortality 1n the high-dose groups of
both male and female mice was 5-10% higher than In the control groups.
There was a significant decrease (7-13%) 1n body weight 1n male and female
mice receiving the high flose. The high-dose males exhibited a 30% decrease
1n thymus weight, while females exhibited a decrease In spleen weight and an
Increase 1n lung weight. Gross pathology but not hlstopathology was
performed. No biologically relevant changes were noted 1n treated animals
for the following measures: hematology, clinical chemistry, hexabarbltal
sleeping time, or Immune function (humoral Immune response, lymphocyte
responsiveness, popliteal lymph node response, and bone marrow function).
For the 90-day study, five groups of 112 male and 112 female mice were
given doses of 0, 5.3, 53 or 133 mg/kg/day. A high mortality was seen among
all groups of male mice, but appeared to be due to the aggressive behavior
of group-housed male mice.
No significant effects on body weight were noted for males or females.
A significant decrease In the absolute weight of the brain, spleen and liver
was noted for females, receiving 133 mg/kg; however, organ-to-body weight
ratios were significantly different only for the spleen. Of the changes
noted In the clinical chemistry data, the Increase 1n blood protein content
1n males and females receiving 53 or 133 mg/kg, the decrease In BUN In all
04420 V-23 05/14/91
-------
treated female groups, and the decrease In calcium 1on concentrations In
males receiving 53 or 133 mg/kg were considered to be treatment related. No
significant changes were noted 1n hematology, 1n HFO activity, or 1n Immune
function for either sex. Hlstopathology data were not presented and H 1s
not known 1f naphthalene caused bronchlolar lesions.
In a subchronlc oral toxlclty study performed for the NTP (1980b),
naphthalene 1n corn oil was administered by gavage to male and female F344
rats (10/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 mg/kg/day, 5
days/week for 13 weeks. 'At 400 mg/kg, two males died during the first week;
this treatment dose caused diarrhea, lethargy, hunched posture and roughened
halrcoats 1n rats of both sexes. A significant (I.e., >10%) decrease 1n
body weight gain was observed among males and females at 200 and 400 mg/kg
and In females at 100 mg/kg. Food consumption was not affected.
All the rats In the study were necropsled and comprehensive hlstopatho-
loglc examinations were performed on rats from the 0 and 400 mg/kg groups.
Hlstopathologlc examinations of the kidneys and thymus were performed on
rats from the 200 mg/kg group (according to the hlstopathology tables; the
100 mg/kg group according to the text). The authors stated that lesions of
the kidney 1n males and thymus 1n females of the 400 mg/kg group may have
been compound-Induced, and that no eye lesions were found. The Incidences
of lesions of kidney and thymus were, however, very low. The renal lesions,
which did not occur 1n females, were observed at Incidences of 0/10 In
controls, 2/10 In the 200 mg/kg group and 1/10 In the 400 mg/kg group.
These renal lesions consisted of focal cortical lymphocytlc Infiltration or
focal tubular regeneration 1n the two 200 mg/kg males and diffuse tubular
degeneration 1n the one 400 mg/kg male. Lymphold depletion of the thymus
04420 V-24 05/14/91
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occurred 1n 2/10 females of the 400 mg/kg group, In none of the control or
200 mg/kg females, and In none of the males of these groups. Hematologlc
analyses revealed marginal decreases 1n hemoglobin and hematocMt 1n males
and females of the 400 mg/kg group, and a moderate Increase In the number of
mature neutrophlls and a decrease In the number of lymphocytes 1n males of
the 400 mg/kg group, relative to controls. No hematologlc changes were
observed at the lower dosages.
In a similar study, naphthalene was administered 1n corn oil by gavage
at 0, 12.5, 25, 50, 100 or 200 mg/kg/day, 5 days/week, to B6C3F1 mice
(10/sex/dose) for 13 weeks (NTP, 1980a). Seven mice (three males and two
females of the 200 mg/kg group, one female of the 25 mg/kg group and one
control male) died during the second, third and fourth weeks of the study
from gavage trauma or accident. Transient signs of toxlclty (lethargy,
rough halrcoats and decreased food consumption) occurred at weeks 3-5 1n the
200 mg/kg groups. All treated groups of male mice gained somewhat more
•
'weight than did control males. Dose-related decreases In body weight gain
were se'en 1n females, but were not statistically significant. All the mice
were necropsled and comprehensive hlstopathologlc examinations were
performed on the mice from the 0 and 200 mg/kg groups. No compound-related
lesions were observed 1n any organs, Including kidneys, thymus, eyes and
lungs. Hematologlc analyses, performed on all groups, revealed no
significant, compound-related changes.
FUzhugh and Buschke (1949) noted the formation of cataracts, within 3
weeks of treatment, In rats fed diets containing 2% naphthalene or one of
several naphthalene derivatives. The effects of pigmentation on cataract
04420 V-25 03/20/91
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formation 1n rats and rabbits have been summarized by van Heynlngen (1979):
Albino rats do not readily convert naphthalene to 1,2-dlhydroxynaphthalene,
which auto-ox1d1zes to form 1,2-naphthoqulnone (the naphthalene metabolite
known to bind to the lens of the eye), possibly because polyphenol oxldase
1s found only In plgmented tissues. Secondary effects occur In the retinas
of both albino and plgmented rats. For rabbits, pigmentation Is not as
Important as a modifier of toxldty. Rather, the depletion of antloxldants
1s a critical step In rabbits since the reserve of ascorbic add and other
antloxldants Is considerably less than 1n the rat.
The critical nature of the depletion of antloxldants 1n rabbits, as
compared with the Importance of pigmentation, was demonstrated by van
Heynlngen and P1r1e (1967) In a gavage study In which naphthalene (1
mg/kg/day) was administered to Dutch and two strains of albino rabbits. In
more than half of the treated rabbits, lens opacities and degeneration of
the retina were observed. This occurred concomltantly with a depletion of
ascorbic acid 1n the aqueous and vitreous humours. Some of those rabbits
that received 10 or more doses of naphthalene showed a general yellowing of
the eye fluids and yellow or brown cortical areas 1n the cataractous lens,
suggesting the presence of 1,2-naphthaqulnone and 1,2-dlhydroxynaphthalene,
respectively. Considerable variation exists among rabbits 1n their response
to naphthalene. Cataract formation 1n .rabbits was not noted following
topical application of a 10X solution of naphthalene In oil or l.p.
Injection of 500 mg/day for 60 days (Ghettl and Mar1an1, 1956).
The effect of pigmentation on the development of cataracts 1n rats
exposed to naphthalene was substantiated by Koch et al. (1976) using five
04420 V-26 03/20/91
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strains of Rattus norveglcus of different pigmentation. Groups of six
animals of each strain were gavaged with either 0 or 1000 mg/kg naphthalene,
dissolved 1n liquid parafflne, every second day for 75 days. No opacities
or lens abnormalities were observed 1n any of the control groups. All
animals of the plgmented strains (E3, BOE, OA) developed zonular cataracts
between 16 and 28 days. Among the albino strains (Wlstar, Sprague-Dawley)
only some of the animals developed changes 1n the eyes. These changes were
less pronounced In the albino strains and occurred after longer latencies of
32-61 days.
Cataract formation 1n C57B1/6J and DBA/2N mice was evaluated by Sh1ch1
et al. (1980) and Sh1ch1 and Nebert (1982). The C57B1/6J mice are
"responsive" to the Induction of AHH activity while the DBA/2N mice are
"nonresponslve" to the Induction of AHH activity (primarily P..-450 enzyme
activity that 1s believed to be Involved 1n the tox1f1cat1on of PAHs).
Groups of 15 mice were fed laboratory chow ad libitum that had been soaked
for at least 24 hours 1n corn oil containing 5 or 10 mg/ma naphthalene.
The feeding regimen was continued for 60 days. Dally Ingestlon was not
calculated by the authors. A concomitant dose of B-naphthoflavone was given
twice weekly as an Inducer of AHH activity. A 6.7X Incidence 1n cataract
formation was observed In C57B1/6J mice at each dose. In addition to
cataract formation, tlsssue degeneration 1n the chorold, ciliary body, and
1r1s occurred. In support of the theory that the mechanism of naphthalene-
Induced cataract formation Involves Us metabolism by P,-450 enzymes to
toxic Intermediates with subsequent binding to lens tissue, no cataracts
were observed 1n DBA/2N mice.
04420 V-27 10/08/91
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Phenanthrene. Pertinent data regarding the subchronlc and chronic
oral toxldty of phenanthrene could not be located 1n the available litera-
ture.
Pyrene. Pyrene was fed at a concentration of 2000 mg/kg diet to young
male rats for 100 days. An Inhibition of growth was observed, which was
reversible upon addition of cystlne or methlonlne. The authors noted that
livers of treated animals (Including rats consuming benzo[a]pyrene or
3-methylcholanthrene 1n the diet) were enlarged and had a fatty appearance
(White and White, 1939).
The U.S. EPA (1989b) conducted a 90-day subchronlc toxldty study 1n
which groups of male and female CD-I mice (20/sex/group) were gavaged with
either 0, 75, 125 or 250 mg/kg/day pyrene 1n corn oil for 13 weeks.
Parameters used to assess toxldty Included body and organ weights, food
consumption, mortality, hematology and serum chemistry and gross and
hlstopathology. Statistically significant (p<0.01) decreases In absolute
kidney weights were observed In males of all treatment groups and 1n females
receiving 250 mg/kg/day. Statistically significant (p<0.01) decreases 1n
relative liver weights were observed 1n males receiving either 125 or 250
mg/kg/day and 1n females receiving 250 mg/kg/day. Nephropathy, character-
ized by the presence of multiple foci of renal tubular regeneration, often
accompanied by Interstitial lymphocytlc Infiltrates and/or foci of Intersti-
tial flbrosls, was observed 1n 4, 1, 1 and 9 male mice 1n the control, low,
medium and high dosage groups, respectively. SlmlHar lesions were seen In
2, 3, 7 and 10 female mice 1n the 0, 75, 125 and 250 mg/kg/day treatment
groups, respectively. The kidney lesions were described as minimal or mild
04420 V-28 10/08/91
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In all Instances. Statistically significant (p<0.01) decreases 1n erythro-
cyte numbers, hematocrHs and hemoglobin levels were observed 1n male mice
receiving 250 mg/kg/day. Statistically significant (p<0.01) Increases 1n
absolute liver weights were observed 1n females receiving 250 mg/kg/day and
In relative liver weights of females receiving either 125 or 250 mg/kg/day
•
and In males receiving 250 mg/kg/day. Based on nephropathy, accompanied by
changes 1n absolute and relative kidney weights, the U.S. EPA (1991a)
1dent1- fled 125 mg/kg/day as the LOAEL; the corresponding NOAEL 1s 75
mg/kg/day.
Subchronlc and Chronic Toxldty By Other Routes
Acenaphthylene. In a study reported by Rotenberg and Hashblts (1965),
white rats were exposed to acenaphthylene dust at 0.5-1.25 mg/m3 for 4
hours/day for 4 months. After 3 weeks of exposure, a delay In weight gain
and a tendency toward decreased blood pressure were observed. Hlstopatho-
loglc examination revealed various degrees of malignancy 1n the lungs of
almost all treated rats. Focal bronchitis and per1bronchH1s with bronchlo-
*•
Hzatlon of the alveolar and metaplasia of the bronchial epithelium were
observed 1n the mildest cases. Advanced cases showed desquamatlon of the
bronchial and alveolar epithelium, paplllar growths 1n the epithelium and,
1n three rats, Isolated regions of carcinoma 1n the form of strands of
epithelial cells. Further details of this study were not provided.
In a study by Reshetyuk et al. (1970) published 1n Russian, -100 white
male rats were exposed to vapors of acenaphthylene at a concentration of
18+2.5 mg/m3, 4 hours/day, 6 exposures/week for 5 months. In exposed
rats, reflexes of the upper airways were altered and an Increase 1n the
concentration of nucleic acids In the liver was observed. H1stopatholog1c
04420 V-29 11/12/91
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examination of the lungs revealed aspedflc pneumonia as the major pathology
of Inhalation exposure to acenaphthylene. Changes observed In the lungs
Included desquamatlon of the cells In the alveolar epithelium and focal
bronchitis accompanied by hyperplasla and metaplasia of the bronchial
epithelium. No signs of malignant growth were observed 1n this study. No
.further details of this study were available. .
Anthracene. Pertinent data regarding the subchronlc and chronic non-
oral toxldty of anthracene could not be located 1n the available literature.
Benzfalanthracene. An early study (Hoch-Llgetl, 1941) demonstrated
lymphold system effects as a consequence of benz[a]anthracene Injection,
Mice received a total dose of 10 mg In weekly s.c. treatments for 40 weeks.
Lymph glands removed at weekly Intervals showed treatment-related Increases.
In retlculum cells and Iron accumulation.
Benzo[a]pyrene. Male white carneau pigeons were given weekly 1njec-r
tlons 1n the pectoral muscle of 0.1, 10 or 100 mg/kg benzo[a]pyrene In corn
oil {Revls et al., 1984). Controls were either not treated or were Injected
with corn oil. Four pigeons/group were cannulated at 3 months and at 6
months for recording of blood pressure and sampling of blood for determina-
tion of plasma cholesterol and Upoprotelns. Atherosclerotic plaques were
counted and sizes measured. By 6 months pigeons treated with 10 or 100
mg/kg/week benzo[a]pyrene were observed to have Increased LDL protein and
cholesterol (p<0.01), HDL protein and cholesterol (p<0.01) and plasma
cholesterol (p<0.05). This was by comparison with corn oil-treated
controls. Benzo[a]pyrene treatment did not produce changes 1n various
04420 V-30 - 03/20/91
-------
cardiovascular parameters Including systolic and diastollc blood pressure,
arterial pulse, left or right ventricular or central venous pressure, heart
rate or relative heart weight.
Among pigeons treated with benzo[a]pyrene coronary artery plaques
ranging In size from 0.6-0.85 mm were observed. Incidence of coronary
artery plaques was 5/24 for benzo[a]pyrene-treated birds by comparison with
1/24 for combined corn oil and untreated controls. It was noted 1n this
study that benzo[e]pyrene administered 1n the same fashion as was
benzo[a]pyrene did not Induce plaque formation.
Penn and Snyder (1988) tested benzo[a]pyrene, anthracene and dlbenz-
[a,h]anthracene to determine whether artherosclerotlc plaque formation 1s
related to mutagen1c1ty or carcinogenic potency 1n chickens. In their study
male White Leghorn chickens received weekly Intramuscular Injections with
one of the test compounds for 16 weeks. The dosage and number of each group
varied as follows: benzo[a]pyrene at 40 mg/kg with 6 chickens, anthracene
at 20 mg/kg with 6 chickens, and d1benz[a,h]anthracene at 20 mg/kg with 5
chickens. At the end of the Injection period, the cockerels were killed and
the aortas Isolated. The number of plaque-containing aorta segments/chicken
was 6.8, 4.3 and 6.2 for the benzo[a]pyrene-, anthracene- and
d1benz[a,h]anthracene-treated groups, respectively. The largest plaque
volumes were noted 1n the animals treated with d1benz[a,h]anthracene.
Benzofblfluoranthene. Pertinent data regarding the subchronlc and
chronic nonoral toxldty of benzo[b]fluoranthene could not be located 1n the
available literature.
04420 V-31 10/24/91
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Benzo[k]fluoranthene. Pertinent data regarding the subchronlc and
chronic nonoral toxldty of benzo[k]fluoranthene could not be located 1n the
available literature.
Benzorq.h.npervlene. Pertinent data regarding the subchronlc and
«
chronic nonoral toxldty of benzo[g,h,1]perylene could not be located 1n the
available literature.
Chrvsene. Pertinent data regarding the subchronlc and chronic nonoral
^^^•Mirf^^^^^^H^H ^
toxldty of chrysene could not be located 1n the available literature.
Dlbenzfa.hlanthracene. Chronic exposure to d1benz[a,h]anthracene has
been associated with gross changes 1n the lymphold system (Hoch-L1get1,
1941). Weekly s.c. Injections of d1benz[a,h]anthracene (0.5 ml of a 0.05X
colloidal solution 1n IX gelatine) were given to a total of 40 albino male
and female mice for 40 weeks. The lymph glands were removed for examination
from 2 mice/week over the study period. Splenectomles were ddne during
weeks 21-30. Among the effects noted were an Increase 1n retlculum (stem)
cells, accumulation of Iron, reduced lymphold cells and dilated lymph
sinuses. Reduction of lymphold cells was more pronounced among d1benz[a,h]-
anthracene-treated mice by comparison with anthracene and benz[a]anthracene
treated animals. Moreover, the weight of the spleens 1n the treated mice
were significantly lower than the spleen weights In the controls.
H1stolog1c examination of spleens from d1benz[a,h]anthracene-treated
animals showed d1m1n1shment of lymphold and retlcular elements. Livers were
pale and soft and showed evidence of fatty degeneration and deposition of
04420 V-32 10/08/91
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Iron In Kupfer cells. Adrenals were marked by Iron deposition In the
cortical zone retlculaMs, and kidneys showed signs of degeneration of
tubules and Malph1g1an bodies. There was some degeneration of spermatogenlc
cells and two female mice had "many big corpora lutea 1n the ovaries"
(Hoch-L1get1, 1941).
Lasn1tzk1 and Woodhouse (1944) conducted a more detailed study of the
effects on the lymphatic system of long-term exposure to d1benz[a,h]anthra-
cene. Subcutaneous Injections (0.278 mg/1nject1on) were given to male rats
5 times weekly for several weeks. The lymph nodes 1n the treated rats
underwent hemolymphatic changes, Including the appearance of extravascular
red blood cells In the lymph spaces and large plgmented cells.
Malmgren et al. (1952) reported that 2- to 6-month-old homozygous strain
C mice (3-4 mice/group) receiving three subcutaneous Injections of either
50, 100 or 400 mg/kg/1nject1on of d1benz[a,h]anthracene spread evenly over a
12-day period, which Included the 5 days following antigen challenge, had
/
reduced serum antibody levels.
Fluoranthene. Limited data are available concerning the toxic effects
of fluoranthene produced by repeated administration. These consist of
reports of mortality produced 1n mice by repeated dermal application or
subcutaneous Injection.
Shear (1938) administered four doses, each consisting of 10 mg of
fluoranthene In glycerol, by subcutaneous Injection to strain A mice. Six
of 14 mice survived for 18 months.
04420 V-33 10/08/91
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Barry et al. (1935) applied a 0.3X solution of fluoranthene In benzene
twice weekly to the Interscapular region of mice. Mortality ranged from
60-70% after 6 months and 70-90X after 1 year.
Hoffman et al. (1972) applied 50 yl of a 1% fluofanthene solution 1n
acetone 3 times weekly for 12 months to the backs of Swiss albino mice. No
mortality had resulted after 15 months.
Fluorene. Pertinent data regarding the subchronlc and chronic nonoral
toxlclty of fluorene could not be located 1n the available literature.
Indeno[1.2.3-cdlpyrene. Pertinent data regarding the subchronlc and
chronic nonoral toxlclty of 1ndeno[l,2,3-cd]pyrene could not be located 1n
the available literature.
Naphthalene. A chronic Inhalation study of naphthalene 1n mice has
recently been conducted by the National Toxicology Program (NTP, 1991). The
f
exposure phase of the 2-year Inhalation study has been completed, but the
Mstopathology data and the final report are not yet available. No other
chronic toxlclty data were found.
Phenanthrene. Pertinent data regarding the subchronlc and chronic
nonoral toxlclty of phenanthrene could not be located In the available
literature. .
Pyrene. Pertinent data regarding the subchronlc and chronic nonoral
toxlclty of pyrene could not be located In the available literature.
04420 V-34 10/08/91
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Target Organ ToxUUy
The target, or preferred, organs for the toxic action of PAHs tend to be
diverse. This 1s a consequence of two propensities of the PAHs. First,
there 1s the preferential association for normally proliferating tissues
such as bone marrow, lymphold organs, gonads and Intestinal epithelium (U.S.
EPA, 1980d). Second, PAHs are distributed extensively throughout the body 1n
general.
The toxldty of various PAHs seems to center on the hematopoletlc and
lymphold systems of different animal species. Robinson et al. (1975)
observed hemopoletlc and lymphold effects of benzo[a]pyrene 1n mice (of
certain strains), and Lasnltzk! and Woodhouse (1944) found dlbenz-
[a,h]anthracene to affect the lymph nodes of rats. While the hematopoletlc
and lymphold systems were also shown to be affected 1n dogs treated with
naphthalene (Zuelzer and Apt, 1949), the two major target organs for this
PAH are nondllated bronchlolar epithelial cells (Clara cells) and eye
tissue. Cataracts have been shown to develop 1n several species of
laboratory animals following exposure to naphthalene by routes other than
Inhalation (FUzhugh and Buschke, 1949; Shlchl et al., 1980; Sh1ch1 and
Nebert, 1982).
Immunotoxlc effects as a consequence of PAH exposure have been studied
by a number of researchers. Malmgren et al. (1952) first reported reduced
hemolysln tHres to sheep red blood cells (SRBC) 1n mice exposed to dlbenz-
[a,h]anthracene and benz[a]anthracene. Subsequently, 1t was reported that
exposure to carcinogenic levels of benzo[a]pyrene resulted 1n a depressed
Immune response to SRBC that persisted for 90 days (Stjernsward, 1966,
04420 V-35 10/08/91
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1969). Both humoral and cell mediated immunity have been shown to be
k/k
depressed 1n C3H/Anf(H-2 ' ) mice Injected with 150 wg benzo[a]pyrene/kg
bw during days 11-17 of gestation. Production of plaque-forming colonies
against SRBC (as measured by the Jerne plaque assay) was depressed severely
from 1-4 weeks of age and persisted from 5-18 months of age. An Ijn vitro
graft vs. host assay also showed 1mmunodepress1ve effects. Lymphocytes from
Immature mice exposed to benzo[a]pyrene (1-4 weeks) were severely limited 1n
their ability to show mixed lymphocyte responses (Urso and Gengozlan, 1984).
B6C3F1 mice were treated s.c. dally for 14 days with 5>, 20 or 40 mg/kg
benzo[a]pyrene (Blanton et al., 1986). Polyclonal antibody responses to
I1popolysacchar1de (IPS) and purified protein derivative (PPD) measured by
Jerne plaque assay were decreased 50-66% after 7 days exposure. Exposure of
B6C3F1 mice to s.c. Injections of 40 mg/kg for 7 or 14 days resulted 1n a 73
and 98X suppression of the T-cell-dependent antibody response, respectively,
as measured 1n a similar fashion.
\
s
Similar results were reported by Dean et al. (1983) for female B6C3F1
mice Injected s.c. for 14 days with 5, 20 or 40 mg/kg bw. Exposure to
benzo[a]pyrene resulted 1n decreased numbers of IgM and IgG plaque-forming
cells In response to SRBC antigen and reduction 1n IgM plaque-forming cells
1n response to IPS. In this assay cell-mediated Immunity (as measured by
delayed cutaneous hypersensUlvlty to keyhole limpet hemocyanln), allograft
rejection and susceptibility to LIsteMa monocytogenes was unaffected 1n
benzo[a]pyrene treated*mice.
04420 V-36 10/08/91
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A comparative study of the ability of PAHs to depress antibody response
was undertaken by White et al. (1985). PAHs were administered s.c. 1n
either single or multiple doses to B6C3F1 and DBA/2 mice. IgM response to
SRBC was measured 1n a hemolytlc plaque assay. Single exposures of B6C3F1
mice to 1 mmol/kg d1benz[a,h]anthracene and benzo[a]pyrene resulted 1n
depression of antibody response. Fourteen days of exposure to a dally dose
of 160 ymol/kg of the following PAHs also resulted 1n suppression (-60%)
of the IgM response: benz[a]anthracene, d1benz[a,h]anthracene and
benzo[a]pyrene. Neither anthracene nor chrysene exposure significantly
affected response. Immunosuppresslon by benzo[a]pyrene was observed to a
greater extent 1n DBA/2 mice. The B6C3F1 mouse strain Is highly Indudble
for aryl hydrocarbon hydroxylase; the DBA/2 mouse strain 1s not.
The Immunocytotoxlc effects of benzo[a]pyrene were studied by Wojdanl et
al. (1984). Elght-week-old, Inbred C3H/FCUM and C57B1/6CUM mice In groups
of six animals were Injected 1.p. with P-815 tumor target cells. This was
followed 10 days later by 1.p. Injection with either 0, 0.5, 5 or 50 mg/kg
/
bw of benzo[a]pyrene In corn oil. After 24 hours splenic lymphocytes and
peritoneal exudate lymphocytes were collected and assayed for target cell
binding and target cell killing effects. A consistently decreasing rela-
tionship was noted between the dose of benzo[a]pyrene and both the binding
and killing of target cells for splenic and peritoneal lymphocytes. At the
two highest* dosages of benzo[a]pyrene (5 and 50 mg/kg bw), significant
decreases 1n the percent of lymphocytes binding to target cells or killing
target cells existed as compared with the controls. This study also
compared lymphocyte function of animals treated with 3-methylcholanthrene
04420 V-37 10/08/91
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and the weak carcinogen benzo[e]pyrene. Lymphocytes from benzo[e]pyrene-
treated animals were essentially similar to those from control animals
regarding target cell binding and cytotoxlc effects. The authors Indicated
that lymphocyte mediated Immunity may be Inhibited by PAHs and that this
Immunosuppressent effect can contribute to their carc1nogen1c1ty.
The effect of PAH exposure on Interferon production In vivo was studied
by Griffin et al. (1986). Female C3H mice were treated l.p. with 0.046,
0.46 or 4.6 mg benzo[a]pyrene 1n corn oil. At 12, 24, 48, 72 or 120 hours
animals were Injected with Sendal virus to Induce Interferon production.
Eight hours after the virus was Introduced mice were bled by cardiac punc-
ture and serum was tHred for Interferon by a cytopathlc effect Inhibition
assay 1n mouse L929 cells. Mice receiving 4.6 mg (180 mg/kg bw) were
significantly depressed In their ability to produce Interferon at 12, 48 and
120 hours after benzo[a]pyrene treatment. At the 48-hour challenge mice
receiving the lower 0.46 mg dose were also significantly Impaired with
respect to their Interferon production.
Carc1noqen1c1ty. Oral
Acenaphthylene. Pertinent data regarding the oral carc1nogen1c1ty of
acenaphthylene could not be located 1n the available literature. In an
abstract Knobloch et al. (1969) reported on the effects associated with
Ingestlon of acenaphthylene. An oral dose of 0.6 g/kg bw of acenaphthylene
1n olive oil administered for 40 days to a group of seven rats yielded the
following physiologic effects: "considerable" body weight loss, changes 1n
the peripheral blood pattern, changes 1n renal function, and Increased serum
amlnotransferase activities. No carcinogenic effects were reported; the
mice were dosed for a short period of time.
04420 V-38 10/08/91
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Anthracene. Studies of orally administered anthracene have been
negative for carcinogenic effects. Druckrey and Schmahl (1955) administered
a diet containing anthracene 1n oil, 6 days/week to 28 BOI or BDIII rats of
unspecified sex for a period of 78 weeks. The total dose was 4.5 g
anthracene/rat. No tumors as a consequence of treatment were found to have
•
developed. Similarly, 1n an unpublished report, Schma'hl and Relter (n.d.)
administered a total dose of 4.4 g of anthracene orally to 31 rats during a
33-month study. Again, no tumors were reported to have occurred.
Benzfalanthracene. Klein (1963) 1n a study of ma-le B6AF1/J mice
provides evidence of the carcinogenic potential of Ingested benz[a]anthra-
cene. A 3X solution of the compound 1n Methocel-Aerosol O.T. was admin-
istered by gavage at the rate of 3 doses/week for 5 weeks. Control animals
received oral doses of Methocel-Aerosol O.T. alone. After 340-440 days and
at 547-600 days, the animals were assessed for tumor development. The Inci-
dence of pulmonary adenomas and hepatomas was Increased at both assessments;
the hepatoma Incidences at 547 -days were higher than at 437 days. No
/
statistical treatment of the data was reported. The findings are summarized
1n Table V-l.
The Klein (1963) paper cites an earlier study by White and Eschenbrenner
(1945) wherein 2/6 rats receiving benz[a]anthracene In the diet developed
multiple hepatomas.
A single gavage dose of 0.5 mg benz[a]anthracene 1n mineral oil produced
no tumors 1n 13 mice after 16 months. Multiple gavage administration, 8 or
16 treatments at 3-7 day Intervals resulted 1n forestomach paplllomas 1n
04420 V-39 10/08/91
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TABLE V-l
Oral Cardnogenlclty Testing of Benz[a]anthracene Administered by Gavage
to Male B6AF1 Mice*
Duration
Administered Doseb of Study
(days)
0.5 ml of vehicle 444
0.5 ml 437
3% solution
0.5 ml of vehicle 547
0.5 ml 547
3X solution
Target
Organ
lung
liver
lung
liver
lung
liver
lung
liver
Tumor
Type
adenoma
hepatoma
adenoma
hepatoma
adenoma
hepatoma
adenoma
hepatoma
Tumor
Incidence
(X)
10/38
0/38
37/39
18/39
7/20
2/20
19/20
20/20
(26)
(0)
(95)
(46)
(35)
(10)
(95)
(100)
aSource: Adapted from Klein, 1963
bBenz[a]anthracene In Methocel-Aerosol or vehicle only.
treated 15 times (3 treatments/week for 5 weeks).
All animals were
04420
V-40
05/14/91
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2/27 treated mice compared with 0/16 mineral oil-treated controls (Bock and
King, 1959).
Benzofalpyrene. A relationship between the 1ngest1on of benzo[a]-
pyrene and the development of tumors has been documented 1n several studies
1n mice. Both benign and malignant tumors of the forestomach as a conse-
quence of oral benzo[a]pyrene treatment have been reported by a number of
authors (Hartwell, 1951; Shublk and Hartwell, 1957, 1969; Thompson and Co.,
1971; Tracor/JUco, 1973a,b; Wattenberg, 1972, 1974).
*•
Berenblum and Haran (1955) examined tumor Induction 1n the forestomach
of male C3H and Swiss mice using a number of carcinogenic substances,
Including benzo[a]pyrene. Mice were starved for 18 hours prior to
treatment, which consisted of 0.3 ma of a 0.5X benzo[a]pyrene solution 1n
PEG-400 administered by stomach tube on a weekly basis. No concurrent
controls were reported. In one part of this study, treatment was for 30
weeks to C3H mice, resulting 1n the formation of one papllloma and 16
carcinomas of the forestomach. In a second experiment, Swiss mice were fed
only milk and water for 3 days prior to the customary 18-hour fasting period
to eliminate the effects of undigested food 1n the stomach at the time of
dosing. Under these conditions, 85X (17/20) of the animals had tumors of
the forestomach. In neither experiment was any tumor of the glandular
mucosa of the stomach found.
As. part of an experiment Investigating tumor promotion by dtrus oils,
albino mice (sex not stated) were administered a single oral gavage of
either 0, 12.5, 50 or 200 vg benzo(a)pyrene 1n polyethylene glycol 400
04420 V-41 10/08/91
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after food had been withheld overnight. The Intestinal tracts of one-half
of the surviving mice 1n each group were examined 280 days post-treatment.
The Incidences of benign forestomach tumors were 0/9, 2/10, 0/9 and 5/11 1n
the control, low-, mid- and high-dose groups, respectively (Pierce, 1961).
The remaining mice were examined upon natural death or 569 days after
treatment. The Incidences of benign forestomach tumors were 0/17, 3/17,
0/19 and 8/17 1n the control, low-, mid- and high-dose groups,
respectively. The total number of tumors found 1n each group was 0, 3, 0
and 27, respectively. No carcinomas were observed and no tumors at other
sites were noted by the authors (Field and Roe, 1965).
As part of a study on the effect of hormonal state on PAH tumor
Induction, virgin female BALB/c/Ch/Se mice were gavaged twice weekly with
almond oil solutions of benzo[a]pyrene (B1anc1f1or1 et al., 1967).
Treatment was for 15 weeks resulting 1n a total dose of 15 mg. Treatment
groups consisted of -25 each Intact and ovarlectomlzed animals receiving
benzo[a]pyrene alone or 1n conjunction with 500-1000 yg/l esterone 1n
the drinking water. No concurrent untreated controls were reported. Among
Intact animals receiving benzo[a]pyrene alone forestomach tumors (5/25),
mammary tumors (2/25) and lung tumors (no Incidence reported) were observed.
Fedorenko and Yansheva (1967) administered benzo[a]pyrene In trlethylene
glycol by gavage to the "antlum of the stomach" of CC5_ mice (sex not
specified). The authors used this solvent based on Us purported ability to
enhance the carcinogenic effects of hydrocarbons. The experimental period
was stated to be 19 months. As H was also stated that the mice were kept
until natural death, there Is some confusion as to the length of exposure
04420 V-42 10/08/91
-------
and observation periods. Gavage doses of 100, 0.01, 0.1, 1 and 10 mg/an1ma1
were given 10 times. Incidence data were not reported for solvent
controls. Tumors were found In the liver, mesentery, peritoneum and stomach
(Table V-2).
Roe et al. (1970) Investigated the use of sodium cyclamlde saccharin and
sucrose on the carcinogenic activity of benzo[a]pyrene 1n female Swiss
mice. A single gavage dose of 50 yg benzo[a]pyrene 1n polyethylene glycol
(PEG) was given and the animals were killed after 18 months. Tumor
Incidence 1s given In Table V-3.
As part of a study on the effects of caffeine 1n tumor Induction 1n
rats, Brune et al. (1981) administered 0.15 mg/kg/treatment of
benzo[a]pyrene to Sprague-Dawley rats for 2 years. Benzo[a]pyrene was
administered to three groups by 1.5X caffeine gavage and to two other groups
through diet. Untreated and caffeine gavage (5 times/week) groups were used
as controls (32 rats/sex/group). Three different gavage treatments were
administered to 32 rats/sex/group; 1n the first treatment rats were gavaged
with benzo[a]pyrene 5 times/week for a total annual dose of 39 mg/kg
benzo[a]pyrene, 1n the second rats were gavaged every third day for a total
annual dose of 18 mg/kg benzo[a]pyrene and 1n the final every ninth day for
a total annual dose of 6 mg/kg benzo[a]pyrene. Rats that died spontaneously
or were killed when moribund underwent hlstologlc examination of various
organs. The combined Incidences of benign and malignant tumors were
significantly different from controls (p<0.05, test) for the 5 times/week
and the every third day gavage groups and at p<0.10 for the every ninth day
gavage group. (For other results see Table V-4.)
04420 V-43 10/08/91
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TABLE V-2
Tumor Incidence 1n "Atrium of Stomach" Following Gavage
Administration of Benzo[a]pyrene*
Total Benzo[a]pyrene Dose (mg) Tumor Incidence
Carcinoma Papllloma
0.01 0/16 0/16
0.10 0/26 2/26
1.00 0/24 5/24
10.00 11/30 12/30
100.00 16/27 7/27
*Adapted from Fedorenko and Yarisheva, 1967
04420 V-44 10/08/91
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TABLE V-3
Incidence of Tumors Observed After a Single Gavage Treatment
of Mice with Benzo[a]pyrene 1n Polyethylene Glycol*
Tumor Incidence
Treatment Forestomach
Papnioma Carcinoma
PEG 2/65 0/65
PEG * 20/61 1/61
0.25 mg
benzo[a]
pyrene
Lung Liver Malignant
Lymphoma
15/65 5/65 3/65
18/61 9/61 0/61
*Adapted from Roe et al., 1970
04420
V-45
10/08/91
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o
*fc
4k
o
TABLE V-4
Carc1nogen1c1ty of Oral Benzo[a]pyrene 1n Sprague-Dawley Rats3
Mode of Application
Untreated control
Gavage caffeine control
Gavagec (5 times/week)
Gavagec (every 3rd day)
Gavagec (every 9th day)
Diet (5 days/week)
Diet (every 9th day)
Total
Dose B[a]P
(mg/kg)
0
0
39
18
6
39
6
Fores tomach Tumor
Benign
2/64
3/64
14/64
25/64
11/64
9/64
1/64
Incidence0
Malignant
0/64
0/64
0/64
1/64
1/64
0/64
0/64
Total Tumors
3/64
6/64
14/64
26/64
13/64
10/64
3/64
CO
10
aSource: Adapted from Brune et al., 1981
°Comb1ned Incidence for males and females. Effective number of animals was assumed to be 64.
C1.5X aqueous caffeine plus benzo[a]pyrene
-------
Two other groups of 32 rats/sex were fed benzo[a]pyrene either 5
times/week [total annual dose 39 mg/kg benzo[a]pyrene or every ninth day
[total annual dose of 6 mg/kg benzo[a]pyrene. Rats that died spontaneously
or were killed when moribund underwent hlstologlc examination of various
organs. The combined Incidences of benign and malignant tumors were
significantly different only 1n the group fe'd benzo[a]pyrene 5 times/week
(p<0.05) (see Table V-4).
Demi et a "I. (1983) dosed five groups of four female Sprague-Dawley rats
with various combinations of benzo[a]pyrene and polychlorjnated blphenyls
(PCBs) dissolved 1n olive oil by means of gastric Intubation for 12 weeks.
After 12 weeks, the livers were examined to determine the number and area of
enzyme-altered Islands. Rats treated with PCBs before Intubation with
benzo[a]pyrene and promoted afterwards with PCBs showed a 3-fold Increase 1n
the number of enzyme-altered Islands present. The total area of these
Islands was also 3 times larger than those found 1n the other test groups.
PCB or benzo[a]pyrene alone appeared to produce no significant changes, but
the pretreatment with PCB along with the administration of benzo[a]pyrene
followed by PCB caused an alteration 1n benzo[a]pyrene metabolism that led
to the formation, of enzyme-altered foci.
As part o/ t. study of Inhibition of tumor formation by organoselenlum
compounds^ CD-I mice (25/group) were gavaged with benzo[a]pyrene. Corn oil
solutions deHveMng 1 mg benzo[a]pyrene/0.2 ml were administered twice
weekly for 4 weeks. Controls were treated with corn oil only. Nice began
treatment at 9 weeks of age and were killed at 28 weeks. At this time no
control animals had developed forestomach papHlomas, whereas 85X (17/20) of
04420 V-47 10/24/91
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benzo[a]pyrene treated animals developed an average of S.3+.2.9 tumors/mouse
(El-Bayoumy, 1985).
Robinson et al. (1987) administered benzo[a]pyrene to A/J mice by gavage
as a positive control on a study of coal tar paints. Forty female mice were
gavaged with 0.25 mg benzo[a]pyrene 1n 2.2 ma 2% emulphor twice weekly for
8 weeks (total dose 6 mg). Animals were killed at 8 weeks and were examined
for lung adenomas and forestomach tumors. 8enzo[a]pyrene treatment produced
lung adenomas 1n 61% (22/36) of mice with an average of 1.42^0.40 tumors/
mouse that was significantly Increased by comparlslon with controls (29% or
11/38 Incidence, 0.32+0.09 tumors/mouse). No forestomach tumors were
observed In controls. Benzo[a]pyrene treatment resulted 1n 92% Incidence;
67% of animals had paplllomas and 61% carcinomas.
As part of a dietary carclnogenesls study, benzo[a]pyrene (1.5 mg, twice
weekly for 4 weeks) was administered by gastric Intubation to 25-30 female
ICR mice/group .(Benjamin et al., 1988). Three groups Intubated with
benzo[a]pyrene had different feeding regimens. The first two groups were
/
administered a basal diet supplemented with 20% soy sauce and ±0.05%
nitrite. The third group received only the basal diet and water. The
feeding regimens were started 1 week before the Initial gastric Intubation
and were continued throughout the experiment. After 21 weeks on study all
mice were sacrificed. The tumor Incidences of the mice 1n the three groups
were similar (91%); however, mice receiving the soy supplemented diet and
nitrite supplemented water had a significantly lower number of neoplasms per
mouse (3.4) than the other two groups (5.2 and 4.0 for the group receiving
no supplementation and the group receiving only the soy supplemented diet,
respectively). In a follow-up experiment, a fourth group was added; this
04420 V-48 10/24/91
-------
group was fed the basal diet and received nitrite supplemented water. After
the 4-week benzo[a]- pyrene dosing, the feed and water of some groups were
changed to allow further evaluation of the effects. This experiment
Indicated that the Inhibition of neoplasla by soy sauce and nitrite probably
occurred during tumor promotion. In a subsequent study soy sauce at a
dietary concentration of 20% was found to produce a significant reduction In
forestomach neoplasms In benzo[a]pyrene-dosed female ICR mice (Benjamin et
al., 1991; see also Benjamin et al., 1989).
Dietary benzo[a]pyrene at various doses was administered to mice (Neal
and Rlgdon, 1967; Rlgdon and Neal, 1966, 1969). These findings are summa-
rized 1n Tables V-5 and V-6.
Using male and female CFW-SwIss mice. 17-180 days old, Neal and Rlgdon
(1967) found that a dose-response relationship existed between the Incidence
of stomach tumors (paplllomas and carcinomas) and long-term, oral exposure
to benzo[a]pyrene 1n the diet. Animals were fed a diet containing .0-250 ppm
/
of benzo[a]pyrene for <197 days. No tumors were found In the control group
of 289 animals or 1n the groups treated with 1, 10 or 30 ppm benzo[a]pyrene.
The Incidence of tumors, however, Increased between the 40 ppm and 250 ppm
benzo[a]pyrene dosages (see Table. V-5). In a second experiment by Neal and
Rlgdon (1967)y mice were fed 100 or 250 ppm benzo[a]pyrene for 1 day with no
>-**;,•..,, -
gastric tuaort developing within 105 days. However, SOX of mice fed 5000
:$vs**-v£.
ppm benzp{»]pyrene for 1 day did have gastric tumors upon examination -113
••'* _ r
days later. Groups of mice were also given food containing 250 ppm benzo-
[a]pyrene for 1, 2, 4 or 30 days. Upon sacrifice 77-104 days later, the
tumor Incidences were 0/10 (1 day), 1/9 (2 days), 1/10 (4 days) and 26/26
(30 days). It was concluded that the development of gastric tumors In these
04420 V-49 10/24/91
-------
TABLE V-5
Incidence of Forestomach Paplllomas and Carcinomas 1n Hale and
Female CFW Mice Administered Benzo[a]pyrene 1n the Diet*
Dose 1n
Diet
(ppm)
0
1
10
20
30
40
45
50
100 '
250
Total
Consumed
(mg)
0.48
4.48
8.88
13.32
17.76
19.8
21.4-29.4
39.2-48.8
70-165
Duration of
Treatment
(days)
110
110
no
110
no
no
107-197
98-122
70-165
Duration
of Study
(days)
70-300
140
140
226
143-177
143-211
141-183
124-219
118-146
88-185
Tumor
Incidence
(X)
0/289 (0)
0/25 (0)
0/24 (0)
1/23 (5)
0/37 (0)
1/40 (3)
4/40 (10)
24/34 (71)
19/23 (83)
66/73 (90)
*Source: Adapted from Neal and Rlgdon, 1967
04420
V-50
10/08/91
-------
o*t
250+
TABLE V-6
Carc1nogen1c1ty of Benzo[a]pyrene Administered
In the Diet to Male and Female Swiss M1cea
Oose
(ppm)
Duration of
Treatment1* Target Organ Tumor Type
(days)
Tumor
Incidence
(X)
38-210+
80-140
stomach
lung
hematopoletlc
system
stomach
lung
hematopoletlc
system
papl1loma/carc1noma
adenoma
leukemia
papllloma/carclnoma
adenoma
leukemia
2/175 (1)
33/151 (19)
0/175 (0)
69/108 (64)
52/108 (48)
40/108 (37)
250*
250*
1000*
1000*
72-99
147-196
73-83
127-187
stomach
lung
stomach
lung
stomach
lung
stomach
lung
papllloma/carclnoma
adenoma
papl 1 lomas/cardnoma
adenoma
papllloma/carclnoma
adenoma
papllloma/carclnoma
adenoma
12/52 (23)
26/52 (50)
9/13 (69)
10/13 (77)
5/9 (56)
7/9 (78)
13/13 (100)
3/13 (23)
aSource: Adapted from Rlgdon and Neal, 1966*, 1969+
bThe duration of the treatment 1s equal to the duration of the study.
04420
V-51
10/08/91
-------
mice Is Influenced both by the amount of benzo[a]pyrene consumed per day and
the number of days It 1s fed.
In another set of studies, Rlgdon and Neal (1966, 1969) also found
evidence of an association between chronic dietary exposure to benzo[a]-
pyrene and tumor Incidence In the stomach and lung, as well as the develop-
ment of leukemia In mice (see Table V-6). Rlgdon and Neal (1966) fed male
and female Swiss mice dietary concentrations of benzo[a]pyrene of 0, 250 and
1000 ppm for different periods of time. The Incidence of stomach tumors
(paplllomas and carcinomas) was related to both dose and length of exposure.
•
All the mice In the 1000 ppm group were found to have gastric tumors after
86 days of benzo[a]pyrene consumption. A similar set of relationships
between tumor Incidence, dose and time held fairly well for lung adenomas as
well. Rlgdon and Neal (1969) also found that the occurrence of leukemia was
related to 1ngest1on of benzo[a]pyrene. Of mice fed 250 ppm benzo[a]pyrene
In their diet over an 80-140 day period, 69/108 (63.9X) developed paplllomas
or carcinomas of the stomach. Similarly, lung adenomas occurred 1n 52/108
(48.IX) mice fed diets with 250 ppm benzo[a]pyrene. Finally, 40/108 (37%)
of the treated mice developed leukemlas. The ability of dietary benzofa]-
pyrene to produce mouse lung adenomas was also confirmed by the work of
Hattenberg and Leong (1970) and Wallenberg (1974).
Previously, a single oral dose of 100 mg benzo[a]pyrene 1n the diet was
shown to produce mammary tumors In 8/9 female Sprague-Dawley rats (Hugglns
and Yang, 1962). Sprague-Dawley rats of both sexes were treated dally with
2.5 mg benzo[a]pyrene. Forestomach and esophageal paplllomas developed In
3/40 rats (61bel, 1964).
04420 V-52 10/08/91
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Chouroullnkov et al. (1967) administered diets containing benzo[a]pyrene
dissolved 1n olive oil to two groups of -80 male albino mice for a period of
14 months. Based on an assumed food consumption of 5 g/day, the authors
calculated a total dietary dose to be 8 mg. One group of treated animals
was permitted water ad_ libitum while the second drank a 3% ethanol solution.
Controls consisted of 40 mice given the standard diet supplemented with
olive oil and a group of 81 fed the untreated diet only. Forestomach tumors
(paplllomas) were found 1n 5/81 surviving animals receiving benzo[a]pyrene
and water and 1n 8/81 treated mice given the 3% ethanol solution. No
gastric tumors were observed among controls.
As part of a study of the effects of phenolic antloxldants and ethoxy-
quln on PAH carclnogenldty, benzo[a]pyrene In sesame oil was administered
1n the diet to female Ha/ICR and A/HeJ mice (Hattenberg, 1972). The Ha/ICR
mice received 0.4 mg/day for 28 days before being returned to a normal diet
for 27 weeks or 1.26 mg/day for 28 days before being returned to a normal
diet for 14 weeks. Forestomach tumors were observed 1n 11/20 low-dose and
13/19 'high-dose animals. Data on untreated control Ha/ICR mice were not
reported. The A/HeJ mice consumed 0 or 4.8 mg benzo[a]pyrene/day for 2
weeks, beginning at age 9 weeks, and were killed 10 weeks after the last
treatment. All treated (12/12) but no control (0/12) animals developed
forestomach tumors.
In a similar study of sulfur-containing compounds, female Ha/ICR and
A/HeJ mice consumed diets containing 300 ppm benzo[a]pyrene 1n sesame oil
(Wattenberg, 1974). The Ha/ICR mice were maintained on the diet from age 9
weeks to 15 weeks and observed until 29 weeks at which time they were killed
and examined for forestomach tumors. These were observed In 8/20 animals;
04420 V-53 10/08/91
-------
no untreated controls were reported. A/HeJ mice started on treatment at 9
weeks of age and remained on the diet for 25 days. On days 7 and 21 of
treatment animals were gavaged with 3 mg benzo[a]pyrene 1n 0.5 ma sesame
oil. At 31 weeks the mice were killed and evaluated for pulmonary adenomas.
These were present 1n 100X (12/12) of the animals with a mean number of 7.8
tumors/mouse. No untreated controls were reported.
THolo et al. (1977) studied production of forestomach tumors 1n mice as
related to IndudbllKy of aryl hydrocarbon hydroxylase. Female Ha/ICR mice
9 weeks of age were fed diets containing 5X corn oil or 5% corn oil with
benzo[a]pyrene to constitute 0.2 or 0.3 mg/g diet. Treatment was continued
for 12 weeks at which point the animals were killed and stomachs only were
examined hlstologlcally for tumors. Results are summarized 1n Table V-7.
Tumors were of the squamous papHloma type. Gross observation of glandular
stomach, lung and liver revealed no tumors 1n either control or treated
animals.
•
Further evidence of cardnogenlclty associated with 1ngest1on of
benzo[a]pyrene 1s found 1n the work of McCormlck et al. (1981). A group of
20 Inbred virgin female LEM/Ha1 rats received a single IntragastMc dose of
50 mg benzo[a]pyrene 1n sesame oil, while a second group received the same
total dose 1n 8 weekly fractions of 6.25 mg. Mammary carcinoma Incidence
after 90 weeks was 77X In the 50 mg benzo[a]pyrene single dose group and 67X
1n the fractionated dose group. Mammary tumors were observed 1n SOX of
untreated rats.
Adrlaenssens et al. (1983) Investigated the effect of dietary butylated
hydroxyanlsole, a phenolic antloxldant, on the formation of benzo[a]pyrene
04420 V-54 10/08/91
-------
TABLE V-7
Induction of Forestomach Tumors In Ha/ICR Mice Fed Dietary Benzo[a]pyrenea
Dose
(mg/g diet)
0
0.2
0.3
Tumor Incidence
0/9
6/9
9/9
Tumors/Mouse
0.0
1.8
4.0
Carcinogenic
Indexb
0.0
121.9
400.0
aSource: Trlolo et al., 1977
bPercentage of tumor-bearing mice x mean number of tumors/mouse
04420 V-55 10/08/91
-------
metabolHe-DNA and -protein adducts 1n the lung, liver and forestomach of
female A/HeJ mice. The mice were fed benzo[a]pyrene (from 2-1351 ymol/kg)
In Identical diets to which butylated hydroxyanlsole (5 mg/g diet, ~1
g/kg/day) was or was not added. Adduct formation, thought by the authors to
be a necessary, but not a sufficient, step 1n the development of
.benzo[a]pyrene Induced tumors, was examined. 48 hours after Ingestlon of
benzo[a]pyrene. The major DNA adduct Identified In each tissue at each dose
was the (+)-7,8-d1hydroxy-9,lO-epoxy 7,8,9,10-tetrahydrobenzoa)pyrene:
deoxyguanoslne; other adducts, Including 7,8-d1hydroxy-9,lO-epoxy 7,8,9,10-
tetrahydrobenzo(a)pyrene:deoxyguanos1ne and (-)-7,8- d1hydroxy-9,lO-epoxy
7,8,9,10-tetrahydrobenzo(a}pyrene:deoxyguanoslne, were Identified.
Formation kinetics of the major adduct 1n lung and liver ONA from
animals on the control diet showed a sigmoldal curve; forestomach adduct-DNA
complexes exhibited no saturation over the levels tested. As the benzo[a]-
pyrene dose approached 0, the dose-response curves became linear; however,
1n the three organs examined, no threshold dose appeared to exist below
which benzo[a]pyrene metabolite adducts were not observed.
Dietary butylated hydroxyanlsole treatment Inhibited the formation
benzo[a]pyrene metabolite adducts to forestomach, lung and liver DNA over a
wide dietary benzo[a]pyrene range. Adduct formation In DNA from the fore-
stomach of butylated hydroxyanlsole-treated animals was 45X lower than the
control group, but demonstrated the same binding kinetics as the animals In
the control diet. The maximum Inhibition of lung and liver DNA adduct
formation 1n butylated hydroxyanlsole-treated animals was 68 and 82% lower
than the control group, respectively. As the benzo[a]pyrene dose approached
04420 V-56 10/08/91
-------
these values declined to 40 and 55X, respectively. Butylated hydroxyanlsole
treatment also diminished the curvilinear nature of the dose-response curve.
The level of benzo[a]pyrene metabolites binding to DNA was -1% of the
amount of binding to proteins 1n the three organs examined. The
dose-response curves for benzo[a]pynene metabolites binding to lung and
liver proteins was parallel for both the control and butylated
hydroxyanlsole-treated groups. Inhibition of metabolite binding was not
dose dependant 1n these organs. No consistent effects of butylated
hydroxyanlsole were noted 1n benzo[a]pyrene metabollte-forestomach protein
binding kinetics.
Hamsters have also been observed to develop paplllomas and carcinomas of
the alimentary tract In response to gavage or dietary exposure to benzo[a]-
pyrene (Dontenwlll and Mohr, 1962; Chu and Malmgren, 1965). Chu and
Halmgren (1965) fed male Syrian hamsters diets containing 500 mg benzo[a]-
pyrene/kg food or 500 mg benzo[a]pyrene plus 5 g vitamin A palmltate or 5 g
/
vitamin A palmltate only. This diet was provided 4 days/week and standard
diet the remaining 3 days. Animals consumed -5 g food/day, thus receiving
~10 mg benzo[a]pyrene and/or 100 mg vitamin A palmltate/week. All 10
animals fed vitamin A free diet died 1-4 weeks after the beginning of the
assay, whereas those consuming vitamin A survived <5 months. Hamsters
consuming benzo[a]pyrene with vitamin A lived longer (that 1s, from 6-14
months) than animals fed benzo[a]pyrene and no vitamin A. Nine of 13
treated animals developed tumors described by the authors as "cancer" 1n the
forestomach (9) and Intestine (2). Vitamin A 1n the diet eliminated
Intestinal tumors and decreased the severity of the forestomach lesions to
04420 V-57 10/08/91
-------
lesions described as papHlomas and dyskeratoses. Tumor Incidence In the
group receiving only vitamin A was 0/27 for esophageal lesions; the
Incidence of forestomach tumors was not described.
BenzoTblfluoranthene. Pertinent data regarding the oral carcinogenic-
•
Hy of benzo[b]fluoranthene could not be located 1n the available literature.
BenzoTklfluoranthene. Pertinent data regarding the oral cardnogenlc-
Ity of benzo[k]fluoranthene could not be located 1n the available literature.
Benzofg.h.1]perylene. Pertinent data regarding the oral cardnogenlc-
Hy of benzo[g,h,1]perylene could not be located 1n the available literature.
Chrysene. Pertinent data regarding the oral carclnogenlcHy of
chrysene could not be located 1n the available literature.
D1benzTa.hlanthracene. Mice (strain not specified) were fetl a diet
/
with added d1benz[a,h]anthracene. After 5-7 months exposure the total
received doses of 9-19 mg resulted 1n the Induction of forestomach tumors In
7/22 survivors at 1 year (Larlnov and Soboleva, 1938).
Lorenz and Stewart (1948) administered strain A mice 0.4 mg d1benz[a,h]-
anthracene/day 1n an aqueous mineral oil suspension given In place of drink-
Ing water. At 406 days exposure, 2 squamous cell carcinomas and 11 papll-
lomas of the forestomach were observed (Lorenz and Stewart, 1948). The
authors also conducted experiments 1n which C57B1, C3H, DBA/2 and A strain
04420 V-58 10/08/91
-------
mice were exposed to d1benz[a,h]anthracene In an olive oil water emulsion
provided to the animals 1n Heu of drinking water (Lorenz and Stewart,
1947). The DBA/2 mice developed tumors Including pulmonary adenomatosls,
that Snell and Stewart (1962a) concluded were similar to adenomatous lesions
found 1n humans. Snell and Stewart (1962a, 1962b) were thus prompted to
•
undertake a similar experiment 1n mice. Groups of 21 male and female mice
of the DBA/2 strain were given 0.2 mg/ml d1benz[a,h]anthracene In an
aqueous olive oil emulsion ad libitum 1n place of drinking water. Twenty-
five male and 10 female control animals received the olive oil emulsion 1n
Heu of drinking water. Neither treated nor control mice tolerated the
olive oil vehicle. Animals lost weight after a few weeks exposure,
eventually becoming emaciated and dehydrated. Hales were estimated to
receive a dally dose of 0.85 mg/day while females received 0.76 mg/day of
d1benz[a,h]anthracene. Duration of the experiment was 279 and 237 days for
male and female mice In the experimental group and 351 and 226 days for
controls. Treated mice developed pulmonary adenomatosls, alveologenlc
carcinomas, mammary tumors (females only), precancerous growths of the small
/
Intestine and hemang1oendothel1omas of the pancreas, mesentery and abdominal
lymph nodes. A pulmonary adenomatosls 1n a male mouse was the only lesion
observed 1n either male or female control animals. Tumor Incidences from
the Snell and Stewart (1962a) study are compared with those of the Lorenz
and Stewart (1947) study 1n Table V-8.
A series of assays was done wherein mice of several strains were gavaged
twice weekly with preparations of 0.5X d1benz[a,h]anthracene In almond oil.
After a 15-week treatment period, the total dose was 15 mg/anlmal. Mammary
carcinomas were observed 1n 1/20 BALB/c females and In 13/24 pseudo-pregnant
04420 V-59 11/12/91
-------
o
4»>
4k
O
TABLE V-8
Oral Cardnogen1c1ty of D1benz[a,h]anthracene 1n DBA/2 M1cea
Duration Duration
Sex Feeding Study
(days) (days)
M 177 281
F 158 265
M 279 279
F 237 237
aSource: Adapted from Snell and
^Compound was administered as an
cNo tumors were observed 1n
pulmonary adenomatosls was the
Doseb
(mg/day)
0.56
0.48
0.85
0.76
Tumor Type
Pulmonary adenomatosls
Alveblogenlc carcinoma
Carcinoma, small Intestine
Precancer, small Intestine
Hemang1oendothe11oma
Pulmonary adenomatosls
Alveologenlc carcinoma
Carcinoma, small Intestine
Precancer, small Intestine
Hemangloendothelloma
Mammary card nomad
Pulmonary adenomatosls
Alveologenlc carcinoma
Carcinoma, small Intestine
Precancer, small Intestine
Hemangloendothelloma
Pulmonary adenomatosls
Alveologenlc carcinoma
Carcinoma, small Intestine
Precancer, small Intestine
Hemangl oendothel 1 oma
Mammary carcinoma**
Tumor
Incidence0
4/10
8/10
3/10
6/10
4/10
3/9
7/9
0/9
5/9
2/9
3/9
14/14
14/14
0/14
2/14
10/14
13/13
10/13
0/13
4/13
6/13
12/13
Reference
Lorenz and
Stewart, 1947
Lorenz and
Snell, 1947
Snell and
Stewart, 1962a
.
Snell and
Stewart, 1962a
Stewart, 1962a
aqueous olive oil emulsion provided 1n 11eu of drinking
10 female
only tumor
\}} and Stewart (1962a) study.
°KO mammary carcinomas were observed 1n the
control mice 1n the Lorenz
observed 1n 25 male and 10
male m1ct~
and Stewart (
female control
water.
1947) study. One
mice used 1n fjie
-------
BALB/c females (B1anc1f1or1 et al.t 1976). A single 1.5 mg dose of
d1benz[a,h]anthracene In PEG-400 produced forestomach paplllomas In 2/42
male Swiss mice after 30 weeks (Berenblum and Haran, 1955).
Fluoranthene. Pertinent data regarding the oral cardnogenlclty of
fluoranthene could not be located 1n the available literature.
Fluorene. The carcinogenic potential of fluorene was studied by
Wilson et al. (1947) and Morris et al. (1960). In the Morris et al. (1960)
study, female buffalo rats were administered 0.05% fluorene. 1n their diets
containing either 3% added corn oil or propylene glycol. This resulted 1n
the consumption of either 4.3 mg/day of fluorene for ~6 months or 4.6 mg/day
for -18 months. The Incidence of tumors 1n the treatment and control groups
was essentially the same (Table V-9). The authors of the study described
fluorene as "slightly carcinogenic."
HUson et al. (1947) studied the effect on tumor development 1n albino
rats of exposure to various concentrations of fluorene In the diet over
various periods of time. One set of rats was exposed to several concentra-
tions (number not specified) ranging from 0.062-1.OX fluorene 1n the diet
for 104 days while a second set received either 0.125, 0.25 or 0.5% fluorene
1n the diet for 453 days. Animals of the short-term group maintained on
diets with fluorene concentrations of 0.5 and 1.0% experienced significant
decreases 1n their rate of growth.. In other aspects they appeared normal.
The Internal organs of rats exposed for 104 days were essentially normal 1n
appearance and histology. Livers of rats consuming the 0.25% and higher
dose diets In the longer study were significantly heavier than normal.
04420 V-61 10/24/91
-------
t\>
o
TABLE V-9
Carclnogenlclty Testing of FlUorene Administered In the Diet to Female Buffalo Rats3
Dose or Duration of
Exposure Treatment
0 mg/day NA •
4.6 mg/dayb 18.1 months
(range, 4.1-19.2)
0 mg/day NA
4.3 mg/dayb 6.1 months
(range, 5.0-6.2)
aSource: Morris et al., 1960
••Reported dally Intake of 0.05X dietary
NA = Not applicable
o
o
CD
Duration of Study
15.5 months
(range. 9.4-19.9)
19.0 months
(range, 5.1-20.1)
13.9 months
(range. 7.8-18.2)
10.2 months
(range. 8.2-10.7)
fluorene
Vehicle or
Physical State
diet with 3X added
corn oil
diet, above plus
fluorene In corn oil
diet with 3X added
propylene glycol
diet, above plus
fluorene added In
corn oil
t
Target
Organ
uterus
uterus
adrenals
pituitary
Inguinal
region
uterus
uterus
R-E system
pituitary
kidney
pituitary
R-E system
kidney
ureter
Tumor Type
adenocarclnoma
flbroeplthellal polyp
cortical adenoma
adenomas
fibroma
flbrosarcoma
carclnosarcoma
granulocytlc
leukemia
chromophobe adenoma
adenoma
chromophobe adenoma
granulocytlc leukemia
squamous-cell
carcinoma
squamous-cell
carcinoma
Tumor
Incidence
(X)
1/18 (6)
2/18 (11)
5/18 (28)
6/18 (34)
1/18 (6)
1/18 (6)
1/18 (6)
1/18 (6)
4/18 (22)
1/18 (6)
2/18 (11)
1/18 (6)
1/11 (6)
1/11 (6)
-------
Spleens of all treated animals weighed less -than normal as did testes of the
highest dosed rats. In the longer-term exposure group, squamous metaplasia
of the bronchial epithelium was noted In three rats while one rat exposed to
0.125X fluorene 1n the diet had developed a small benign kidney tubular
adenoma. Total number of antmals treated was not Indicated, nor was a
control group described.
Indenon.2.3-cdlpyrene. Pertinent data regarding the oral carclno-
genldty of 1ndeno[l,2,3-cd]pyrene could not be located 1n the available
literature.
Naphthalene. There 1s only very limited Information available on the
carcinogenic potential of naphthalene following oral administration to
laboratory animals.
Schmahl (1955) reported that naphthalene administered 1n food was not
carcinogenic 1n rats (In-house strains BDI and BDIII). Naphthalene was
/
dissolved 1n oil and given 6 times/week 1n food. The dally dose was between
10 and 20 mg. After reaching a total dose of 10 g/rat (food Intake was not
reported), treatment was stopped and animals observed until death, between
700 and 800 days of age.
Tsuda et al. (1980) administered a single gavage dose of 100 mg/kg
naphthalene 1n corn oil to a group of 10 young adult F344 rats (sex not
specified) at 12 hours after partial hepatectomy. A vehicle control group
of 10 rats was Included. At 2 weeks after surgery, 2-acety1am1nof1uorene
was added to the diet at 200 ppm to Inhibit proliferation of "nonreslstant"
04420 V-63 10/08/91
-------
hepatocytes. After 1 week of dietary 2-acetylamlnofluorene, a single 2.0
ma/kg dose of carbon tetrachloMde was given to necrotlze "nonreslstant"
hepatocytes and permit proliferation of "resistant" hepatocytes. Feeding of
2-acetylam1nofluorene continued for 1 week, followed by a basal diet for 1
week. The rats were then sacrificed and Hvers were sectioned and hlsto-
chemlcally examined for the number and size of y-glutamyl transpeptldase
(GGT)pos1t1ve fod. These foci contain cells that are "resistant" to the
necrotlzlng effects of carbon tetrachlorlde and to the proliferation-
Inhibiting effects of 2-acetylam1nofluorene and are considered to represent
an early stage 1n the process of neoplastlc transformation.' Neither the
number nor the size of 6GT fod appeared to be Increased 1n naphthalene-
treated rats compared with vehicle controls. The use of GGT as a biochemi-
cal marker of preneoplastlc foci 1s generally accepted (Hendrlch and PHot,
1987).
Phenanthrene. A single oral dose of 200 mg phenanthrene dissolved In
sesame oil was administered to ten, 50-day-old, female Sprague-Dawley rats.
/
No mammary tumors were produced within 60 days. In a positive control group
of 700 animals given 20 mg of 7,l2-d1methylbenz[a]anthracene administered
under the same conditions, mammary tumor Incidence was 100X (Hugglns and
Yang, 1962).
Pyrene. Pertinent data regarding the oral carclnogenldty of pyrene
could not be located 1n the available literature.
Carc1noqen1c1ty. Other Routes
Acenaphthylene. Hartwell (1951) cited a study wherein 20 mice (strain
not specified) were skin painted with 0.25X acenaphthylene In benzene. At 6
04420 V-64 10/08/91
-------
months 13 animals were alive; 7 were alive at 1 year. No tumors were
observed.
. Rotenberg and MashbHs (1965) reported "various degrees of malignancy"
1n the lungs of almost all of an unspecified number of rats exposed to
acenaphthylene dust at 0.5-1.25 mg/m3 for 4 hours/day for 4 months.
Further details were not provided. In another study (published In Russian)
with male white rats, hlstopathologlc lesions Including hyperplasla and
metaplasia of the bronchial epithelium, but no signs of malignancy, were
reported following Inhalation of acenaphthylene vapors 4t 18 mg/m3, 4
hours/day, 6 days/week for 5 months (Reshetyuk et al., 1970).
Anthracene. The carcinogenic potential of anthracene has been tested
by skin application with and without UV radiation 1n mice, 1n skin Initia-
tion-promotion assays with mice, by s.c. or 1.p. routes 1n rats, by Implan-
tation Into the lungs of rats, and by Implantation Into the brain or eyes 1.n
rabbits. These studies are summarized 1n Table V-10. The results of the
/
skin painting bloassays for both complete carclnogenldty and for Initiating
activity do not provide evidence of carclnogenldty, but contradictory
results were obtained when anthracene was applied to skin together with
exposure to UV radiation. The Injection and Implantation studies do not
provide evidence of carclnogenldty, but these results cannot be regarded as
conclusive due to Inadequacies In experimental design (e.g., small number of
animals, limited number of exposures. Inadequate controls).
Benzfalanthracene. Shlmkln and Stoner (1975) demonstrated negative
results 1n the strain A mouse lung adenoma assay after a single 1.v. Injec-
tion of 0.25 mg benz[a]anthracene and an observation period of 6 months.
04420 V-65 11/12/91
-------
o
Jfc
4k
O
TABLE V-10
Dermal, Injection and Implantation Carctnogenlclty Assays of Anthracene
Route
Skin
Species/
mouse/NR
No.VSex
100/NR
Purity
MR
Treatment Duration
dose and number of appUca- NR
tlons not specified; 40X solu-
tion In lanolin
Effects/Comments
No skin tumors; 45 and 6 mice sur-
vived >6 months and >160 days.
respectively
Reference
Kennaway,
1924a,b
Skin
Skin
Skin
Skin
Skin
Skin
Skin
mouse/NR 41/NR
mouse/NR SO/NR
mouse/Swiss 5/F
mouse/NR
mouse/NR
mouse/NR
mouse/NR
44/NR
44/MR
100/NR
NR/NR
NR
NR
NR
NR
NR
NR
NR
o
CO
)2 Skin
mouse/Skh: 24/m1xed
halrless-1
NR
dose and number of applied- life (133
tlons not specified; unsped- days average)
fled solutions In water,
benzene or sesame oil
dose not specified; 0.3X solu- 732 days
tlon In benzene twice weekly
dose not specified; 10X solu- life
tlon In acetone 3 times/week (10-20 months)
on the back
dose not specified; 5X solu- life
tlon In petroleum jelly-olive (11 months)
oil 3 times/week on the ear
treatment as above but with life
ultraviolet radiation (>320 nm) (10 months)
for 40 or 60 minutes, 2 hours
after skin application
treatment as above but mice life
received ultraviolet radiation (10 months)
for 90 minutes
dose and number of appUca- NR
tlons not specified; 10X solu-
tion 1n petroleum Jelly-olive
oil followed by unspecified
exposure to ultraviolet
(320-405 nm) alone or with
visible light
0 or 4 iig 1n methanol once 38 weeks
dally, followed by ultraviolet
(>290 nm) for 2 hours, 5 days/
week
No skin tumors; d1benz[a.h]anthra-
certe was tumorlgenlc In same study
No skin tumors; 34 and 16 alive at
6 months and 1 year, respectively
No skin tumors; benzo[a]pyrene
Induced high Incidences of skin
paplllomas and carcinomas under
the same conditions
No skin tumors; l/44*a11ve after
11 months; primary report not
available
No skin tumors; 5/44 alive after
7 months
No skin tumors; 7/100 alive after
7 months
"High Incidence* of skin tumors.
Including many carcinomas, was
observed after 5-8 weeks; no tumors
In controls treated with anthracene,
ultraviolet or ultraviolet with
visible light; primary report not
available; unusually short latency
and Inadequate hlstopathology
reporting noted by IARC (1983)
Incidence of skin tumors not sig-
nificantly Increased In treated
group; survival was 20/24 and 19/24
In controls and treated groups
Pollla. 1939
Bachmann
et al.. 1937
Wynder and
Hoffmann.
1959a
Hlescher. 1942
Hlescher. 1942
Hlescher, 1942
Heller. 1950
Forbes et al.,
1976
-------
TABLE V-10 (cont.)
Route
Species/ No.VSex
Purity
Treatment
Duration
Effects/Comments
Reference
Skin
mouse/S 20/NR
NR
Skin
mouse/CO-1 30/F
Skin
mouse/CRI: 20/F
CD/1(ICR)BR
chromato-
graphic-
ally
purified
NR
two applications (0.3 ml of
0.5X solution 1n acetone per
application) with a 30-mlnute
Interval 3 times/week for a
total of 20 applications (30
rag/animal total); IB weekly
applications of croton oil In
acetone (0.3 mt) consisting of
1/0.17X, 2/O.OB5X and 15/0.17X
croton oil, beginning 25 days
after the first anthracene
application; controls received
the same treatment with croton
oil only.
single application of 10
(1782 ng) In benzene followed
1 week later by 5 umol TPA
twice weekly for 34 weeks;
controls received the same
treatment with TPA only
ten applications (0.1 mg In
0.1 ml acetone per application)
applied every other day. Ten
days later 0.25 tig TPA In 0.1
ml acetone was applied 3 times/
week for 20 weeks
25 weeks
35 weeks
25 weeks
Incidence of skin tumors not sig-
nificantly Increased In treated
groups; survival was 19/20 and
17/20 1n the control and treated
groups, respectively
Salaman and
Roe. 1956
Incidence of skin paplllomas was
4/2B (14X) In treated and 1/30 (3X)
In controls
No significant Increase 1n skin
tumor Incidence In treated groups
ScMbner. 1973
LaVole et al..
1985
s.c.
o
CD
s.c.
vS.C.
rat/NR 10/NR NR weekly injections of 2 ml of >18 months
0.05X suspension In water for
. life (103 mg/an1mal maximum
total dose)
rat/Wlstar 5/NR NR 5 mg In sesame oil, once 10 months
weekly for 6 or 7 weeks
rat/BOI 10/NR highly 20 mg In unspecified oil once life
and BDIII purified weekly for 33 weeks
No subcutaneous sarcomas; survival Boyland and
was 7/10 after 12 months and 8/10 Burrows, 1935
after 18 months; d1benz[a,h]-
anthracene was tumoHgenlc under
the same conditions
No tumors; examinations apparently Pollla, 1941
Included viscera; d1benz[a,h]-
anthracene Induced subcutaneous
tumors In 2/5 similarly treated rats
Injection site tumors (Hbromas Schmahl, 1955
with sarcomatous areas) In 5/9
rats, mean latency, 26 months; no
controls, but rats treated'simi-
larly with naphthalene In oil did
not develop local tumors
-------
TABLE V-10 (cont.)
«•
ro
o
<
Ch
CO
Route Species/ No.VSex Purity
1.p. rat/801 or 10/NR
BOIII
Cerebral rabbi t/NR 5/NR
cortex
Implant
Cerebellar rabbi t/NR 2/NR
Implant
Optic rabbi t/NR 2/NR
Implant
Lung Implant rat/Osborne- 60/F
Mendel
'Numbers In treatment and control
highly
purified
NR
NR
NR
refined
recrystal-
11 zed
(If used) group(s)
Treatment Duration
Effects/Comments
20 mg In unspecified oil once life Tumor In one rat (spindle-cell
weekly for 33 weeks (mean -2 years) sarcoma In abdominal cavity); no
control group
10 mg (1 rabbit) or 20 mg 4.5 years
(4 rabbits) pellets
12 mg pellets 4.5 years
4 or 5 mg pellets 4.S years
0.5 mg In 0.05 ml warm soft 55 weeks
1:1 beesway: trlcaprylln
unless specified otherwise.
No tumors; survival was 4/5 after
4 years and 2/5 at 4.5 years
No tumors; survival was 2/2 at
4.3 years
No tumors; survival was 2/2 at
4.5 years
No tumors In treated or control
animals
Reference
Schmahl, 1955
Russell, 1947
Russell. 1947
Russell. 1947
Stanton
et al.. 1972
NR = Not reported; s.c. = subcutaneous; 1.p. *> Intraperltoneal
o
00
-------
As part of a large study of PAHs and their nitrated derivatives, newborn
CD-I mice were treated 1.p. with a total of 2800 ymol benz[a]anthracene In
OMSO. Liver tumors were not observed 1n treated female mice, but 31/39
males developed Hver tumors of which 25/39 were carcinomas. This was a
significant Increase compared with controls. By contrast, female mice were
•
observed to have a significantly Increased Incidence of lung tumors (6/32
compared with 0/31) while the males did not (6/39 compared with 1/28)
(H1slock1 et al., 1986).
Benz[a]anthracene 1s' a well-documented complete skin carcinogen. Both
the parent compound and the 3,4-d1hydrod1ol and the 3,4-d1o1-l,2-epox1de
have produced tumors on mouse skin (IARC, 1973; Santodonato et al., 1981).
By contrast neither Oonryu rats nor Syrian golden hamsters developed tumors
after topical benz[a]anthracene application (Tawflc, 1965; Shublk et al.,
1960).
Subcutaneous administration of benz[a]anthracene 1n tr1capryl1h to mice
resulted In Injection site sarcomas. Incidences of sarcomas 1n C57B1 mice
observed 9 months after being given graded doses of benz[a]anthracene were
as follows: 0.05 mg, 5/43; 0.2 mg, 11/43; 1.0 mg, 15/31; 5.0 mg, 49/145; 10
mg, 5/16 (Stelner and Falk, 1951; Stelner and Edgecomb, 1952). Klein (1952)
showed that Intramuscular Injection of albino mice derived from strain A
with benz[a]anthracene In a 1 or 3% combination with croton oil produced
Injection site Hbrosarcomas and hemangloendothellomas.
Benzofalpyrene. Benzo[a]pyrene Is known to produce tumors when admin-
istered by Inhalation or Intratracheal Instillation. It 1s generally more
04420 V-69 10/08/91
-------
effective as a lung carcinogen when accompanied by a resplrable participate
or a cocarclnogenlc gas. Laskln et al. (1970) exposed 21 rats to 10 mg/m3
benzo[a]pyrene for 1 hour/day for 1 year. Two animals developed squamous
cell carcinomas 1n this group. When rats were exposed to 10 ppm S0« for
an additional 6 hours/day, carcinoma Incidence was 5/21. No animals
receiving SO. only developed tumors. Intratracheal Instillation of
benzo[a]pyrene accompanied by India Ink resulted 1n a dose-dependent
Increase 1n lung tumors 1n rats (Yanlsheva, 1971). Rats that received
14C-benzo(a)pyrene along with carbon black or asbestos through
Intratracheal Instillation had a higher incidence of lung tumors than those
receiving only this PAH (Pylev et al., 1969).
Intratracheal Instillation studies of partlculate and benzo[a]pyrene In
hamsters have also shown Increased Incidences of respiratory tumors. An
Increased Incidence of respiratory tract tumors was reported 1n Syrian
golden hamsters that had been administered benzo[a]pyrene along with ferric
oxide particles (Safflottl et al., 1965, 1968). In a similar experiment,
FarreVl and Davis (1974) showed that carbon black and ferric oxide were more
effective tumor promoters than aluminum oxide when these three types of
particles were bound to benzo[a]pyrene. The percentage of respiratory tract
tumor-bearing hamsters 1n each group was 49, 49 and 22% for the carbon
black, ferric oxide and aluminum oxide groups, respectively. Intratracheal
Instillation of radlolabeled benzo[a]pyrene-coated carbon, aluminum oxide
and ferric oxide resulted In an. Increase 1n the retention time of the
radlolabel 1n the hamster lung when compared with administration of only the
radlolabeled benzo[a]pyrene (Henry and Kaufman, 1973).
04420 V-70 10/08/91
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Hamsters have provided a useful model for the study of benzo[a]pyrene
lung cardnogenlcHy. Concurrent exposure with sesame oil resulted 1n
trachea! paplllomas and carcinomas (Mohr, 1971). Saff1ott1 et al. (1972)
conducted several assays wherein a saline suspension of benzo[a]pyrene
coated Fe-O- particles were repeatedly Instilled In hamster treacheas.
In one assay the lowest dose employed, 0.25 mg benzo[a]pyrene/week, Induced
respiratory tumors 1n 10/88 hamsters. At the high-dose, 3 mg/week, the
Incidence was 34/57 with multiple tumors observed In some animals (Saff1ott1
et al., 1972). Using the same model, Sellakumar and Shublk (1972) reported
a 3054 Incidence of respiratory tract tumors 1n animals receiving 20 weekly
treatments of 0.5 mg benzo[a]pyrene.
In research by Thyssen et al. (1981), groups of 24 hamsters Inhaled
benzo[a]pyrene at concentrations of 2.2, 9.5 or 46.5 mg/ma, 4.5 hours/day,
7 days/week for 10 weeks followed by an exposure for 3 hours/day, 7 days/
week for a maximum of 675 days. Neither control animals nor animals receiv-
ing the lowest dose developed respiratory or upper digestive tract tumors.
/•
Above 2.2 mg/m3 benzo[a]pyrene, the Incidence of respiratory and upper
digestive system tumors Increased with dose. Table V-ll summarizes the
findings.
A later report Indicated that concurrent exposure of Syrian golden
hamsters to SO. and benzo[a]pyrene-coated sodium chloride resulted 1n an
enhancement of tumor response and a decreased latency period (Pauluhn et
al., 1985).
Feron and Kruysse (1978) and Ketkar et al. (1978) also studied the
Incidence of respiratory tract neoplasms 1n male and female hamsters
04420 V-71 10/08/91
-------
TABLE V-ll
CardnogenlcHy of Benzo[a]pyrene to
Male Syrian Golden Hamsters By Inhalat1ona»b
Dose
(mg/m3)
0.0
2.2
9.5
46.5
mg/Hamster
0
29
127
383
Duration of
Treatment0
(weeks)
96.4
95.2
96.4
59.5
Target Organ
respiratory tract
upper digestive tract
respiratory tract
upper digestive tract
respiratory tract
upper digestive tract
respiratory tract
upper digestive tract
Tumord.
Incidence
(X)
0/27 (0)
0/27 (0)
0/27 (0)
0/27 (0)
9/26e (35)
7/26e (27)
13/25f (52)
14/25^ (56)
aSource: Thyssen et a!., 1981
^Exposure was for 4.5 hours/day for the first 10 weeks, 3 hours/day there-
after for 7 days/week as NaCl vapor (>99X of the particles had diameters
between 0.2 and 0.5 yM) 1n air.
cThe duration of the study 1s equal to the duration of the treatment
^Tumors were paplllomas, papillary polyps, and squamous cell carcinomas
e3 nasal cavity, 8 laryngeal, 1 tracheal, 6 pharyngeal and 1 forestomach
tumor
fl nasal cavity, 13 laryngeal, 3 tracheal, 14 pharyngeal, 2 esophageal and
1 forestomach tumor
04420
V-72
10/08/91
-------
associated with Intratracheal administration of benzo[a]pyrene. Ketkar et
al. (1978) found the mean survival time of both male and female hamsters was
positively related to the dose of benzo[a]pyrene administered as weekly
Instillations In bovine serum albumin (Table V-12). For male hamsters, the
Incidence of respiratory tract tumors tended to Increase with dose except at
the highest level where the tumor Incidence rate declined slightly. For the
female hamsters, tumor Incidence was also Increased by benzo[a]pyrene, but
an Inverse relationship held between dosage and tumor Incidence. Why this
Inverse relationship was observed Is not readily apparent.
Kobayashl (1975) treated 32 male and 28 female Syrian golden hamsters
with Intratracheal Instillations of 1 mg of benzo[a]pyrene 1n saline weekly
for 30 weeks. The control group (20 mice/sex) was administered an
equivalent dose of saline. After 60 weeks the mice were sacrificed and
complete necropsies performed. Survival rates appeared to be equivalent 1n
the benzo[a]pyrene-treated and control groups (both sexes combined). The
Incidences of respiratory tract tumors -were 11/26 1n treated males and 14/26
1n treated females. The majority of these tumors were found 1n the
peripheral areas of the lung. No Incidences of respiratory tract tumors
were found 1n the control mice.
Stenback and Rowland (1978) studied the role of talc and benzo[a]pyrene
In respiratory tumor formation. Two groups of 24 Syrian golden hamsters/sex
received either 18 Intratracheal Instillations of 3 mg of talc 1n saline or
3 mg benzo[a]pyrene an'd 3 mg talc In saline weekly. Control groups received
saline or remained untreated. Hamsters were autopsled upon spontaneous
death. Animals treated with talc and benzo[a]pyrene had a shorter Hfespan
04420 . V-73 10/08/91
-------
TABLE V-12
CarclnogenlcHy of Benzo[a]pyrene Administered
by Intratracheal Instillation to Syrian Hamsters3
Duration of
Doseb Treatment0
(mg/week) (weeks)
0 41
0.10 40
0.33 24
1.0 10
Incidence of Respiratory Tract Neoplasms^
(X)
Hales
0/29
5/26
7/29
6/27
(0)
(19)
(24)
(22)
Females
•0/30
12/30
10/28
6/20
(0)
(40)
(36)
(30)
aSource: Ketkar et al.f 1978
^Benzo[a]pyrene (97X pure) was delivered
Controls received vehicle only.
1n bovine serum albumin.
cHean survival time. Survival time was also equal to study duration.
^Carcinomas, adenomas, adenocarclnomas and paplllomas were reported.
04420
V-74
10/08/91
-------
(52 weeks) than the untreated controls (55 weeks), and these deaths were
primarily attributed to pulmonary dysfunctions caused by either Interstitial
flbrosls or neoplastlc Involvement of the respiratory system. Only the
hamsters treated with the talc and benzo[a]pyrene mixture displayed
paplllomas, squamous cell carcinomas, and undlfferentlated tumors of the
lung, larynx and trachea (Incidence 33/48). Talc appeared to have no
carcinogenic activity alone, but Increased the cardnogenldty of benzo[a]-
pyrene.
In an Intratracheal Instillation study, male Syrian golden hamsters were
administered benzo[a]pyrene, arsenic (as arsenic trloxlde)' or arsenic and
benzo[a]pyrene together. They were Introduced with 3 mg of charcoal carbon
dust carrier. The control group received only the carrier dust. The
animals were dosed weekly. The benzo[a]pyrene group (50 hamsters) received
0.44 mg/exposure (~6 mg/kg), and the arsenic group (67 hamsters) received
0.25 mg/exposure (~3 mg/kg). The group receiving both chemicals (90
hamsters) received 0.45 mg and 0.23 mg of benzo[a]pyrene and arsenic*,
respectively. The 50-week survival rates of the control, benzo[a]pyrene,
/
arsenic and combined groups were 40, 54, 52 and 41X, respectively. The
100-week survival rates of the control, benzo[a]pyrene, arsenic, and arsenic
and benzo[a]pyrene combined groups were 13, 14, 25 and 13X, respectively.
The Incidences of carcinomas of the larynx, trachea, bronchll or lungs were
0/53, 3/47, 17/40 and 25/54 In the control, arsenic, benzo[a]pyrene, and
arsenic and benzo[a]pyrene combined groups, respectively (Pershagen et al.,
1984).
Male Sprague-Oawley rats were exposed to either [sH]-benzo[a]pyrene
mlcrocrystals, [8H]-benzo[a]pyrene mlcrocrystals In Iron oxide or
04420 V-75 10/24/91
-------
chrysotlle asbestos, or [3H]-benzo[a]pyrene absorbed to Iron oxide or
chrysotlle asbestos participates by Intratracheal Instillation. Rats were
sacrificed at times ranging from 2 minutes to 72 hours after administration
and the distribution of the radlonucUde examined. Absorption of
benzo[a]pyrene administered as mlcrocrystals was rapid. Addition of
asbestos decreased the absorption rate of the mlcrocrystals, and addition of
Iron oxide had. little or no effect .on absorption. Partlculates did not
appear to Increase the cardnogenldty of benzo[a]pyrene by particle
enhanced uptake or Increased retention time 1n the lungs (Bevan et al.,
1988).
Two studies by Feron and co-workers showed benzo[a]pyrene Instilled In
saline suspension produced respiratory tumors In hamsters. Feron et al.
(1973) reported that male Syrian golden hamsters receiving a total adminis-
tered dose of 0, 3.25, 6.5, 13, 26 or 52 mg benzo[a]pyrene had the following
Incidences of respiratory tract tumors: 0/29, 3/30, 4/30, 9/30, 25/29 and
26/28, respectively. Feron and Kruysse (1978) found a positive dose-
response relationship for tumor Incidence 1n male and female hamsters given
<
a total dose of 0, 18.2 or 36.4 mg of benzo[a]pyrene In saline 1ntra-
tracheally for 52 weeks. The Incidence of respiratory tract tumors
corresponding to these doses was 0, 13.8 and 63.3X In males, while 1n
females the corresponding tumor Incidences were 0, 11.1 and 29.2X.
PapHlomas and carcinomas of the trachea and pulmonary adenomas were most
often observed.
Benzo[a]pyrene, 1 mg/mi 1n phosphate buffer solution, was administered
Intratracheally once a week for 15 weeks to 8-week-old female Syrian golden
04420 V-76 10/24/91
-------
hamsters. Treated animals and controls survived 443 and 614 days, respec-
tively, after the Initial Instillation. No control animals were observed to
develop lung or trachea! tumors whereas 6/13 treated animals had trachea!
tumors (2 malignant, 4 benign) (Yamamoto et al., 1985).
Benzo[a]pyrene has been shown to produce, tumors at various sites by a
number of modes of administration. Intraperltoneal Injection of newborn
BLU:Ha(ICR) mice with benzo[a]pyrene produced lung adenomas. A dose of 280
vg/mouse resulted 1n a 94X tumor Incidence (Busby et al., 1984). Individ-
ual Swiss mouse fetuses were treated with a single 1.p. Injection of benzo-
[a]pyrene or derivatives In 1 yi trloctanoln and acetone. Doses of 0,
0.4, 4.0, 9.9 or 19.8 vmol/fetus were given on day 15 of Intrauterlne
growth and animals were carried to term, weaned and kept until 12 weeks of
age. At this time they were killed and examined for lung adenomas. Inci-
dence of adenomas 1n the control group was 4/37 and In the treated groups at
the stated dosages were 1/39, 10/42, 10/38 and 12/31. A racemlc mixture of
benzo[a]pyrene 7,8-d1ol-9,lO-epox1des was more tumorlgenlc producing an
Incidence of 27/37 at a dose of 4.0 ^mole/fetus (Rossi et al., 1983).
Male Wlstar rats were given an 80 mg/kg bw IntrapeMtoneal Injection of
benzo[a]pyrene 18 hours after partial hepatectomy (Dock et al., 1988). The
rats were then placed on a specific diet schedule that consisted of 2 weeks
of a basal diet, followed by 2 weeks of a diet supplemented with 0.02%
2-acetylam1nofluorene (2-AAF). A single Injection of carbon tetrachloMde
(2 ml/kg bw) was administered midway through the 2 weeks. The rats were
then administered a basal diet for 2 weeks. The benzo[a]pyrene and 2-AAF
04420 V-77 10/08/91
-------
produced a significant number of enzyme-altered fod 1n the regenerating
liver. There was a 50% reduction 1n the metabolism of benzo[a]pyrene In the
proliferating liver.
Benzo[a]pyrene 1s carcinogenic when administered s.c. to mice, rats,
hamsters, guinea pigs and some primates (IARC, 1983). Bryan and Shlmkln
(1943) attempted to establish dose-response curves for Induction of tumors
following s.c. administration 1n mice. Tr1capryl1n solutions (0.25 or 0.5
ml) were Injected once only Into the right axilla of male C3H mice, and
animals were observed throughout their lifetime. No controls were reported,
but several no effect doses were observed (Table V-13). In addition to
Injection site sarcomas, newborn mice administered benzo[a]pyrene s.c.
developed hepatomas or lung adenomas (Pletra et al., 1961; Roe and Waters,
1967. Toth and Shublk, 1967; Grant et al., 1968).
Peralno et al. (1984) treated newborn (day 1) Sprague-Dawley rats by a
single 1.p. Injection of 0.59 vmole benzo[a]pyrene/kg bw. At day 21 the
animals were weaned and placed on a SOX casein diet containing 0.05X pheno-
barbltal, a known promoter of hepatic neoplasms. Animals were killed at
Intervals up to ~500 days, and their livers were examined for hlstocheml-
cally detectable foci of altered hepatocytes as well as for hepatic tumors.
Hepatic tumor Incidence 1n females was -57% and ~37X In males. Focus
Incidence had reached 100% 1n both males and females by day 200.
Benzo[a]pyrene Is among the most potent and best documented of skin car-
cinogens. It 1s routinely used as a positive control 1n skin painting bio-
assays of other materials. Skin tumors have been produced by benzo[a]pyrene
04420 V-78 10/08/91
-------
TABLE V-13
Sarcomagenlc Activity of Benzo[a]pyrene 1n Male C3H Mice
Following a Single Subcutaneous Injection*
Dose (mg)
0.00195
0.0078
0.0156
0.031
0.062
0.125
0.25
0.5
1.0
2.0
4.0
8.0
Tumor Incidence
Number Tumor -Bear Ing
Animals/Number
Effective Animals
2/81
0/40
0/19
0/16
4/20
15/19
14/21
19/19
18/20
19/19
16/19
20/21
Percentage
2
0
0
0
20
79
67
100
90
100
84
95
*Source: Bryan and Sh1mk1n, 1943
04420
V-79
10/08/91
-------
1n mice, rats, rabbits and to a lesser extent 1n guinea pigs. In mice the
tumoMgenlc dose 1s dependent upon the solvent used for delivery and on the
strain of mice (IARC, 1973). Benzo[a]pyrene Is both an Initiator and a
complete carcinogen. For example, Nesnow et al. (1986) studied the effects
of dermal treatment with benzo[a]pyrene on 7- to 9-week-old SENCAR mice.
The benzo[a]pyrene was applied as a single topical treatment 1n 0.2 ml of
acetone and as five dally doses of 2 mg each. One week after treatment, 2.0
vg of the tumor promoter TPA was administered topically twice weekly. The
results are presented 1n Table V-14.
Perelra et al. (1979) applied [3H]benzo[a]pyrene to shaved female HA/ICR
mice; the mice were sacrificed 7 hours later. [8H]Benzo[a]pyrene
metabolite conjugated epidermal DMA, Isolated from these treated mice, was
found to contain two major benzo[a]pyrene-DNA adducts. The maximum
concentrations of both aducts occurred 7 hours after a single dermal
application. Benzo[a]pyrene-DNA adduct formation occurred 1n proportion to
dose at doses several orders of magnitude below doses that normally yield a
carcinogenic response.
f r
In an analysis of two separate sk1n-pa1nt1ng experiments using benzo[a]-
pyrene 1n acetone, Lee and O'Neill (1971) showed that the Incidence rate of
both tumors and Infiltrating carcinomas could be described by the equation
2 k
d (t-w) where t Is time from first application, w and k are dose
Independent constants and d Is the applied dose. In the first experiment 75
female albino mice/group (16 total groups) received either 6, 12, 24 or 48
yg of benzo[a]pyrene/week 1n either 2 applications/week, 4 applications/
week and 2 groups that were administered 3 applications/week. One group was
04420 V-80 10/24/91
-------
ro
o
i
03
TABLE V-14
SENCAR Mouse Skin Tumor1genes1s, Benzo[a]pyrene - Tumor Initiation3
Dose
(Mg/mouse)
0
0
2.52
2.52
12.6
12.6
50.5
50.5
101
101
Sex
M
F
M
F
M
F
H
F
M
F
No. Mice
Surviving
37
39
40
39
40
37
39
40
38
38
Mice with
Pap1l1omasb
(X)
8
5
45
31
73
57
100
75
95
97
Paplllomas/
Mouse1*
0.08
0.05
0.50
0.44
1.8
1.1
5.8
2.8
10.2
7.9
Mice with
Carcinomas
(X)
5
0
5
5
20
23
25
20
30
25
Carcinomas
per Mouse0
0.05
0
0.07
0.05
0.20
0.23
0.25
0.20
0.33
0.25
o
CO
aSource: Nesnow et al., 1986
bScore at 6 months
cCumulat1ve score after 1 year
-------
dosed on MWF and the second on TWF. In the second experiment, four groups
of 40 albino female mice were treated with either 1, 3, 9 or 27 vg of
benzo[a]pyrene every fourth day.
Several electrophlUc metabolites of benzo[a]pyrene are skin carcino-
gens. These Include dihydrodlols, dlol-epoxldes and some phenolic deriva-
tives (Gelboln, 1980).
Benzo[a]pyrene has also been reported to be carcinogenic when adminis-
tered by the following routes: 1.v.; transplacentally; Implantation 1n the
»
stomach wall, renal parenchyma, and brain; Injection Into the renal pelvis;
and vaginal painting (IARC, 1983).
Groups of partially hepatectomlzed male Sprague-Oawley rats were given
either a single 30 mg benzo[a]pyrene 1.p. Injection 24 hours after the
partial hepatectomy (10 rats), repeated Intragastrlc Injections of 4 mg
benzo[a]pyrene for 6 days (15 rats) or a single olive oil Injection 24 hours
after .the partial hepatectomy (10 rats In the control group) (KUagawa et
a!., 1980). All groups were fed a diet containing 0.05X phenobarbltal
beginning 2 weeks after the partial hepatectomy. The animals were
sacrificed at 52 weeks and were examined for hepatic tumors. In both groups
receiving benzo[a]pyrene, many rats died of abdominal wall sarcomas prior to
52 weeks. In* fact 0/10 rats Injected with benzo[a]pyrene survived until
week 52. Of the rats that received Intragastrlc Injections, 6/7 had hepatic
tumors and 0/9 In the control group had hepatic tumors. It was reported
that there were many enzyme-altered foci In the livers of rats receiving
Intragastrlc benzo[a]pyrene Injections.
04420 V-82 10/08/91
-------
active as the parent compound, whereas the 9,10-dlol of benzo[J]fluoranthene
was less active than Its parent compound (see Table V-15). The 8,9-d1ol of
benzo[k]fluoranthene was not tumorlgenlc. The 9,10-dlol of benzo[b]fluor-
anthene 1s a potential precursor to a bay region dlhydrodlolepoxlde. The
benzo[j]fluoranthene 9,10-dlol could be metabolized to d1ol-epox1de with the
epoxlde ring In a four-sided "pseudo bay region." Taken together, these
results suggest that the formation of bay region dlhydrodlol epoxldes may
not be the major activation mechanism In benzofluoranthene tumor 1 genesis.
This work Is corroborated ,by the reports of Amln et al. (1985a,b).
Initiation/promotion protocols using benzo[b]fluoranthene and benzo[k]-
fluoranthene were carried out In Crl:CD-1(ICR)BR albino female mice. Each
Initiating compound was applied every other day In 10 doses as acetone
solutions to the shaved backs of 20 mice/treatment group. Ten days after
completion of the Initiating treatment promotion was begun using thrice
weekly applications of 2.5 yg of TPA (dissolved In 0.1 ml acetone).
This was continued for 30 weeks. Results summarized 1n Table V-18 Indicate
that both benzo[b]fluoranthene and benzo[k]fluoranthene can serve as
Initiators of cardnogenesls. Benzo[b]fluoranthene, however. Is more potent
producing the same Incidence of tumors with 1/100 the dose necessary to
Initiate tumors with benzo[k]fluoranthene.
Injection site sarcomas were observed 1n 18/24 survivors of a total
group of 16 male and 14 female strain XVIInc/Z mice given three s.c. Injec-
tions of 2.6 mg benzo[-b]fluoranthene over a period of 2 months (Lacassagne
et al., 1963).
04420 V-83 05/14/91
-------
TABLE V-15
Cardnogenldty of Benzofluoranthenes by Implantation 1n Rat Lungs3
Treatment
Untreated
Pelletb
Benzofb]-
fluoranthene
Benzo[k]-
fluoranthene
Benzo[g,h,1.]-
perylene
Dose
(mg)
Total ,
—
—
0.1
0.3
1.0
0.16
0.83
4.15
0.16
0.83
4.15
Median
Survival
Time
(weeks)
118
104
110
113
112
114
95
98
109
114
106
Ep1dermo1d
Carcinomas
0/35
0/35
0/35
1/35
9/35
0/35
3/31
12/27
0/35
1/35
4/34
Sarcomas
0/35
0/35
1/35
2/35
4/35
0/35
0/31
0/27
0/35
0/35
0/34
Tumor
Incidence
0/35
0/35
1/35
3/35
13/35
0/35
3/31'
12/27
0/35
1/35
4/34
aSource: Deutsch-Wenzel et al., 1983
bRefers to beeswax and trloctanoln Implanatatlon medium
04420
V-84
10/08/91
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TABLE V-16
Carc1nogen1c1ty Assay of PAH 1n Newborn M1cea
I
oo
en
O
OO
Tumor Inc1denceb
Compound
DMSO
Benzo[a]pyrene
Benzo[b]f luoranthene
Benzo[J ]f luoranthene
Benzo[k]f luoranthene
Indenofl ,2,3-cd]pyrene
Total
Dose
(iimol)
1.1
0.5
1.1
2.1
2.1
Sex
H
F
M
F
M
F
H
F
H
F
H
F
Hepatic Tumors
Adenomas
0/17
0/18
9/17
0/14
6/15
0/17
8/21
0/18
2/16
0/18
0/11
0/9
Hepatomas
1/17
0/18
4/17
0/14
2/15
0/17
3/21
0/18
1/16
0/18
0/11
0/9
Total
1/17
0/18
13/17C
0/14
8/1 5C
. 0/17
11/21C
0/18
3/16
0/18
0/11
0/9
Lung
Adenomas
0/17
0/18
14/17C
9/14
2/15
3/17
11/21C
4/1 8d
1/16
3/18
1/11
0/9
aSource: LaVole et al., 1987
^Denominator denotes animals surviving until at least 35 weeks.
cp<0.005
dp<0\05
-------
controls were run (Wynder and Hoffman, 1959b). As part of the same study,
20 Swiss mice were treated with benzo[k]fluoranthene. No tumors developed
1n animals painted with the 0.1% solution, but skin paplllomas were observed
1n 2/20 mice treated with 0.5X benzo[k]fluoranthene. By contrast to this
study, no significant Increase 1n tumor Incidence was observed when 40
female NMRI mice were given skin applications of 3.4, 5.6 or 9.2 yg
benzo[k]fluoranthene 2 times/week for their lifetime. No effect on
mortality was noted as a consequence of this treatment (Habs et al., 1980).
A single application of 11 mg benzo[k]fluoranthene did not Induce tumors
1n 20 Swiss mice 1n a 63 week study. When this Initiating Hose was followed
by promoting treatments with croton resin, 18/20 animals developed papll-
lomas and 5/20 carcinomas (Van DQQren et al., 1966).
The tumor Initiating activities of benzo[b]fluoranthene, benzo[j]fluor-
anthene and benzo[k]f!uoranthene and three of their dlhydrodlols (9,lO-d1-
hydro-9,lO-d1hydroxybenzo[b]fluoranthene, 9,lO-d1hydro-9,lO-d1hydroxybenzo-
[Jjfluoranthene and 8,9-d1hydro-8,9-d1hydroxybenzo[k]fluoranthene) were
evaluated after application to the shaved backs of Crl:CO-l mice (LaVole et
al., 1982a). Each compound was applied 1n acetone solution (0.1 ma,) to
the backs of 20 animals/group. Controls received acetone alone. Three
Initiating dose levels, 10, 30 and 100 yg for benzo[b]fluoranthene and 30,
100 and 1000 yg for the other two compounds (10 doses, every other day),
were used followed by 2.5 yg TPA (3 times weekly for 20 weeks) (Table
V-17). This study demonstrated that of the compounds tested, benzo[b]-
fluoranthene was the most potent tumor Initiator followed by benzo[J]fluor-
anthene. Benzo[k]fluoranthene also showed tumor Initiating activity but was
not a complete carcinogen. The 9,lO-d1ol of benzo[b]f!uoranthene was as
04420 V-86 10/08/91
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TABLE V-17
Tumor Initiating Activity of Benzofluoranthenes 1n Crl:CD-l M1cea
Compound
Benzo[b]f luoranthene
B[b]F-9,lO-d1ol
Benzo[ j ]f luoranthene
B[J]F-9,lO-d1ol
-\
Benzo[k]f luoranthene
B[k]F-8,9-d1ol
Acetone
Total
Initiating .
Dose
(yg)
100
30
10
100
30
10
1000
100
30
1000
100
30
1000
100
30
1000
100
30
-
Percent
Skin Tumor -
Bearing
An1malsb
80
60
45
95
63
26
95
55
30
84
20
5
75
25
5
10
10
15
0
Skin
Tumors/
Animal
7.1
2.3
0.9
8.8
3.8
1.0
7.2
1.9
0.6
4.5
0.3
0.1
2.8
0.4
0.1
0.4
0.1
0.1
0
Other
Tumors
0
"|C
id
0
0
ld
lc
0
0
0
le
0
ld
0
0
0
0
0
0
aSource: LaVole et al., 1982a.
times/week for 20 weeks.
Promoting treatment was 2.5 >ig TPA, 3
bSk1n tumors were predominantly squamous cell paplllomas
cMa!1gnant lymphoma
dEndometr1al carcinoma of uterus
eLung adenoma
04420
V-87
10/08/91
-------
active as the parent compound, whereas the 9,lO-d1ol of benzo[J]f1uoranthene
was less active than Us parent compound (see Table V-17). The 8,9-d1ol of
benzo[k]fluoranthene was not tumorlgenlc. The 9,lO-d1ol of benzo[b]fluor-
anthene Is a potential precursor to a bay region dlhydrodlolepoxlde. The
benzo[j]fluoranthene 9,lO-d1ol could be metabolized to d1ol-epox1de with the
•
epoxlde ring In a four-sided "pseudo bay region." Taken together, these
results suggest that the formation of bay region dlhydrodlol epoxldes may
not be the major activation mechanism In benzofluoranthene tumor 1 genesis.
In another skin painting assay benzo[b]fluoranthene> 1n acetone was
applied to the skins of 20 female CD-I mice/group for a total dose of either
0, 1.0 or 4.0 ymol (a total of 10 subdoses were applied every other day)
(Weyand et al., 1990). Ten days after the final dose, 2.5 yg of
tetradecanoylphorbol acetate was applied 3 times/week for 20 weeks. In the
acetone control group, 2/20 mice developed skin tumors; the average number
of skin tumors/mouse was 0.1. In both benzo[b]fluoranthene groups 20/20
mice developed skin tumors; the average number of skin tumors/mouse was 8.5
and 11.0 In the low- and high-dose groups, respectively.
This work 1s corroborated by the reports of Am1n et al. (1985a,b).
Initiation/promotion protocols using benzo[b]fluoranthene and benzo[k]-
fluoranthene were carried out 1n Crl:CD-l(ICR)BR albino female mice. Each
Initiating compound was applied every other day 1n 10 doses as acetone
solutions to the shaved backs of 20 mice/treatment group. Ten days after
completion of the Initiating treatment promotion was begun using thrice
weekly applications of 2.5 wg of TPA (dissolved In 0.1 ml acetone).
This was continued for 30 weeks. Results summarized 1n Table V-18 Indicate
04420 V-88 11/12/91
-------
TABLE V-18
Tumor Initiating Activity of Benzofluoranthenes 1n Crl:CD-l(ICR)BR M1cea
Compound1*
Acetone
Benzo[b]fluoranthene
Acetone
Benzo[b]fluoranthene
Acetone
Benzo[k]fluoranthene
Total
Initiating
Dose
(ymol)
—
40
100
—
40
100
—
4000
Percent
Animals
with Tumors
10
45
95
5
42
53
0
37
Skin Tumors/
Animal
0.2
0.9
3.3
0.1
0.5
0.9
0
. 0-7
aSource: Am1n et al., 1985a,b
bEach treatment Is presented with Us concurrent control. Promoting
treatment consisted of thrice weekly applications of 2.5 yg TPA for 30
weeks.
04420 V-89 10/08/91
-------
that both benzo[b]fluoranthene and ,benzo[k]fluoranthene can serve as
Initiators of cardnogenesls. Benzo[b]fluoranthene, however. Is more potent
producing the same Incidence of tumors with 1/100 the dose necessary to
Initiate tumors with benzo[k]fluoranthene.
Injection site sarcomas were observed In 18/24 survivors of a total
group of 16 male and 14 female strain XVIInc/Z mice given three s.c. Injec-
tions of 2.6 mg benzo[b]fluoranthene over a period of 2 months (Lacassagne
et al., 1963).
Benzofg.h.l.lperylene. Benzo[g,h,1]perylene did not 'produce a sig-
nificant Increase 1n tumor Incidence (see Table V-15) when Implanted as
beeswax trloctanoln pellets 1n lungs of female OH rats (Deutsch-Wenzel et
al., 1983). Although a few pulmonary tumors were observed 1n rats after
Intrapulmonary Injection, the data were considered Inadequate for evaluation
(IARC, 1983).
No Increased Incidence of tumors was reported from two sk1n-pa1nt1ng
bloassays of benzo[g,h,1]perylene conducted In female Swiss or Ha/ICR/mll
Swiss mice (Hoffman and Wynder, 1966; Wynder and Hoffman, 1959b). Three
Initiation promotion assays for skin tumorlgenesls 1n mice were likewise
negative. Benzo[g,n,1]perylene did not produce Injection site tumors 1n
either of two studies wherein mice were exposed subcutaneously (IARC, 1983).
Van DQQren et al. (1973) reported some evidence that benzo[g,h,1]-
perylene served as a cocardnogen with benzo[a]pyrene when both were applied
simultaneously to the skin of ICR/Ha Swiss mice.
04420 V-90 10/08/91
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Chrvsene. As part of a study of nitrated PAHs, chrysene 1n DMSO was
administered 1.p. to CD-I mice on days 1, 8 and 15 of age. Tumor Incidences
were recorded after 1 year. A total dose of 2800 nmol/mouse resulted In
hepatic and lung tumors In 41 and 21X of the males, respectively. A total
dose of 700 nmol Induced liver tumors In 29% of the males; females were
unaffected (W1slock1 et al.. 1986).
A series of early skin painting assays conducted 1n mice produced
variable results. It was noted that 1n these bloassays the chrysene was
likely to be contaminated with methylchrysene or other materials not
detectable by the technology then available (IARC, 1983).
As part of a study of tobacco constituents, female Swiss mice were
treated by brushing a IX acetone solution of chrysene on the skin 3
times/week. No solvent control was reported, but 45X of the treated animals
developed paplllomas and 40% were observed to have carcinomas (Wynder and
Hoffman, 1959a).
/
"Specially purified" chrysene was applied topically to 20 male C3H mice
as either a decahydronaphthalene solution or as a 50:50 mix with n-dodecane.
Applied alone, chrysene Induced a papllloma 1n 1/12 mice at 76 weeks. In
combination with n-dodecane 5/19 mice were observed to have paplllomas and
12/19 bore carcinomas at 49 weeks (Norton and Christian, 1974).
Chrysene was assayed In a mouse skin Initiation/promotion assay using
ICR/Ha Swiss mice. Paplllomas were Induced In 5/20 animals treated with
croton resin only and In 16/20 mice receiving croton oil preceded by a
single application of 1 mg chrysene 1n acetone (Van DQQren et al., 1966). A
04420 V-91 10/08/91
-------
second study using pure TPA as the promoter yielded similar results. Female
CD-I mice received a single topical application of 1 mg of chrysene In
acetone, followed by 25 yg TPA (1n 0.1 ml acetone) 2 times/week. At 30
weeks, 3% of the promoter-only mice were observed to have papHlomas,
whereas 73X of the chrysene-treated animals developed these growths
(ScMbner, 1973).
Further evidence of chrysene's potential as an Initiator of skin
cardnogenesls comes from studies by Hecht et al. (1974) and Levin et al.
(1978). In the first study, 20 female Swiss mice (Ha/ICR/M1l) were given 10
dally treatments with 100 yg chrysene In acetone. After 'a 10-day resting
period, 2.5 yg TPA In acetone was applied 3 times/week for 20 weeks. A
group receiving chrysene only was observed at 72 weeks to have a carcinoma
Incidence of 4/11 as compared with an Incidence of 11/18 (carcinomas and
paplllomas) 1n the chrysene plus TPA group (Hecht et al., 1974).
Levin et al. (1978) used female CD-I mice, 30/group, In an assay wherein
a single topical application of a 0.4, 1.25 or 4.0 ymol solution of
chrysene 1n tetrahydrofuran:DMSO (95:5) was followed by twice weekly appli-
cations of TPA (200 pi of a 16 ymol solution In acetone). A second
group receiving TPA alone had a papllloma Incidence of 7X (0.07 tumors/
mouse). Tumor Incidence for the low, medium and high chrysene dose groups
receiving TPA were the following: 25, 43 and 52X with 0.32, 0.97 and 1.45
tumors/mouse, respectively.
Three Initiation promotion studies 1n female CD-I mice also were
positive for chrysene Initiating activity. Wood et al. (1979) reported an
Incidence of 21/30 for observation of paplllomas In mice given a single
04420 V-92 10/08/91
-------
application of 200 vl of a 2 ymol chrysene solution In acetonerDMSO:
ammonium hydroxide (1000:100:1), followed by twice weekly applications TPA
(200 pi of 16 ymol 1n acetone). Equivalent doses of the bay region
3,4-epoxy-l,2,3,4-tetrahydrochrysene and Us precursor metabolite l,2-d1-
hydrochrysene produced essentially the same papllloma Incidence. Control
animals treated with acetone followed by TPA had a tumor Incidence of 1/30.
These data support the bay-region theory of cardnogenesls that suggests
epoxldes on saturated angular benzo rings which form part of a bay-region
are particularly susceptible to undergoing ring opening to an electrophHlc
carbonlum Ion.
A second experiment by Wood et al. (1980) employed chrysene of higher
purity (98X). Thirty mice were treated with 200 yl of an acetone solution
of this chrysene preparation and subsequently with TPA (200 yi of 16
ymol solution) 2 times/week for 25 weeks. The acetone plus TPA controls
were observed to have a tumor Incidence of 4X (0.04 papHlomas/mouse); the
chrysene plus TPA group Incidence was SOX (2.16 tumors/mouse).
/
In a third study, Rice et al. (1985b) Initiated 25 CD-I mice by appli-
cation of 100 yg chrysene 1n acetone every other day for a total of 10
times (1.0 mg total dose). This was followed by thrice weekly applications
of TPA beginning 10 days after completion of the Initiation phase and
continuing for 20 weeks. All animals thus treated survived, and 92% were
observed to bear tumors.
Sencar mice were likewise sensitive to chrysene Initiation. Paplllomas
were observed 1n 21/29 mice treated once with 2 ymol chrysene followed by
04420 V-93 10/08/91
-------
twice weekly TPA treatments (2 yg/treatment). TPA only animals had a
papllloma Incidence of 3/30 (Slaga et al., 1980).
Early studies with chrysene reported no Injection site tumors as a
consequence of 1.m. or s.c. administration (Bottomly and Twort, 1934; Barry
and Cook, 1934; Shear and Lelter, 1941). Small numbers of sarcomas at the
site of chrysene Injection were reported for C57B1 male and female mice when
trlcaprylln or arachls oil was used as the vehicle (Stelner and Falk, 1951;
Stelner, 1955; Boyland and S1ms, 1967). PolUa (1941) reported no tumors to
be Induced In rats receiving repeated Injections of chrysene 1n water or
sesame oil, while Barry and Cook (1934) reported four sarcomas 1n 10 rats
repeatedly Injected with 2-6 mg chrysene compared with two sarcomas In
solvent control rats.
Perinatal exposure of mice to chrysene has resulted 1n tumor Induction.
Male and female Swiss mice were Injected s.c. with 100 vg chrysene 1n PEG
on the day of birth and the next 2 days. After 70 weeks tumor Incidences
were the following: one Injection of PEG caused liver tumors 1n 5/20 males
and 3/21 females and lung tumors 1n 2/20 males and 3/21 females; three
Injections of PEG caused liver tumors In 10/30 males and 0/15 females and
lung tumors In 4/30 males and 1/15 females; chrysene caused liver tumors In
13/27 males and 0/21 females and lung tumors 1n 2/27 males and 1/21 females
(Gi-over et al., 1975).
In a similar experiment, Swiss Webster BLuVHa(ICR) mice received l.p.
Injections of a DMSO solution of chrysene on days 1, 8 and 15 of life.
Total administered dose was 1.4 vmol. By 38-40 weeks treated animals had
04420 V-94 10/08/91
-------
developed lung tumors (5/24 males, 2/11 females) and hepatic tumors (6/24
males), and one lymphosarcoma was observed 1n a male mouse. DMSO-treated
animals developed only lung tumors (2/21 males, 7/38 females) (Buenlng et
al., 1979a). The same protocol was applied 1n a graded dose bloassay
wherein the three 1.p. Injections were of 0.2, 0.4 and 0.8 pmol repurlfled
chrysene. There was no Increase 1n pulmonary tumors, but 22% of the treated
male mice developed hepatic tumors compared with no hepatic tumors observed
1n the DMSO controls (Chang et al, 1983).
D1benz[a.hlanthracene. Pulmonary administration of d1benz[a.h]anthra-
cene has Induced lung adenomas 1n mice (Andervont, 1937; Rask-N1e1son, 1950,
Kuschner et al., 1956). Intratracheal Instillation of a d1benz[a,h]anthra-
cene suspension 1n protein blood substitute with powdered India Ink resulted
1n the development of squamous cell carcinomas (Yan1sh1va and Balenko,
1966). Single 1.v. Injections of a colloidal dispersion of this material
were also tumorlgenlc 1n a dose-dependent fashion In strain A mice (Heston
and Schnelderman, 1953).
/
Kennaway (1930) was the first of many researchers to report Induction of
skin tumors by d1benz[a,h]anthracene. In another study, a 0.2% solution of
d1benz[a,h]anthracene 1n acetone/benzene was painted twice weekly on the
skin of Swiss mice (38 yg/dose). Skin tumors were observed 1n 16/20
animals (Lljlnsky et al., 1965). Van OQQren et al. (1967) topically exposed
ICR/Ha Swiss mice 3 times/week to acetone d1benz[a,h]anthracene solutions at
the following concentrations: 0.001, 0.01 and 0.1X. Tumor Incidences were
1/30 (1 carcinoma), 43/50 (39 carcinomas) and 39/40 (32 carcinomas),
respectively. The authors noted that latency was also dose related.
04420 V-95 10/08/91
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D1benz[a,h]anthracene was compared with benzo[a]pyrene as to potency 1n
Inducing skin tumors 1n Swiss mice. Repeated skin exposure to a 0.001%
acetone solution of benzo[a]pyrene Induced paplllomas 1n 43% of the mice and
carcinomas 1n 3%. The same d1benz[a,h]anthracene exposure resulted In a
papllloma Incidence of 30% and a carcinoma Incidence of 30%. Exposure of
flilce to a 0.01% solution of either compound resulted 1n papllloma and
*
carcinoma development In >90% of the animals. Latent periods for Induction
were similar for both carcinogens (Wynder and Hoffman, 1959a).
D1benz[a,h]anthracene has been reported to Initiate skin tumor develop-
ment In mice at doses as low as 0.02 pg given once (Klein, .1960). Other
tumors such as paplllomas and pulmonary tumors may also arise as a conse-
quence of skin exposure to d1benz[a,h]anthracene (Buenlng et al., 1979b).
Virgin C3H (Jax) mice were painted twice weekly with 0.25% d1benz[a,h]-
anthracene 1n benzene (thlophene-free) for their lifetime. A 50% Incidence
of mammary tumors was observed 1n the control animals as compared with 10/11
1n the treated mice (Ranadlve and Karande, 1963).
In one study hamsters appeared to be somewhat more resistant to the skin
tumor!genie properties of d1benz[a,h]anthracene. A group of 10 Syrian
golden hamsters received 20 applications of 0.2% d1benz[a,h]anthracene In
mineral oil over a 10 week period. At 50 weeks, 5 animals survived, but no
tumors were observed (Shublk et al., 1960).
Several assays have been reported wherein d1benz[a,h]anthracene has been
administered s.c. or 1.m. Tumors at Injection sites have been reported 1n
rats, guinea pigs (low Incidence), pigeons and unspecified fowl (Roussy et
al.. 1942; Shabad, 1938; Shabad and UMnson, 1938; PMchard et al., 1964;
04420 V-96 10/08/91
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Peacock, 1935). Mice are the animals most studied 1n this regard. Reports
Included those by Hartwell (1951), Shublk and Hartwell (1957, 1969) and
Thompson and Co. (1971).
The effective dose for s.c. Induction of tumors was established by Bryan
and Shlmkln (1943). TMcaprylln solutions of d1benz[a,h]anthracene were
administered once to groups of >19 C3H mice so as to deliver the following
doses: 0.0019, 0.0078, 0.016, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4 or 8
mg. Incidences of Injection site sarcomas were as follows: 2/79, 6/40,
6/19, 16/21, 20/20, 21/23, 19/21, 20/21, 22/22, 19/19, 17/20 and 16/21,
respectively. It was noted that the lowest effective dose for. d1benz[a,h]-
anthracene was 0.0019 mg while that for 3-methylcholanthrene was >0.0039 mg.
The authors also observed that the average latent period for d1benz[a,h]-
anthracene sarcoma development was 3.7 months as compared with 2-5 months
for 3-methylcholanthrene and 3 months for benzo[a]pyrene. Similar results
were reported by Oobrovolskala-Zavadska'ia (1938) and Lettlnga (1937).
/
In an abstract, Platt et al. (1983) reported that d1benz[a,h]anthracene
showed stronger carcinogenic responses when administered s.c. to NMRI mice
than did the following metabolites: 3,4-d1ol, 5-phenol, 5,6-ox1de. The
l,2-d1ol and 5,6-d1ols of d1benz[a,h]anthracene were reported to be non-
carcinogenic by this treatment regimen.
Newborn mice (general purpose/NIH) were exposed by s.c. Injections of
d1benz[a,h]anthracene doses between 0.003 and 6.7 yg/mouse. A dose-
related Increase In sarcoma Incidence was noted for doses of >0.08 yg, and
an Increase In lung adenomas was observed for doses >0.2 yg (O'Gara et
al., 1965).
04420 V-97 10/08/91
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Female NMRI mice were observed to develop Injection site sarcomas after
one s.c. treatment of as little as 2.35 yg d1benzo[a,h]anthracene 1n
trlcaprylln (Pfelffer, 1977). No vehicle controls were reported but a group
of."non-carcinogenic" PAHs tested concurrently were observed to have sarcoma
Incidences of 4-13X. Table V-19 compares sarcoma Induction for dlbenzo-
[a,h]anthracene and benzo[a]pyrene.«
Lubet et al. (1983a) found that s.c. Injections of d1benz[a,h]anthracene
were associated with flbrosarcoma development 1n mice, but only for some
strains. Four strains of mice used Included two, C3H/HeJ and C57B1/6J, that
respond to 3-methylcholanthrene treatment by Increased levels and types of
hepatic enzymes Including AHH. Two strains, AKR/J and DBA/2J, were non-
responders. Groups of 30 animals were Injected with a single dose of 150
vg d1benz[a,h]anthracene 1n 0.05 ml trloctanoln and observed for 9
months. A control group for each strain, consisting of 10 animals each,
received a s.c. Injection of 0.05 ml trloctanoln alone. A summary of the
findings Is given 1n Table V-20. The tumor Incidence 1n the treated animals
varied between 0 and 80%, depending on the strain. Tumor Incidences were
higher 1n the C3H and C57B1 mice, which also were readily Indudble for AHH.
Likewise, the average latency period (In days) for flbrosarcoma development
varied with the strain and tended to be Inversely correlated with the tumor
Incidence rate. The authors concluded that, as for benzo[a]pyrene, the Ah
receptor was Involved 1n the process of tumor Induction by s.c. Injection
for d1benz[a,h]anthracene.
Fluoranthene. Fluoranthene was first tested for carcinogenic activity
more than 5 decades ago (Barry et al., 1935). The results from that
04420 V-98 10/08/91
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TABLE V-19
Sarcomagenlc Activity of Subcutaneously Injected D1benzo[a,h]anthracene
and Benzo[a]pyrene 1n Female NMRI Mice*
Compound
D1benzo[a,h]anthracene
Benzo[a]pyrene
Dose (tig)
2.35
4.7
9.3
18.7
37.5
75.0
3.12
6.25
12.5
25.0
50.0
100.0
Tumor Incidence
37/100
39/100
44/100
56/100
65/100
69/100
9/100
35/100
51/100
57/100
77/100
83/100
*Source: Pfelffer, 1977
04420
V-99
10/08/91
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TABLE V-20
Incidence of Flbrosarcomas 1n Mice Associated with Subcutaneous
Injections of D1benz[a,h]anthracenea
Strain
C3H/HeJ
C57B1/6J
AKR/J
DBA/2J
Treatmentb
D1benz[a,h]anthracene
Control
D1benz[a,h]anthracene
Control
01benz[a,h]anthracene
Control
D1benz[a,h]anthracene
Control
Tumor
, Incidence
24/30
0/10
16/30
0/10
0/30
0/10
1/30
0/10
X
80
0
33
0
0
0
3
0
Average
Latency
(days)
165
0
242
0
0
0
230
0
aSource: Lubet et al., 1983a
bAn1mals In the d1benz[a,h]anthracene group received a single Injection of
150 yg of d1benz[a,h]anthracene In 0.05 ml of trloctanoln. Control
animals received an Injection of 0.05 mi trloctanoln alone.
04420
V-100
10/08/91
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Investigation, and from several studies conducted since that time, Indicated
that fluoranthene had virtually no activity as a complete carcinogen.
Suntzeff et al. (1957) administered a 10% solution of fluoranthene In
acetone 3 times weekly by topical application to CAP, Jackson, Swiss and
MUlerton mice. No tumors were found by 13 months.
Wynder and Hoffmann (1959a) administered a 0.1X solution of fluoranthene
In acetone to the backs of 20 female Swiss (Mlllerton) mice 3 times/week for
life. No tumors were found.
Hoffmann et al. (1972) administered 50 vl of a IX fluoranthene
solution to the backs of 20 female Swiss-Albino Ha/ICR/MUl mice 3 times/
week for 12 months. All treated mice survived, and no tumors were
observed. As part of the same study, 30 mice received 0.1 mg fluoranthene
1n 50 yl acetone every second day for a total of 10 doses; 10 days later
promotion with 2.5X croton oil 1n acetone was started and continued for 20
/
weeks. A single papllloma was noted 1n 29 surviving mice.
Morton and Christian (1974) administered 50 mg fluoranthene 1h decalln
or In decal1n:n-dodecane (50:50) to the backs of 15 male C3H mice. The mice
were treated 2 times/week for 82 weeks. No skin tumors were observed.
Van Ouuren and Goldschmldt (1976) administered 40 yg fluoranthene 1n
acetone- 3 times weekly for 440 days to female ICR/Ha Swiss mice. No skin
tumors were observed.
04420 V-101 10/08/91
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Barry et al. (1935) administered four doses of 10 mg fluoranthene In
glycerol by subcutaneous Injection to strain A mice. Six out of 14 mice
survived for 18 months; no tumors were found by 19 months.
Shear (1938) administered four doses of 10 mg fluoranthene 1n glycerol
by subcutaneous Injection to strain A mice. Six out of 14 mice survived for
18 months; no tumors were found by 19 months.
Fluoranthene was tested by Busby et al. (1984) 1n a mouse lung adenoma
assay. Newborn male and female mice of Swiss-Webster BLU:Ha strain were
Injected IntraperHoneally with a total dose of 700 yg (163 mg/kg) or 3.5
mg (815 mg/kg) fluoranthene In three Installments on days 1, 8 and 15 (1/7,
2/7 and 4/7 of the dose, respectively). H1stopatholog1c examination of the
lungs of the mice sacrificed at 24 weeks showed a significant Increase 1n
the high-dose group 1n the total Incidence and number/mouse (28/48 or 58%,
1.08 tumors/mouse) of lung tumors compared with vehicle controls (5/55 or
9%, 0.09 tumors/mouse). Tumor response 1n the low-dose group (10/51 or 20%,
0.24 tumors/mouse) was not statistically significant. A positive response
In a lung adenoma assay 1n the absence of corroborating studies Is generally
considered Insufficient evidence of carc1nogen1c1ty.
Fluorene. Studies of fluorene as a complete mouse skin carcinogen and
as a cocardnogen with 3-methylcholanthrene were negative or Inconclusive
(Kennaway, 1924a; Rlegel et al., 1951). IARC (1983) considered both reports
Inadequate for evaluation.
04420 V-102 10/08/91
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Fluorene was also found to be Inactive both as a tumor Initiator (with
TPA as a promoter) and a complete carcinogen 1n skin-painting bloassays
performed 1n female Ha/ICR mice by LaVole et al. (1979, 1981b). No
Injection site tumors were produced 1n 10 strain A mice after seven s.c.
Injections of 10 mg fluorene 1n glycol. The study was terminated at 18
months (Shear, 1938).
IndenoH,2.3-cdlpyrene. Indeno[l,2,3-cd]pyrene was administered to
female OH rats by Implantation 1n the lung of beeswax-tMoctanoln pellets
containing 0.16, 0.83 or 4.15 mg of the compound. Results summarized In
Table V-21 Indicate a dose-dependent Increase In keratlnlzed epldermold
carcinomas capable of Invading the extrapulmonary chest wall. There was no
Increase In ple1omorph1c sarcomas. A calculated potency from this assay
relative to that of benzo[a]pyrene (=1.00) was 0.08 (Deutsch-Uenzel et al.,
1983).
By contrast to these results, 1ndeno[l,2,3-cd]pyrene was not tumorlgenlc
/
1n newborn CD-I mice treated 1.p. on days 1, 8 and 15 of life (total dose
2.1 vmol, see Table V-16) (LaVole et al., 1987).
Groups of 20 female Swiss albino Ha/ICR/MH mice were given topical
applications of 1ndeno[l,2,3-cd]pyrene prepared as dloxane or acetone solu-
tions. Dloxane preparations of 1ndeno[l,2,3-cd]pyrene at concentrations of
0.05 or 0.1X did not Induce skin tumors whereas benzo[a]pyrene at the same
concentration produced -tumors In 90X of the treated mice In 7 months. By
contrast, acetone solutions of 1ndeno[l,2,3-cd]pyrene produced skin tumors
In a dose-related fashion. No tumors were observed In animals painted with
04420 V-103 10/08/91
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TABLE V-21
CardnogenlcHy of Indeno[l,2,3-cd]pyrene upon Implantation 1n Rat Lungs3
Treatment
Untreated
Pelletb
Indeno[l,2,3-cd]-
pyrene
Dose
(rug)
—
—
0.16
0.83
4.15
Median
Survival
Time
(Weeks)
118
104
116
109
92
Tumor
Ep1dermo1d
Carcinomas
0/35
0/35
3/35
8/35
21/35
Incidence
Sarcomas
0/35
0/35
1/35
0/35
0/35
Total
0/35
0/35
4/35
8/35
21/35
aSource: Deutsch-Wenzel et al., 1983
t
^Refers to beeswax and trloctanoln Implantation medium
04420
V-104
10/08/91
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the 0.01 and 0.05X solutions. The 0.1X treatment Induced six paplllomas and
three carcinomas beginning at 9 months; the 0.5X concentration of Irideno-
[l,2,3-cd]pyrene resulted 1n seven paplllomas and five carcinomas with the
first tumor appearing at 3 months. This paper also reported that a total
dose of 250 yg 1ndeno[l,2,3-cd]pyrene delivered 1n 10 applications was a
sufficient Initiating dose when followed by croton oil promoting treatment
(Hoffman and Wynder, 1966).
Indeno [l,2,3-cd]pyrene was not shown to be a complete carcinogen when
applied to the skin of'female NMRI mice twice a week for their lifetime.
Total doses applied 1n acetone were 0, 3.4, 5.6 or 9.2 mg/an1mal (Habs et
al., 1980).
Solutions of 4.0 yrnol 1ndeno[l,2,3-cd]pyrene and two fluoridated
metabolites, 2-fluoro1ndeno[l,2,3-cd]pyrene and 8,9-d1fluoro1ndeno-
[l,2,3-cd]pyrene, were applied to the shaved backs of 30 female CD-I
•
mice/group every other day for a total of 10 doses. Mice treated with
benzo[a]pyrene and acetone served as positive and negative controls. Ten
days after the last Initiating dose 12-0-tetradecanoylphorbol-13-acetate
(TPA) was applied to the backs of the mice as a promoter 3 times/week for 20
weeks. At the end of the promotion period 1ndeno[l,2,3-cd]pyrene and TPA
treatments Induced tumors 1n 72X of the mice (2.1 tumors/mouse), the
2-fluoro1ndeno[l,2,3-cd]pyrene and TPA treatments Induced tumors In 76X of
the mice (3;9 tumors/mouse) and the 8,9-d1fluoro1ndeno[l,2,3-cd]pyrene and
TPA treatments Induced tumors 1n 40X of the treated mice (0.6 tumors/mouse).
At the end of the Initiation period, DNA from the skins of 5 mice/group was
Isolated. [32]P-postlabel1ng analysis of the hydrolyzed DNA showed that
04420 V-105 10/08/91
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1ndeno[l,2,3-cd]pyrene and 2-fluoro1ndeno[l,2,3-cd]pyrene each form a single
major DNA adduct when analyzed by thin-layer chromatography. These adducts
have different retention behaviors. 8,9-01fIuoro1ndeno[l,2,3-cd]pyrene does
not have this adduct; the authors speculated that this could be one factor
1n the variance of the tumor Incidences of the three compounds (R1ce et al.,
1990). •
As part of a study of Initiating capability of Us major metabolites 1n
mouse skin, 1ndeno[l,2,3-cd]pyrene was applied to the shaved backs of 20
Crl:CD-l(ICR)BR female mice. Acetone solutions were applied, every other day
for 10 days for a total Initiating dose of 1 mg. This was followed 10 days
.later by thrice weekly TPA applications (0.0025X 1n 100 yl acetone) for 20
weeks. Tumor Incidence was essentially 100X. Indeno[l,2,3-cd]pyrene-l,2-
dlol and -l,2-ox1de treatment both resulted 1n 80% tumor Incidence by
comparison with 8-hydroxy (~25X) and the acetone-treated controls (~5X)
(R1ce et al., 1986).
/
An earlier Initiation-promotion bloassay performed by Rice et al.
(1985c) showed a pronounced dose-response relationship. Following the same
protocol described above, there was an 80% tumor Incidence In mice receiving
a total Initiating dose of 1 mg 1ndeno[l,2,3-cd]pyrene with an average of
about four tumors/mouse after 22 weeks of promotion. However, when the
total Initiating, dose was decreased to 100 or 300 yg, the number of
tumor-bearing mice was below statistical significance.
Injection site sarcomas were reported In 10/14 male and 1/14 female
XVIInc/Z mice administered three Injections at 1 month Intervals of 0.6 mg
1ndeno[l,2,3-cd]pyrene 1n olive oil (Lacassagne et al., 1963).
04420 V-106 10/24/91
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Naphthalene. In an Inhalation study, naphthalene (scintillation
grade, purity >99%) vapor was administered at 0 and 10 ppm to 75 B6C3F1
mice/sex/dose for 6 hours/day, 5 days/week for 103 weeks (NTP, 1991b). Two
groups of 75 B6C3F1 mice/sex were administered 30 ppm naphthalene for the
same amount of time as the 0 and 10 ppm groups. In each group 25 mice/sex
were designated for hematology evaluation; 5 mice/sex were to be sacrificed
after 14 days and 3, 6, 12 and 18 months. Only the 14-day hematology mice
were sacrificed 1n the control male mice because of high mortality. When
compared with survival In exposed male groups, survival was decreased 1n the
male control group; the decrease was the result of wound trauma from
fighting among animals caged together. Survival was equivalent In all
female groups. Final body weights were equivalent In all groups and no
clinical observations could be attributed directly to naphthalene.
*
There was a significant Increase 1n the Incidence of alveolar or
bronchlolar adenomas 1n the high-dose females by palrwlse comparison with
controls. The Incidences were 5/68 (7X), 2/64 (3X) and 28/134 (21X) 1n the
t
control, low- and high-dose groups, respectively. The recent historical
control Incidence for females 1n this laboratory 1s 91/1166 (7.8X, range
0-16X) and the recent historical control Incidence for females specifically
In Inhalation studies Is 39/466 (8.4%, range 0-12%). The first Incidence of
alveolar or bronchlolar adenomas was reported on experiment day 714, 736 and
656 In the control, low- and high-dose groups, respectively. In the
high-dose females there was a single Incidence of alveolar or bronchlolar
carcinoma; this was not observed In the other groups. There was also a
statistically significant Increase In the Incidence of respiratory
epithelial cell hyperplasla In the naphthalene-treated groups. Because the
04420 V-107 10/24/91
-------
Incidence of alveolar or bronchlolar adenomas was statistically significant
and above historical control ranges, NTP judged the Increased adenoma
Incidence to be directly related to naphthalene exposure.
There was a statistically significant Increase In the Incidence of
respiratory epithelial cell hypefplasla 1n the naphthalene-treated male
groups. There was no significant Increase In the Incidence of alveolar or
bronchlolar adenomas or carcinomas (either separately or combined). The
Incidences of alveolar or bronchlolar adenomas or carcinomas (combined) were
7/69 (10%), 17/69 (25%) and 31/135 (23%) In the control, low- and high-dose
groups, respectively. The male control Incidences were 94/478 (19.7%, range
10-30%) 1n recent Inhalation studies and 229/1172 (19.5%, range 6-42%) In
all carcinogen studies conducted recently 1n this laboratory. As of this
writing the U.S. EPA has not evaluated this study.
Adklns et al. (1986) exposed groups of 30 six- to elght-week-old female
A/J strain mice by Inhalation to naphthalene at concentrations of 0, 10 or
30 ppm, 6 hours/day, 5 days/week for 6 months. After the 6-month exposure
period, excised lungs were examined for tumors. Naphthalene exposure did
not significantly Increase tumor Incidence, but did cause a statistically
significant Increase (p<0.05) In. the number of adenomas per tumor-bearing
mouse lung. There was no dose-response.
Schmahl (1955) administered naphthalene by 1.p. Injection to a group of
10 rats (In-house strains BDI and BOIII) with another group of 10 rats
serving as controls. The dally dose was 20 mg/rat and Injections were given
weekly for 40 weeks. Animals were observed until spontaneous death.
Hlstologlc examination offered no evidence of carcinogenic effects.
04420 V-108 10/08/91
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Boyland et al. (1964) Implanted naphthalene Into the bladder of stock
Chester Beatty mice (23) In an effort to determine the suitability of
naphthalene as a potential vehicle for cardnogenldty testing. The mice
were observed for 30 weeks. Tumor Incidence was as low as when paraffin wax
was used and lower than with cholesterol. Naphthalene was judged to be
Inert but to have no advantage over cholesterol as a base for Implantation
pellets.
Coal tar-derived naphthalene that contained -10% unidentified Impurities
was tested for cardnogenldty by Knake (1956). White, rats (40, sex
unspecified) were given seven subcutaneous Injections of 500 mg/kg naphtha-
lene In sesame oil at 2-week Intervals. Lymphosarcomas were found 1n 5/34
surviving rats at 18 months (14.7%), whereas vehicle controls had a 2%
Incidence of these tumors. Mice (25, Inbred black) were painted with 0.5%
naphthalene 1n benzene 5 days/week for life. Four treated mice developed
leukemlas In contrast to 0/21 vehicle controls; the untreated control Inci-
dence was 0.4%. The value of these studies for assessing cardnogenldty 1s
very limited due to the presence of potentially carcinogenic Impurities.
Moreover, the vehicle 1n the mouse study has been shown to cause leukemlas,
and the site of Injection 1n the rats study was painted, prior to Injection,
with carbofuchsln, a known carcinogen.
Kennaway (1930) reported that naphthalene was not carcinogenic In skin
painting studies 1n mice. The concentration, purity, dosing regimen, and
other details were not provided. The reaction product of naphthalene and
aluminum trichloride was reported to be carcinogenic, but the product was
not Identified.
04420 V-109 10/08/91
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Schmeltz et al. (1978) tested the carcinogenic activity of benzo[a]-
pyrene and naphthalene In female ICR/HA (Sprague-Dawley) mice. A 100 vi
solution containing 0.25% naphthalene and 0.003% benzo[a]pyrene was painted
on the shaved backs of 30 mice 3 times/week for 78 weeks. Naphthalene
Inhibited benzo[a]pyrene-1nduced tumors; -42% of the mice treated with
benzo[a]pyrene alone had skin tumors whereas -20% of mice had skin tumors
when naphthalene and benzo[a]pyrene were administered together.
Phenanthrene. Data on phenanthrene's potential as a skin carcinogen
have been summarized by IARC .(1983). Phenanthrene did .not serve as a
complete carcinogen In two studies 1n mice that were Incompletely reported
(Kennaway, 1924b; Roe and Grant, 1964). Phenanthrene -was reported to be
active as an Initiator 1n female CD-I mice 1n one study (ScMbner, 1973).
Thirty-five weeks after Initiation with 10 ymol phenanthrene and twice
weekly treatments with 5 ymol TPA, 12/30 mice developed paplllomas as
compared with 0/30 TPA-treated controls. Phenanthrene. however, was
Ineffective as an Initiator of skin tumorlgenes 1s In Swiss Ha/ICR, albino, S
*•
and CD-I mice (LaVole et al., 1981a; Roe, 1962; Salaman and Roe, 1956; Wood
et al., 1979) and Inactive as a promoter In mice of unspecified strain (Roe
and Grant, 1964). It was noted by Wood et al. (1979) that 30 female CD-I
mice given one topical application of 10 ymol phenanthrene or the 1,2- or
3,4-d1hydrod1ol followed by twice weekly applications of 16 ymol TPA (In
200 yi acetone) were observed to have papllloma Incidences 2-4 times that
of background. These Incidences were not significantly Increased by
comparison with controls because of the small number of animals tested and
the spontaneous tumor Incidence.
04420 V-110 10/08/91
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Subcutaneous administration of phenanthrene to mice did not result In
treatment-related Increases In tumor Incidence (Stelner, 1955; Roe, 1962).
Likewise, neither s.c. nor 1.p. treatment of neonatal mice resulted In
significant tumor Induction (Grant and Roe, 1963; Buenlng et al., 1979a).
While the 1,2-d1ol-3,4-epox1des of phenanthrene are mutagenlc to Salmonella
typhlmuMum and to mammalian (V79) cells, they did not Induce pulmonary
tumors 1n newborn mice (Wood et al., 1979; Buenlng et al., 1979a).
Pyrene. Intratracheal Instillation of pyrene with Fe_03 particles
did not result 1n Increased numbers of respiratory tumors. In male Syrian
golden hamsters (Sellakumar and Shublk, 1974).
As part of a study of nitrated PAHs (W1slock1 et al., 1986), a dimethyl
sulfoxlde solution of pyrene was administered 1.p. to newborn CD-I mice on
days 1, 8 and 15 of age. Total administered doses were 200, 700 or 2800
nmole. Animals were weaned, separated by sex and observed without further
•
treatment to 1 year. A small but significant Increase 1n liver carcinomas
/
was observed In the mid-dose male mice only (Table V-22). The Incidences of
total liver tumors (adenomas and carcinomas), lung tumors or malignant
lymphomas were not significantly elevated 1n treated animals.
Tests of pyrene as a complete skin carcinogen and as an Initiator of
carclnogenlcHy 1n mice have been negative or Inconclusive (Badger et al.,
1940; Roe and Grant, 1964; Norton and Christian, 1974; Van Duuren and
Goldschmldt, 1976; Salaman and Roe, 1956; Scrlbner, 1973). Co-adminis-
tration of pyrene and benzo[a]pyrene to the backs of ICR/Ha mice resulted 1n
an enhancement of the benzo[a]pyrene tumor1gen1c1ty (Van Duuren and
Goldschmldt, 1976; Goldschmldt et al., 1973).
04420 V-lll 10/08/91
-------
TABLE V-22
Assay of Pyrene In Newborn M1cea
Tumor Incidence
Sex
H
F
M
F
M
F
H
F
Total
Dose
(nmol)
0
0
200
200
700
700
' 2800
2800
Liver
Adenomas
7/73
0/65
0/29
0/31
0/25
0/49
2/14
0/18
Liver
Carcinomas
0/73
0/65
0/29
0/31
3/25b
0/49
1/14
0/18
Lung
Adenomas
3/73
1/65
1/29
1/31
2/25
5/49
1/14
1/18
Lung
Carcinomas
2/73
1/65
0/29
0/31
0/25
0/49
0/14
0/18
aSource: Wlslockl et al., 1986
bS1gn1f1cantly different from controls (p<0.05). Fisher exact test
04420
V-112
10/08/91
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Pyrene did not produce tumors 1n Jackson A mice Injected s.c. and
observed 18 months (Shear and LeUer, 1941).
Reproductlve/Teratoqenlc Effects
Evidence of deleterious reproductive effects, documented for PAHs In
general and benzo[a]pyrene In particular, has teen equivocal. This evidence
1s considered here.
Anthracene. A total of 8 mg anthracene/mouse was administered as
dally s.c. doses or In a single oral dose during the last week of gestation
to dams of the following strains: BALB/C, C3H/A and C57B1 X CBA FI.
Fetuses were removed and the kidney cells established 1n culture. These
cells exhibited enhanced plating efficiency as well as some hyperplastlc
changes by comparison with fetal cells obtained from untreated animals
Indicating the ability of anthracene to pass through the placenta (Shabad et
al.t 1972).
BenzFalanthracene. Wolfe and Bryan (1939) reported fetal death and
resorptlon In two pregnant rats as a consequence of s.c. Injection of 5 mg
benz[a]-anthracene beginning with day 1 of gestation.
Benzofalpyrene. Rlgdon and Rennels (1964) conducted two series of
experiments In rats (strain not specified) to ascertain possible reproduc-
tive consequences of dietary benzo[a]pyrene. In series one, eight females
and an unspecified number of males were fed laboratory chow to which 1 mg/kg
benzo[a]pyrene had been added, and a control group of six females and an
unspecified number of males were fed a standard diet. Treated females were
04420 V-113 10/08/91
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mated with control males, and control females were mated to benzo[a]pyrene-
treated males. Vaginal smears were taken during a 28-day period beginning
with the first day of benzo[a]pyrene feeding. The authors observed no
treatment-related effects on the estrus cycle. Three normal pregnancies
were reported for the control females, and five benzo[a]pyrene-treated
females also became pregnant. Of the treated-females, only one delivered a
total of four pups at day 23. Two of the four pups were stillborn, one of
which was grossly malformed. One pup left with the dam died 3 days
postpartum, presumably of starvation. Of the dams not delivering, one was
found upon autopsy to carry four dead fetuses.
In the second series 6 control male and female rats were mated as were
males and 7 females fed benzo[a]pyrene as described above. The pregnant
control rats had apparently normal pregnancies. Two benzo[a]pyrene-fed
females of seven mated became pregnant. On autopsy. It was found that one
dam carried four dead fetuses and that fetal resorptlon had occurred In the
other. While these data suggest a reproductive effect of benzo[a]pyrene,
the report was unclear as to specifics of experimental design and length and
timing of the feeding period for treated males and females. In this study
the 1 mg/kg dose appears.to be the LOAEL with no NOAEL for reproductive
effects 1n rats.
Rlgdon and Neal (1965) conducted a series of four experiments on repro-
ductive effects In Swiss mice fed diets containing benzo[a]pyrene at concen-
trations of 0, 250, 500 or 1000 ppm. The animals were given the dietary
benzo[a]pyrene for various time spans during mating, gestation and post-
partum. Blue fluorescence. Indicative of the presence of benzo[a]pyrene or
04420 V-114 10/08/91
-------
Its metabolites, was demonstrated In the Internal organs of treated mice.
No teratogenlc, embryotoxlc, fetotoxlc or other reproductive effects were
observed In the treated animals. Fertility of male mice was apparently
unaffected. Males fed the 500 ppm diet for 30 days were shown to have sperm
present In the lumen of testlcular tubules.
In a study of CD-I strain pregnant mice, however, HacKenzle and Angevlne
(1981) found adverse reproductive effects In the offspring of animals dosed
with benzo[a]pyrene. Benzo[a]pyrene 1n corn oil at doses of 0, 10, 40 or
160 mg/kg bw was administered by gavage on days 7-16 of ges.tatlon to groups
of 30 or 60 dams. At these doses benzo[a]pyrene was not toxic to dams or
fetuses. Pregnancies'were carried to term and pups permitted to nurse until
weaning. Number of pups and gross abnormalities were scored on day 1
postpartum, and on day 4 pups were weighed and sexed. At this time Utters
were culled to 8 pups for breeding studies. At 6 weeks of age 10 F, males
and 10 F, females were sacrificed for. hlstologU examination of repro-
ductive organs. At 7 or 8 weeks the remaining F, mice were bred with
/
untreated animals. Females, both those treated \n utero and those mated
with F- males, were sacrificed on days 14-19 of gestation, and numbers of
Implants, fetuses and resorptlons were recorded. Gross abnormalities were
also noted.
Reduced fertility and reproductive capacity were also observed In the
F. mice. The FI males exposed to 10 mg/kg bw in utero showed a marked
reduction In gonadal weight, although no effect on body weight was found.
All control males (45/45) were fertile (sired at least one litter) and as a
group Impregnated 80% of the females to which they were exposed. Fertility
04420 V-115 10/08/91
-------
rates were 20/25, 3/45 and 0/20 for the 10, 40 and 160 mg/kg male dose
groups, respectively, and the percent of Impregnated females was 52, 7 and
OX, respectively.
In the control females the pregnancy rate was 35/35; the pregnancy rate
for the female groups exposed in utero was 23/35, 0/55 and 0/20 for the 10,
0 and 160 mg/kg dose groups, respectively. In this study a 10 mg/kg dose Is
the LOAEL with no NOAEL for a reproductive effects In mice.
To examine embryotoxldty, fetal Swiss mice were mlcrolnjected In utero
on day 10, 12 or 14 of gestation with benzo[a]pyrene, benzo[a]pyrene-4,5-
oxlde, a racemlc mixture of 7B,8a-d1hydroxy-9o,lOa-epoxy-7,8,9,lO-
tetrahydrobenzo[a]pyrene (compounds thought to be carcinogenic metabolites
of benzo[a]pyrene) or 6-methylbenzo[a]pyrene (Barb1er1 et al., 1986). The
vehicle was 1:1 tr1octanon1n:acetone and the mice were administered the test
compounds at doses of 0.4-16 nmol/embryo. An additional group was treated
transplacentally with 47.5 nmole benzo[a]pyrene on day 10, 12 or 14* On day
18, the dams were sacrificed and Inspected for Implantation sites, as well
as the number of dead and live fetuses; live fetuses were further examined
for gross malformations. Results, given 1n Table V-23, show that 1ntra-
embryonal administration of benzo[a]pyrene and benzo[a]pyrene-4,5-ox1de did
not significantly Increase the Incidence of fetal malformations; however,
the 7,8-d1ol-9,lO-epox1de caused significant Increases In the Incidence of
fetal malformations. Specific malformations Included exencephaly,
thoracoschlsls, gastroschlsls, phocomella and edema. 6-Methylbenzo[a]pyrene
also Increased the Incidence of terata; transplacental administration of
benzo[a]pyrene, however, did not.
04420 V-116 10/24/91
-------
TABLE V-23
EmbryotoxIcHy and Malformations 1n Swiss Mice Exposed by
Intraembryonal Injection of Benzo[a]pyrene and Derivatives3
Treatment
Compound
Control5
Benzo[a]pyrene
Benzo[a]pyrene-4,5-
oxlde
Benzo[a]pyrene-7,8-
d1ol-9,lO-epox1de
6-Methy1benzo[a]pyrene
Day
10
12
14
10
12
14
10
12
14
10
12
14
10
12
14
Dose
nmole/Embryo
0.5 (yl)
1 (P*)
2 (vl)
4
8
16
4
8
16
0.4
2
4
4
8
16
Viability Malformations
No. Live/ No. Malformed/
No. Treated Total No. Alive
53/69
30/36
59/82
18/41C
42/78c
28/45
25/28
21/43C
51/66 [sic]
7/46d
59/1 llc
37/58
20/366
13/27C
39/65
11/53
2/30
4/59
6/18
6/42
4/28
8/25
4/21
5/51
7/7d
36/59
-------
LeGraverand et al. (1984) found the extent of embryotoxlclty and terato-
genlclty of benzo[a]pyrene to be related both to the route of administration
and to the affinity of a receptor (AHH) for agents that Induce hepatic
cytochrome P-450 response. Benzo[a]pyrene (120 mg/kg/day) was orally
administered to pregnant mice from day 2 to day 10 of gestation. The mice
were either heterozygous or homozygous for the low-affinity receptor for
Inducers of cytochrome P-450 (the high-affinity receptor 1s dominant).
Pharmacoklnetlc studies Indicated that embryos developing 1n dams homozygous
for the Iow-aff1n1nty receptors were exposed to higher levels of
benzo[a]pyrene. Within this group of embryos, receptor homozygotes had an
Increased Incidence of IntrauteMne toxlclty and malformation when compared
with receptor heterozygotes. Heterozygous dams exhibit an enhanced ability
to metabolize benzo[a]pyrene In their Intestines and liver; therefore, the
developing embryos receive less benzo[a]pyrene. Consequently, less
Intrauterlne toxldty and fewer malformations were seen. Intraper1ton1al
administration of benzo[a]pyrene caused the exact opposite effects,
.Indicating the Importance of both the administration route and genotype of
the exposed animal 1n the potential for a toxic response.
Swartz and Mattlson (1985) showed that acute exposure to benzo{a]pyrene
had an adverse, albeit transient, effect on oocyte follicle growth, ovula-
tlon and formation of corpora lutea. Five female C57B1/6N mice/group were
treated with a single 1.p. Injection of 0, 1, 5, 10, 50, 100 or 500 mg/kg bw
benzo[a]pyrene In corn oil. One week post-treatment numbers of corpora
lutea were significantly reduced In mice treated with 5-500 mg/kg benzo[a]-
pyrene. By week 4 this depression was evident only In the mice In the two
04420 V-118 10/08/91
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highest treatment groups. Ovarian toxldty 1n the highest dose group was
Indicated not only by absences of corpora lutea but also by lack of signs of
folllculogenesls.
Taklzawa et al. (1984) established ED5Q doses for small oocyte
destruction by benzo[a]pyrene 1n several mouse strains. Treatment was by
1ntraovar1an Injection of 0, 0.1, 1.0, 10 or 30 yg/ovary. Two weeks post-
treatment mice were killed and ovarian tissue examined h1stolog1cally. The
ED5Qs for various strains were the following: C57B1/6N, ED5Q = 3.38
vg/ovary; DBA/2N = 36.14; C57B1/6J x DBA/2JF1 = 8.27.
Pregnant C3H/Anf mice were treated 1.p. at either 11-13 or 16-18 days of
gestation with 100 or 150 mg benzo[a]pyrene/kg bw. Body weights of progeny
were comparable between control and treated groups. Offspring of treated
animals, however, were observed to have a severe supresslon of Immune
function as measured by the ability to elicit an antibody response to sheep
red blood cells. This supresslon of response persisted Into adulthood
Indicating permanent 1mmunosuppress1on when prenatal exposures occur (Urso
and Gengozlan, 1980).
Benzo[a]pyrene has been shown to be a transplacental carcinogen
producing liver tumors In HA/ICR, strain A and C57B1 mice and lung tumors 1n
the Ha/ICR mice (Bulay, 1970; Bulay and Wattenberg 1971; Nlkonova. 1977).
Female A strain mice were subcutaneously administered 150 mg/bw benzo-
[a]pyrene 1n sunflower oil on the 18th and 19th day of pregnancy (Turusov et
al., 1990). The offspring were mated 1n a brother-sister system to create
04420 V-119 10/08/91
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the F2-F5 generations. Controls receiving only sunflower oil were
maintained. The offspring were sacrificed at 1 year of age. When compared
with controls, there was a statistically significant Increase 1n the lung
adenoma Incidence 1n both the males and females of the first generation.
The lung tumor Incidences 1n Fl females were 42/78 and 12/78 1n the exposed
and control mice, respectively, and 1n Fl males the Incidences were 52/67
and 6/75 1n the exposed and control mice, respectively. The F2 females
exhibited a statistically significant Increase 1n lung tumor Incidences
also; these were 20/91 and 10/95 1n the female F2 descendants of the treated
dams and 1n the controls, respectively. This Increased Incidence of lung
tumors was not seen 1n the F2 males or 1n later generations, but a
statistically significant Increase 1n tumor multiplicity was seen In the
F2-F5 generations.
Rabbit progeny have also been shown to develop tumors after
transplacental exposure to benzo[a]pyrene during the last third of
preganancy (BenlashvlH, 1978).
f
Chrysene. Mallard duck eggs were painted with 10 yS, of a 0.1X
solution of chrysene 1n an unspecified petroleum hydrocarbon reported to be
of "relatively low embryotox1c1ty" (IARC, 1983). Embryotoxlclty and terato-
genlc effects among ducklings were observed (Hoffman and Gay, 1981).
Fluoranthene. Irvln and Martin (1987) reported, 1n an abstract, a
developmental study 1n which a single 1.p. Injection of fluoranthene (dose
unspecified) was given to pregnant C57/B6 mice on gestatlonal days 6. 7, 8
or 9. A gestation-dependent Increased rate of embryo resorptlon was
observed.
04420 V-120 10/08/91
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Naphthalene. In what appears to be two reports of the same study
(Plasterer et al., 1985; Booth et al., 1983), single oral doses {300 mg/kg)
of naphthalene were administered dally for 8 consecutive days to 50 pregnant
CD-I mice beginning on day 7 of gestation. This dose was estimated to be at
or just below the maximum tolerated dose for acute lethality. A significant
Increase In maternal lethality (p<0.05) and a decrease In mean maternal body
weights as well as the number of live pups per Utter (p<0.05) on postpartum
day 1 were noted when compared with the controls. There was not a concomi-
tant Increase 1n dead pups. There were no effects on pup survival and mean
body weights. No gross congenital abnormalities were detected In the pups
although the method used to examine the pups was not reported.
Hardln et al. (1981) administered naphthalene 1.p. (395 mg/kg) In corn
oil to pregnant Sprague-Dawley rats on day 1 of gestation. Dally Injections
continued through day 15. No treatment-related effects on maternal
toxlclty, fetal toxIcHy, or teratogenesls were reported.
•
/•
In a pilot range-finding study, 20 artificially Inseminated New Zealand
white rabbits (at least 24 weeks of age and weighing 4-5 kg) were orally
dosed with naphthalene (In IX methylcellulose vehicle) at 50-1000 mg/kg from
gestatlonal days 6-18. Maternal lethality and/or abortion were Increased at
doses of >630 mg/kg, but no data were collected. No differences 1n
reproductive parameters were noted, and no malformations or fetal death
occurred at the lower dose levels (Na1smith and Matthews, 1985).
In the main study by Nalsmlth and Matthews (1986), 18 artificially
Inseminated New Zealand white rabbits per group were orally dosed with
naphthalene (1% methylcellulose vehicle) at 0, 40, 200 or 400 mg/kg/day from
04420 V-121 10/08/91
-------
gestation days 6-18. Dams were at least 24 weeks of age (exact ages were
not specified). Maternal body weights and body weight gains were comparable
among all test groups and controls. Food consumption of high-dose (400
mg/kg) animals was significantly greater (p<0.05) than controls during
gestation days 7-15, 23-25 and 27-29. Pharmacotoxlc signs observed during
the study Included decreased activity, dyspnea, weight loss, cyanosis,
salivation, and loose stools or diarrhea; these signs occurred In an
apparent dose-related manner. Gross examination of dams and controls
Indicated no differences 1n the following reproductive measures: number of
corpora lutea, total number of Implantations, viable or nonvlable fetuses,
pre- or postlmplantatlon loss, fetal body weights, and fetal sex
distribution. The malformations and variations were equally distributed
among groups, and no dose-related trends were apparent. These malformations
Included 2 Incidences of fused sternebrae and an Incidence of umbilical
hernlatlon from 3 different Utters 1n the control group, an Incidence of
vlceral malformations 1n the mid-dose group and 3 Incidences of fused
- • •
sternebrae from 3 different Utters 1n the high-dose group. The study
authors concluded that oral administration of naphthalene to pregnant
rabbits did not evoke a teratogenlc effect. The U.S. EPA (1987a) concluded
that the teratogenlc potential could not be adequately assessed. The data
were considered Incomplete because of lack of Information on the methods of
fetal sacrifice and of visceral and skeletal examinations.
Matorova (1982) reported that naphthalene administered by gavage (0.015,
0.15 and 1.5 mg/kg) on a chronic basis (duration not reported) to pregnant
female albino rats was associated with adverse effects on reproductive
function and development of progeny. These effects Included slow fetal
development, an Increase In number of hemorrhages and bleeding of Internal
04420 V-122 10/08/91
-------
fetal organs, and a reduced viability. The reported threshold for effects
was 0.075 mg/kg. A lack of Information on protocol design, tests for
significance, and experimental data renders these results difficult to
Interpret.
flutaqen1c1ty/Genotox1c1tv
The PAHs have long been recognized as having mutagenlc and other geno-
toxlc effects. The literature contains many reports of short-term Ui vitro
and IJN vivo tests of the genotoxldty of these compounds and of their
metabolites. Because of the extensive scope of these reports, their results
are presented 1n tabular form (Table V-24).
Synerqlsm and/or Antagonism
Because PAHs rarely occur 1n Isolation from each other In the environ-
ment, 1t Is Important to understand the potential health effects associated
with mixtures of PAHs. Much research has focused on the promoting or
'Inhibitory effect of noncarclnogenlc PAHs, such as pyrene, on the
carcinogenic potential of known carcinogens, often benzo[a]pyrene. The
route of administration Is typically nonoral (dermal, subcutaneous Injection
or Inhalation).. Some of the reports on cocardnogenlc activity are
considered In this section.
Falk et al. (1964) conducted a series of experiments with C57B1 male
mice (3-4 months old) to assess the potential Inhibitory effects of phenan-
threne and other PAHs considered noncarclnogenlc on the tumor 1gen1c1ty of
d1benz[a,h]anthracene. Groups of 30 animals received single s.c. Injections
of various dosages of d1benz[a,h]anthracene alone and 1n combination with
04420 V-123 10/08/91
-------
TABLE V-24
Results of Short-Term Tests of Polycycllc Aromatic Hydrocarbons
to
Test
BACTERIA
Mutation
MAMMALIAN CELLS
Cell transformation
BAC1ERIA
DNA damage
Mutation
Organism (Assay)
Salmonella typhlmurlure
(reverse mutation, his)
S. typhlmurlum (reverse
mutation, his) (taped-
plate assay)
S. typhlmurlum (forward
mutation. BAG*)
Syrian hamster embryo cells
(morphologic changes)
Escherlchia coll
(2P_LA)
E. coll
(uvrA. recA. lexA, polA)
Bacillus subtllls
(reel
S. typhlmurlum
(reverse mutation, his)
Exogenous Activation*
' System
Rat liver S9 (Aroclor)
Rat liver S9
(3-methylcholanthrene)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor
or phenobarbltal)
None
Rat liver S9 (none)
Rat liver S9. (Aroclor)
Rat liver S9 (Aroclor)
Rat liver $9 (Aroclor or
3-methylcholanthrene)
Results Reported
ACENAPHTHYLENE
Negative
Negative
Negative
Positive
Negative
ANTHRACENE
Negative .
Negative.
Potency
(A minimal
Inhibitory
concentration/
nmole)' <0.0009
Negative
Negative
Comments
Tested up to 250 pg/mt In
strains TA1537 and TA1538
Tested up to 3 pmol/plate In
strains TA98 and TA100
Tested up to 50 pg/plate In
strains TA98 and TA100
Strain TM677 tested at
1 pmol/mt
1-50 pg/mt
Tested up to 250 pg/mt
Minimal Inhibitory concentra-
tion >100 pg/well In all
strains with and without S9
Tested at 62 pg/well
Tested up to 1000 pg/plate In
strains TA1535. TA1537,
TA1538. TA98 and TA100
Reference
Gatehouse, 1980
Florin et al..
1980
Bos et al..
1988
Kaden et al.,
1979
Tu et al., 1986
Rosenkrantz and
Polrler, 1979
OeFlora et al..
1984
McCarroll
et al.. 1981
McCann et al.,
1975; Simmon,
1979b; La Vole
et al., 1979;
Salamone et
al.. 1979; Ho
et al.. 1981;
Florin et al.,
1980
-------
TABLE V-24 (cont.)
(V)
o
Test
Nutation (cont.)
<
«J •
ro
FUNGI
Recombination
MAMMALIAN CELLS
ONA damage
o
t\>
vo
Organism (Assay)
S. typhlmurlum
(reverse mutation, his)
S. typhlmurlum (reverse
mutation, his) (taped-
plate assay)
$_._ typhlmurlum
(forward mutation, 8AG*t)
S. typhlmurlum (forward
mutation. ARA")
Saccharomyces cerevlslae
(mltotlc recombination, 03)
Primary rat hepatocytes
(unscheduled DNA synthesis)
HeLa cells (unscheduled
DNA synthesis)
Exogenous Activation*
System
Hamster or rat liver S9
(Aroclor)
•°Co gamma Irradiation
of anthracene
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor or
phenobarbltol)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None
Rat liver S9 (phenobarbltal
or 3-methylcholanthrene)
Results Reported
Positive
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Comments
In strain TA100 from 50-400
tig/plate
In strains TA98. TA1535,
TA1537 and TA1538
In strain TA1537 at 0.22 mM
Tested 1n TA1535. TA1537.
TA1538, TA95 and TA100
TA97 at 5 and 10 pg/plate
Tested In TA98 and.TAlOO at up
to 200 pg/plate
Tested up to 50 pg/plate In
strains TA98 and TA100
Tested up to 225 nmol/ml
In strain BA9 and 0.5-3 pmol
None
Tested up to 1 pg/ml
Tested up to 100 pg/ml
Reference
Carver et al..
1986
Gibson et al..
1978
Selxas et al..
1982
OeFlora et al.,
1984
Sakal et al.,
1985
La Vole et al..
1985
Bos et al.,
1988
Kaden et al..
1979
Dorado and
Pueyo, 1988
Simmon. 1979a
Williams. 1977;
Probst et al.,
1981
Martin et al.,
1978; Martin
and McDermld,
1981
-------
TABLE V-24 (cont.)
o
iv> Test
o
DNA damage (cont. )
Mutation
rvj
Chromosome effects
o
o
CO
to
Organism (Assay)
F344 rat tracea epithelial
organ culture (unscheduled
DNA synthesis)
Chinese hamster ovary cells
(DNA synthesis)
Primary rat hepatocytes
(unscheduled DNA synthesis)
Chinese hamster V79 cells
(forward mutation. 6TGR)
Mouse lymphoma L5178Y cells
(forward mutation, TFTR)
Human lymphoblastold TK6
cells (forward mutation,
TFTR)
Rat liver epithelial cell line
ARL18 (forward mutation 6TGR)
Chinese hamster V79 cells
(forward mutations; OUAR,
6TGR)
Mouse lymphoma L5178Y cells
(forward mutation. TK-)
Chinese hamster ovary cells
(forward mutation. HGPRT)
Chinese hamster D6 cells
(sister -chromatld exchange;
breaks)
Rat liver epithelial ARL-18
cells (sister -chromatld
Exogenous Activation*
System
None
None <
None
Rat liver S9 (?)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None
Liver and lung cell mediated
systems for OUAR, kidney
and bladder cell mediated
systems for 6TGR
None
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None
None
Results Reported
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Comments
Tested from O.lxlO"* to
10xlO'« M
Tested 1000 pg/ml
Tested at lx!0~» M
, Tested up to 125 pg/ml
Tested up to 100 nmol/mi
Tested up to 200 nmol/mt
Tested at 10'» and 10"«
Tested at 3 pg/mi
Up to 500 pg/mi
Tested at 6-15 pg/ml
Tested at 3.5 pg/ml
Tested up to 1 pmol/mi
Tested up to 1 pmol/mi
Reference
Ide et al..
1981
Garrett and
Lewtas. 1983
Williams
et al.. 1989
Knapp et al.,
1981
Amacher and
Turner, 1980;
Amacher et al.,
1980
Barfknecht
et al.. 1981
Ved Brat
et al.. 1983
Langenbach
et al.. 1983
Mitchell
et al.. 1988
Oshlro et al.,
1988
Abe and Sasaki,
1977
Tong et al..
1981b
exchange)
-------
TABLE V-24 (cont.)
0
CO
to
Test
Chromosome effects
(cont.)
Cell transformation
BACTERIA
ONA damage
Organism (Assay)
Rat liver epithelial cell
line ARL18 (slster-chromatld
exchange)
Chinese hamster bone marrow
cells (111 vivo slster-
chromatld exchange)
Rat hepatocy.tes (ONA single
strand breaks)
Mouse lymphoma LS178Y/TK+/-
cells (ONA strand unwinding
assay)
Human peripheral lymphocytes
(sister -chromatld exchange)
House BALB/3T3 cells
(morphologic changes)
Guinea pig fetal cells
(morphologic changes)
Syrian hamster embryo cells
(morphologic changes)
C3H/10T1/2 cells mouse
embryo Hbroblast
(morphologic changes)
Mouse embryo C3H10T1/2 cells
(morphologic changes)
E. coll (polA)
E. coll
(uyrA. recA. jejcA, polA)
Exogenous Activation* Results Reported
System
None Negative
None . Negative
None Negative
Rat liver S9 (Aroclor) Positive
Intact rat hepatocytes Negative
None Negative
None Negative
None Negative
None Negative
None Negative
None Negative
BENZralANTHRACENE
Rat liver S9 (none) Negative
Rat liver S9 (Aroclor) Negative.
Potency
(A minimal
Inhibitory
concentration/
nmol) >0.0001
Comments
Tested at 10"» and 10~« M
450 mg/kg was Injected 48 and
24 hours prior to test
Tested at 0.003. 0.03 and
0.3 mM
Tested at 4.74x10"* H to
9.48x10"' M
Tested at 10"« to 10'* M
Tested at 10~« to 10~* M
Tested at 10 pg/mi
Tested at 0.5 yg/mi
Tested up to 50 pg/ml
Tested at 4 PH
Tested up to 30 pg/ml
Tested up to 250 pg/ml
Minimal Inhibitory concentra-
tion >1000 pg/well with and
without S9 In all strains
Reference
Ved Brat
et al.. 1983
Roszlnsky-
Kocher et al . ,
1979
Slna et al..
1983
Garberg et al..
1988
Undahl-
Klessllng
et al.. 1989
DIPaolo et al. .
1972
Evans and
DIPaolo. 1975
Plenta et al..
1977
Peterson
et al.. 1981
Lubet et al..
1983b
Rosenkrantz and
Polrler. 1979
Deflora et al..
1984
-------
TABLE V-24 (cont.)
Test
rs>
oo
o
oo
Organism (Assay)
Exogenous Activation*
System
Results Reported
Comments
threne; pyrene; benzo[k]-
fluoranthene; benzo[g,h,1]-
perylene; anthanthrene;
coronene; chrysene; benzo-
[J]fluoranthene; benzo[b]-
fluoranthene; Indeno-
[l,2,3-cd]pyrene; dlbenz-
[a.hjanthracene; clophen
ASO; benzo(c)phenanthrene,
benzo[b]naphthal[2,3-d]-
thlophene; benzo[bj-
naphthol[1,2-d]th1ophene;
trlphenylene; benzo[b]naph-
thol[2.1-djtheophene)
benzo[J]fluor- •
anthene, clophen
ASO, benzo[b]fluor-
anthene, Indeno'-
[1.2.3-cd)pyrene.
d1benz[a.h]-
anthracene
by different PAHs. the weak
carcinogen benz[a]anthracene
exhibited mutagenlc activities
comparable to those of benzo-
[ajpyrene In strain TA100
Reference
Mutation E. coll (expression of
lac Z gene under control
of SOS gene. sfIA)
S. tvphlmurluni (expression
of lac Z gene under control
of urn! gene)
S. typhlmurluni
{reverse nutation, his)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
•
•°Co gamma Irradiation
of benz(a)anthracene
Rat liver S9
(me thy Icholan threne)
Rat liver S9 (f luoranthene;
benzo[e]pyrene; phenan-
Posltlve
Weakly positive
Positive
Negative
Positive
Negative
Positive
Positive
Positive when rats
were treated with
In strain PQ37 up to 100 mM
Tested at 2.4 pg/mi
20 yg/plate In strain TA100
Strain TA1535
In strains TA98. TA1537 and
TA1538 at 250 yg/plate
Tested 1n strain TA1535 up
to 250 pg/plate
In strains TA98 and TA100
up to 3 iimoles/plate
Strains TA97. TA98, TA100.
TA1537, TA1538. Potency
(revertants/nmole)=12
In the presence of various
liver S9 preparations Induced
von der Hude
et al.. 1988
Nakamura
et al., 1987
McCann et al.,
1975; Coombs
et al., 1976;
Simmon, 1979b;
Salamone
et al.. 1979
Gibson et al . ,
1978
Florin et al . ,
1980
DeFlora et al. ,
1984
Norpoth et al. ,
1984
-------
TABLE V-24 (cont.)
f\>
o
Test
Organlsn (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
Nutation (cont.)
S. typhlmurluro
(reverse mutation, his)
o
CD
FUNGI
Recombination
INSECTS
Nutation
NANHALIAN CELLS
ONA damage
S. tvphlmurlum (reverse
mutation, his) (faped-
plate assay)
S. tvphlmurlum
(forward mutation, 8A6R)
S. cerevlslae
(mltotlc recombination, 03)
Drosophlla melanogaster
(lethals, vlslbles. bobbed
mutants)
Chinese hamster ovary cells
(ONA synthesis)
Primary rat hepatocytes
(unscheduled DMA synthesis)
Rat liver S9 Positive
(3-methylcholanthrene)
Rat hepatocytes Intact and Positive
homogenized (Aroclor)
Rat liver S9 (Aroclor) Positive
Rat liver S9 (polychlorl- Positive
nated blphenyl or pheno-
barbltal)
Hamster liver S9 (Aroclor) Positive
Nouse. rat or pig S9 Negative
(Aroclor)
Rat liver S9 (Aroclor) Negative
Rat liver S9 (Aroclor Positive
or phenobarbltal)
Rat liver S9 (Aroclor) Negative
None Positive
None Negative
None Positive
0.07 iimol/plate In strain
TA100
Suspension Incubation TA100
weaker response with Intact
cells
In strains TA98 and TA100
at 50 pg/plate
In strains TA98 and TA100
at SO pg/plate
In strain TA100 at 10-50
wg/plate
In strain TA100 at 10-50
pg/plate
In strains TA9B and TA100
at 50 jig/plate
65 nmol/mt In strain TN677
None
Administered by mlcrolnjectlon
Tested 1000
100 nmol/mt
Bartsch et al..
1980
Utesch et al.,
1987
Bos et al..
1988
Ito et al..
1988
PhlUpson and
lonnldes, 1989
Bos et al..
1988
Kaden et al.
1979
Simmon. 1979a
Fahmy and
Fahmy, 1973
Garrett and
Lewtas. 1983
Probst et al.,
1981
-------
TABLE V-24 (cont.)
09
O
o
CD
IO
Test Organism (Assay)
DNA damage (cont.) HeLa cells (unscheduled
ONA synthesis)
Primary F344 rat trachea!
epithelial organ culture
(unscheduled ONA synthesis)
Primary F344 rat hepatocytes
(unscheduled ONA synthesis)
Mutation Chinese hamster V79 cells
(forward mutation, OUA*)
Chinese hamster V79 cells
(forward mutation, 6T6R)
House lymphoma LS178Y cells
(forward mutation, TFT")
Rat liver epithelial cells
(forward mutation. 6T6")
Chromosomal effects Chinese hamster ovary cells
(slster-chromatld exchange)
Simian virus transformed
Chinese hamster ovary cells
(selective DNA amplification)
Bone marrow cells of Long-
Evans rats (chromosomal
aberrations)
Cell transformation .Syrian hamster embryo cells
(morphologic changes)
Mouse prostate C3HG23 cells
(morphologic changes)
Exogenous Activation*
System
Rat liver S9
(3-methylcholanthrene)
None
None
Syrian hamster embryo cell
feeder layer
Rat liver S9
(3-methylcholanthrene)
Various
None
None
None
None
None
None
Results Reported
Positive
Negative
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Positive
Positive
Comments
Up to 100 pmol/ml
Tested at 0.1x10'* to
10xlO"« M
Tested at lx!0'« M
44 nmol/ml
46 nmol/ml
Small Increase at
40 nmol/ml
Up to 100 nmol/ml
2 tig/ml
Tested at 0.14 pinole
lr\ vivo, rats received 25,
50 or 100 mg/kg
0.1 pg/mi
10 pg/ml
Reference
Martin et al.,
1978
Ide et al..
1981
Williams
et al.. 1989
Slaga et al..
1978
Krahn and
Heldelberger.
1977
Amacher et al..
1980; Amacher
and Turner.
1980
Tong et al. .
1981a'
Pal. 1981
Pool et al..
1989
Ito et al..
1988
Plenta et al. .
1977; OlPaolo
et al., 1969.
1971
Marquardt and
Heldelberger,
1972
-------
TABLE V-24 (cent.)
o
-("• ........
4k
o Test
Cell transformation
(cont.)
Organism (Assay)
House C3H/10T1/2 cells
(morphologic changes)
Exogenous Activation* Results Reported Comments
System
N
None Negative Tested up to 100 nmol/ml
Reference
Nesnow and
Heldelberger .
1976
BACTERIA
DNA damage
i
to
Nutation
GO
vo
E. coll
(uyrA. recA. lexA. polA)
E. coll (expression of
lac Z gene under control
of SOS gene, sf1A)
S. typhlmurlum (expression
of lac Z gene under control
of urou C gene)
'S. typhlmurlum
(reverse mutation, his)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
•°Co gamma Irradiation
of benzo(a)pyrene
•°Co gamma Irradiation
of benzo(a)pyrene
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat or hamster liver S9
(Aroclor)
BENZOfalPYRENE
Negative.
Potency
(A minimal
Inhibition
concentration/
nmole) <0.0002
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Positive
Positive
Minimal Inhibitory concentra-
tion >BOO pg/well In all
strains with and without S9
In strain PQ37 up to 100 mM
Tested at 1
In strain TA100 up to
1000 pg/plate
In strains TA98, TA1537 and
TA1S38 from 50-100 wg/plate
In strain TA153S up to 100
ng/plate
Strain TA1535
Strain TA97, TA98. TA100,
TA1537, TA1538. Potency
(revertants/nmole) = 18S
Tested at 2.5 |ig/plate In
strains TA98 and TA100.
Positive only with metabolic
activation
Tested at 8 nM 1n strain
TA100. Selenium at nontoxlc
concentrations Inhibited the
mutagenlclty of benzo[a]pyrene
In both the rat liver and
hamster liver systems
DeF lora et al.,
1984
von der Hude,
et al., 1988
Nakamura
et al., 1987
Andrews et al.,
1978
Gibson et al..
1978
DeFlora et al.,
1984
Lofroth et al..
1984
Teel, 1984
-------
TABLE V-24 (cont.)
Test
Organism (Assay)
Exogenous Activation*
,. System
Results Reported
Comments
Reference
Nutation (cont.)
FUNGI
Recombination
1, INSECTS
GO
^ Chromosomal effects
Nutagenldty
PLANTS
Chromosome damage
MAMMALIAN CELLS
ONA damage
o
•v
o
CD
S. typhlmurlum
("reverse nutation, his)
S. tvphlimirlum
(^forward mutation. BAG")
S. typhlmurlum
(forward mutation. ARAR)
S. cerevlslae
(mltotU recombination, 03)
D. roelanoqaster
(whole chromosome gain)
D. melanogaster (sex
linked recessive lethal
nutation)
Tradescant Ions clone 4430
(tetrad analysis)
F344 rat trachea epithelial
organ culture (unscheduled
ONA synthesis)
Primary BALB/C mouse
epidermal cells benzo[a]-
pyrene-ONA adduct formation)
Rat liver S9 (Aroclor) Positive
Rat hepatocytes Intact and Positive
homogenized (Aroclor)
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Positive
Liver mlcrosomes Negative
(Aroclor)
None Negative
None Negative
None Positive
None . Positive
None Positive
Strains TA97, TA98 and TA?nn
positive at 1 tig/plate
Suspension Incubation; str
TA100; weaker response wll
Intact cells
In strain TM677 at 4 MM
In strain BA13 at a minimum
dose of 0.77 nMol
None
Larvae and adult flies exposed
1n feed. Oocytes and oogonla
examined for chromosome gain
Fed at 2500 and 50.000 ppm
Dorado and
Pueyo, 1988
Simmon, 1979a
Fabian and
Matoltsy. 1946
Valencia
et al.. 1989
Minimum effect level 12.6 ppm Sandhu et al..
1989
Tested at concentrations that Ide et al..
ranged from 0.1x10'* to 1981
lOxlO'* M
Increase In benzo[a]pyrene-DNA Nakayama
adduct formation with PAH con- et al., 1984
centratlons In range of 50-250
nmol
-------
TABLE V-24 (cont.)
o •
Jfc - -
4k
o Test Organism (Assay) Exogenous Activation* Results Reported Comments
System
OKA damage (cont.) Chinese hamster ovary cells Adult rat hepatocytes Positive
(DMA single strand breaks)
Permeablllzed human dlplold- None Negative
flbroblasts (to which E. coll
DNA polymerase was .added)
(DNA strand breaks)
Primary rat hepatocytes None Positive
(unscheduled ONA synthesis)
Mouse lymphoraa L5178/TK*/- Rat liver S9 (Aroclor) Positive
Dose and exposure time depen-
dent Increases noted at IxlCT4
and 5xlO~» H
Tested at 1 yH for 30 minutes
Tested at lxlO~« M
Tested at 5xlO"« to 50xlO~« M
Reference
Yang et al. ,
19B4
Snyder and
Matheson. 1985
Williams
et al.. 1989
Gar berg et al..
cells (ONA strand-unwinding
assay)
Hutatlon
Chinese hamster
cel
6Tfi
cells (forward mutation.
Ri
CO
CO
Chromosomal effects
o
o>
Chinese hamster ovary cells
(forward mutation. HGPRT)
Mouse lymphoraa L517BY
(forward mutation. TK~)
Mouse Tymphoma LSI78 cells
(forward mutation. TK~)
Mouse embryo cells
C3H/10T1/2 (ONA single
strand breaks)
Rat liver epithelial cells
RL-12 (s1ster-chromat1d
exchange)
Human peripheral lymphocytes
(slster-chromatld exchange)
Chinese hamster epithelial
liver cells (slster-
chromatld exchange)
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Positive
None Negative
None Positive
Intact rat hepatocytes Positive
None Positive
None Positive
Tested up to 6
Tested at 0.5-4.0
Tested at 5xlO"« to
10xlO~» mol/l
Tested from 3.2-10
Tested up to 10 pg/ml
Tested at 1.6xlO~* M to
1.6x10'' M. Cell line can
activate procarclnogen to
genotoxlc form
Tested at 10"« to 10"* M
Tested at 10~« to 10"6 M
Tested at 0.11-1 vg/mi
1988
Reclo and Hsle.
1984
Oshlro et al..
1988
Wangenhelm and
Bolcsfoldl.
1988
Clay and Cross.
1990
Lubet et al..
1983b
Murlson et al.
1984
Llndahl-
Klessllng
et al.. 1989
OeSalvIa
et al.. 1988
-------
TABLE V-24 (cont.)
ro Test
o
Chromosomal effects
(cont.)
Cell transformation
i
BACTERIA
Mutation
i
3
MAMMALIAN CELLS
Mutation
BACTERIA
Mutation
5 BACTERIA
CD
^ Mutation
Organism (Assay)
Simian virus transformed
Chinese hamster ovary cells
(selective ONA amplification)
House BalbC/3T3
(morphologic changes)
C3H/10T1/2 mouse embryo
flbroblast cells
(morphologic changes)
Syrian hamster embryo cells
(morphologic changes)
S. typhlmurluni
(reverse mutation, his)
DPI-3 epithelial cell line
(6-TG)
S. typhlmurlum •
(reverse mutation, his)
S. typhlmurlum
Exogenous Activation* Results Reported
System
None Positive
None Positive
None Positive
None Positive
BENZOfblFLUORANTHENE
Rat liver S9 (Aroclor) Negative
Rat liver S9 (Aroclor) Positive
No data. Negative
BENZOfklFLUORANTHENE
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Positive
BENZOfq.h.nPERYLENE
Rat liver S9 (Aroclor) Positive
Comments
Tested at 0.01 iimol
At 10 and 20 pg/ml
Tested at 4 pM
Tested at 1-5 pg/ml
Tested up to 100 tig/ml In
strain TA100
At 100 pg/p1ate ID strain
TA100. 7 nmol/plate In strain
TA98, 0.5 pmol In TA100
,
Tested up to 4 pM .'
i
'
Tested up to 100 pg/plate In
strain TA100
5 wg/p1ate 1n strain TA98
From 10-50 pg/plate 1n strain
TA100
At 20 pg/plate In strain TA100
Reference
Pool et al.,
1989
DIPaolo et al..
1972
Peterson
et al.. 1981
Tu et al.. 1986
Nossanda
et al., 1979
LaVole et al..
1979; Hermann.
1981; Amln
et al., 1985b
Dooley et al..
1981
LaVole et al..
1980
Hermann et al.,
1980
Meyand et al..
1988
Andrews et al..
(Yeverse mutation, his)
1978; Mossanda
et al.. 1979
-------
TABLE V-24 (cont.)
g Test
Nutation (cont.)
MAMMALIAN CELLS
< ONA damage
j^
S BACTERIA
ONA damage
Nutation
_,
o
o
CO
10
Organism (Assay)
S. typhlmurlum
(reverse nutation, his)
S. typhlitmrluro
(forward mutation, 8AGR)
Chinese hamster ovary cells
(DNA synthesis)
E. coll (polA)
S. -typhlmurlum (expression
of lac Z gene under control
of umu C gene)
S. typhlmurlum
(reverse mutation, his)
(
Exogenous Activation*
System
N
Rat liver S9 (Aroclor)
•°Co gamma Irradiation '
of benzo(g,h,1)pyrene
None
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None noted
Rat liver S9 (none)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor-)
Rat liver mlcrosomes
{Aroclor ) ,
Results Reported
Positive
Positive
Negative
Positive
Positive
Positive
CHRYSENE
Negative
Negative
Positive
Positive
*
Positive
Positive
Positive
Comments
In range 0.1-1000 pg/plate In
strain TA98
In strains TA98. TA1537 and
TA1538 at 400 pg/plate
In strain TA1535 at concentra-
tions up to 800 pg/plate
At 10 pg/plate 1n TA97 and TA98
At 72 nmol/ml In strain TM677
At 1000 pg/ml DNA synthesis
at 3X of controls
Tested up to 250 pg/mi
None
Tested at 15 pg/mi
At 10 pg/plate In strain TA100
At 125 nmol/plate In strain
TA100; dose-response seen
with Increasing concentrations
of mlcrosomes
None
None
Reference
SaTamone
et al.. 1979
Gibson et al . .
1978
Sakal et al..
1985
Kaden et al . ,
1979
Garrett and
Lewtas, 1983
Rosenkrantz and
PolMer, 1979
Lelfer et al..
1981
Nakamura
et al.. 1987
McCann et al..
1975; LaVole
et al.. 1979
Wood et al..
1977
Dunkel and
Simmon, 1980
Toklwa et al. ,
1977
-------
TABLE V-24 (cont.)
4k
o Test
Nutation (cont.)
«c
i
— ,j
CO
^
o FUNGI
w
^v
<£> Recombination
Organism (Assay) Exogenous Activation*
System
S. typhlmurlum Rat liver S9
Treverse nutation, his) (3-methykholanthrene)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Hamster and rat S9
(Aroclor)
None
Rat liver S9 (Aroclor)
.Rat liver S9 (Aroclor)
•°Co gamma Irradiation
In air
S. typhlrourlum (reverse Rat liver S9 (Aroclor)
nutation, his) (taped-
plate assay)
S. typhlmurluro Rat liver .59 (Aroclor)
(forward mutation, 8AGR)
S. .cerevlslae Rat liver S9 (Aroclor)
Results Reported
r
Positive
Negative
Positive
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Negative
Negative
Positive
Negative
Comments
At 1.5 (jmol/plate 1n strain
TA100
At 1.5 (jmol/plate In strain
TA98
Identified In highly mutagenlc
fraction of raw gas condensate.
Nutagenlclty assessed 1n
strain TA98
At 5 vg/plate, strains TA97.
TA98 and TA100
In strain TA100 at 5 ng/plate
In strains TA98 and TA100
In strain TA100 at 100 pg/plate
In strain TA98 at 100 »g/plate
In strains TA98 and TA100 at
50 pg/plate
In strains TA98. TA1537 and
TA1538 at 1000 ug/plate
In strain TA1535 up to 1000
vg/plate
In strains TA98 and TA100 at
50 vg/plate
At 45 nmol/ml In strain TN677
None
Reference
Florin et al. .
1980
Benson et* al. ,
1984
Sakal et al.,
1985
Carver et al.,
1986
Glatt et al.,
1986
Glatt et al.,
1986
Bos et al..
1988
Gibson et al..
1978
Bos et al.,
1988
Kaden et al..
1979
Simmon, 1979a
(mltotlc recombination, 03)
-------
TABLE V-24 (cent.)
ro
O
CO
oo
ID
Test
HAHHALIAN CELLS
DNA damage
Nutation
•
Chromosomal effects
Cell transformation
Organism (Assay)
Primary rat hepatocytes
(unscheduled DNA synthesis)
F344 rat trachea epithelial
organ culture (unscheduled
DNA synthesis)
Primary rat hepatocytes
(unscheduled ONA synthesis)
Chinese hamster V79 cells
(forward mutation OUA",
BAG")
V-79. Chinese hamster cells
(forward mutation. 6-TGR)
Chinese hamster cells
(sister chromatld exchange)
House oocytes
(chromosome aberrations)
Hamster spermatogonla
(chromosome aberrations)
Hamster bone marrow cells
Syrian hamster embryo cells
(morphologic changes)
House prostate C3HG23 cells
(morphologic changes)
House prostate H2 cells
(morphologic changes)
Exogenous Activation*
System
V
None
None
None
Syrian hamster embryo cell
feeder layer
Rat liver S9
None
in vivo (gavage)
in vivo (gavage)
(phenobarbltal)
In vivo (gavage)
(phenobarbltal)
In vivo (gavage)
(phenobarbltal)
None
None
Tested both with and without
rat liver
Results Reported
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Comments
Tested up to 100 nmol/ml
I
Tested from 0.1x10"* to
10xlO~« H
Tested at concentrations up
to lxlO"« H
Tested up to 10 wg/mi
Tested from 2.5-80 vq/mt
Tested from 2.5-80 jig/rot
900 mg/kg
Weak positive at 450 mg/kg
Increase not significant at
total dose of 900 mg/kg
Tested at 10pg/mt
Tested up to 10 yg/mi
Tested from 10-40 ug/mt
Reference
Tong et al..
1981a
Ide et al..
1981
Williams
et al.. 1989
Huberman and
Sachs. 1976
Glatt et al..
1986
Roszlnsky-
Kocher et al..
1979
Easier et al..
1977
Plenta et al. .
1977
Marquardt and
Heldelberger.
1972
Glatt et al..
1986
-------
TABU V-24 (cont.)
2 Test
o
BACTERIA
ONA damage
Nutation
i
CO
CO
PLANTS
Chromosome damage
HAHHALIAN CELLS
ONA damage
o
V.
INJ
V.
• f*
Organism (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
DIBENZfa.hlANTHRACENE
E. coll (recA) .
B. subtllls (recA)
S. typhlrourlum
(reverse mutation, his)
•
S_._ typhlmurlum
(forward mutation. 8AGR)
Tradescantla clone 4430
(tetrad analysis)
Human foreskin epithelial
cells (unscheduled DNA
synthesis)
Hela cells (unscheduled
DNA synthesis)
Syrian hamster embryo cells
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat or guinea pig liver
(Aroclor) or (3-methyl-
cholanthrene) rat liver S9
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
•°Co gamma Irradiation
of d1benz[a,h]anthracene
Rat. hamster, pig or human
S9 (Aroclor)
Rat liver S9 (Aroclor)
•
None
None
3-methylcholanthrene
None
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Positive
Positive
B
Positive
Positive
Negative
At 25 pg/well
Minimal Inhibitory concentra-
tion for rec* 50 pg/well;
for rec- 12 pg/well
At 5 pg/plate 1n strain TA100
At 12 nmol/plate In strain TA98
In strain TA100 up to 1000
pg/plate
In strains TA98. TA1535 and
TA1537
In strain TA100 at 10-50
pg/plate
At 75 nmol/mt In strain TM677
Minimum effective concentration
was 12.5 ppm
In range 1-100 pg/mt
At 100 pmol/ml
Tested up to 20 pg/ml
Ichlnotsubo
et al., 1977
McCarroll
et al.. 1981
McCann et al.,
1975; Andrews
et al., 1978
Baker et al.,
1980; Hermann,
1981
Andrews et al. ,
1978
Gibson and
Smith. 1979
Phlllpson and
lonnldes. 1989
Kaden et al..
1979
Sandhu et al..
1989
Lake et al..
1978
Martin et al..
1978
Casto. 1979
(unscheduled DNA synthesis)
-------
TABLE V-24 (cont.)
CO
to
o
oo
Test
DMA damage (cont.)
Nutation
Cell transformation
BACTERIA
DMA damage
Organism (Assay)
Primary rat hepatocytes
•{unscheduled ONA synthesis)
F344 rat trachea epithelial
organ culture (unscheduled
ONA synthesis)
Chinese hamster V79 cells
(forward mutation, OUA",
8A6«)
Chinese hamster V79 cells
(forward mutation. 6T&")
Syrian hamster embryo cells
(morphologic changes)
Mouse C3H10T1/2 cells
(morphologic changes)
Nouse prostate C3H cells
(morphologic changes)
Nouse prostate C3HG23 cells
(morphologic changes)
B. subtllls (rec A)
S. typhlmurlum (expression
of lac Z gene under control
Exogenous Activation*
v System
None
None
Syrian hamster embryo cell
feeder layer
Rat liver S9
(3-methylcholanthrene)
None
None
None
None
None
With or without rat
liver S9 (polychloMnated
blphenyl)
Rat liver S9 (Aroclor)
Results Reported Comments
Negative Tested up to 100 nmol/mi
Negative Tested from 0.1x10'* to
10xlO'« N
Positive At 1 tig/ml
Positive At 56 nmol/mt
.
Positive At 0.5-10 ng/ml
Positive At 20 pg/mt
Weakly positive At 10 and 30 pg/mi
Positive At 10 iig/mi
Negative . Tested up to 10 wg/ml
FLUORANTHENE
Negative At 10 rag/well
Negative Tested up to 167 ug/ml
Reference
Probst et al.,
1981
Ide et al..
19B1
Huberman and
Sachs. 1976;
Huberman, 1978
Krahn and
Heldelberger,
1977
OlPaolo et al..
1969; Plenta
et al.. 1977;
Casto et al..
1977; Casto.
1979
Reznlkoff
et al.. 1973
Lubet et al..
1983b
Chen and
Heldelberger .
1969
Narquardt and
Heldelberger.
1972
Klnae et al..
1981
Nakamura
et al.. 1987
of umu C gene)
-------
TABLE V-24 (corit.)
o
4fc
O
Test
Organlsn (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
Nutation
L. typhlmurlum
(reverse mutation, his)
I
wj
*•.
o
S. typhlmurlum •
(reverse mutation, his)
o
CD
S. typh1mur1um
(reverse mutation, his)
(taped-plate assay)
S. typhlmurlum
^forward mutation, 8AGR.)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
With or without rat
liver S9 (polychlorlnated
blphenyl)
With or without rat
liver S9 (polychlorlnated
blphenyl)
Rat llvor S9 (Aroclor)
Rat liver S9
(3-methylcholanthrene)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Negative
Negative
Weakly positive
Negative
Positive
Negative
Positive
Positive
Negative
Positive
Positive
Positive
Unknown concentration In
airborne partlculate materials
(strain TA98)
Strains TA98 and TA100
Unknown concentration In
strain TA9B
Tested up to 1000 pg/plate In
strains TA98. TA100, TA1535.
TA1537 and TA1538
At 5 pg/plate In strain TA98
Strains TA98 and TA1537 at
100 pg/plate
Tested up to 200 pg/plate 1n
strain TA100
Tested In strains TA98 and
TA100 at 3 ymol/plate
Tested up to 500 nmol/mt
In strain TA97
Tested up to 100 pg/bottom
plate In strains TA98 and
TA100. Results negative with-
out the addition of S9 mix.
Lowest concentration to show a
significant mutation rate was
5 pM. Not mutagenlc at up to
50 pH without the addition of
S9 mix. Strain TM677
Toklwa et al. .
1977
LaVole et al.,
1979
Epler et al..
1978
Salamone et al.,
1979
Hermann et al.,
I960
K1nae et al.,
1981
Strain TA100 at 100 ng/plate Klnae et al.,
1981 .
LaVole et al..
1982b
Florin et al.,
1980
Hera and
Pueyo. 1988
Bos et al..
1988
Rastetter
et al., 1982
-------
TABLE V-24 (cent.)
Test
Organism (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
Hutatlon (cont.)
MAMMALIAN CELLS
Hutatlon
Chromosomal effects
BACTERIA
DNA damage
Nutation
S. typhlmurluro
(forward mutation, BAG")
S. typhlmurluro
(forward mutation. Ara")
Human lytnphoblast cell line
AHH-1 (forward mutation to
6GT«)
Chinese hamster ovary cells
(differential cytotoxldty
of repair-deficient strains)
Chinese hamster ovary cell
(forward mutation 6GTR)
Chinese hamster ovary cells
(sister chromat1d exchange)
Chinese hamster epithelial
cell strain
S. typhlmurlum (expression
of lac Z gene under control
of umu C gene)
S. typhlrourlum
(reverse mutation, his)
Rat liver S9 (Aroclor)
Rat liver 59 (Aroclor)
Rat liver S9 (Aroclor)
MFO Induced by treatment
with B-naphthaflavone
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None (using metabollcally
competent liver cells)
Rat liver S9 (Aroclor)
•°Co gamma Irradiation
of fluorene
o
o
CO
Positive
Positive
Positive
Negative
Positive
Negative
Weakly positive
Positive
Negative
FLUOREME
Negative
Positive •'
Negative
Strain TN677 at 1 pg/mt Kaden et al.,
1979
Tested up to 10 gg/mt In Babson et al.,
strain TM677 1966
Tested up to 500 nmol/mt Hera and
In strain BA9 Pueyo, 1988
Tested at 10-100 iiN, 48-hour Crespl and
exposure Thllly. 1984
Strains UV4 and UV5 deficient Hoy et al..
In nucleotlde excision repair 1984
Strain EM9 defective 1n DNA-
strand-break rejoining
Tested at 20 wg/mt LI. 1984
Tested at 9. 18 and 36 Mg/ml Pallttl et al..
1986
Tested at 25, 35 and 45 wg/mt DeSalvIa
et al.. 1988
Tested up to 16.7
In strains TA98 and TA1538 up
to 1000 yg/plate
In strains TA1535 and TA1537
up to 1000 pg/plate
Nakamura
et al'., 1987
Gibson et al..
1978
-------
TABLE V-24 (cont.)
ro
Test
Organism (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
Mutation (cont.)
S. tvphlmurluro
(reverse mutation, his)
MAMMALIAN CELLS
DMA damage
Mutation
S. typhlmurluni (reverse
mutation, his) (taped-
plate assay)
S. typhlmurlum
(forward mutation, 8AGR)
Primary rat hepatocytes
(unscheduled DMA synthesis)
Primary mouse or hamster
hepatocytes (unscheduled
DNA synthesis)
Permeablllzed human dlplold
flbroblasts and E. coll DNA
polymerase (DNA strand breaks)
Primary rat hepatocytes
(unscheduled DNA synthesis)
Mouse L5178Y TK+/- lymphoma
cells (forward mutation, TK~)
Chinese hamster ovary cells
(forward mutation, HGPRT)
Rat liver S9 (Aroclor) Negative
Rat liver S9 (Aroclor) Negative
Rat liver S9 (3-MC or TCDD) Negative
Rat liver S9 (Aroclor) Negative
Rat liver S9 (Aroclor) Negative
Rat liver S9 (Aroclor or Negative
phenobarbltal)
None Negative
•None Negative
None Negative
None Negative
Rat liver S9 (Aroclor) Negative
None Negative
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor) Negative
Tested up to 1000 |ig/plate In
strains TA1535, TA1537, TA98
and TA100
Tested at up to 250 ug/plate
strains TA97. TA98 and TA100
Tested up to 1504 nmol/plate
In strain TA100
In strains TA98 and TA100 up
to 50
In strains TA98 and TA100 up
to 50 |ig/plate
Tested up to 300 nmol/ml In
strain TM677
Tested up to 10 nmol/ml
Tested up to 1 pM
Tested at 3 mM for 2 hours
Tested at IxlO'4 M
Tested up to 30 iig/mi
Tested up to 60 pg/ml
Tested at 5.8x10"" to
7.78xlO~« mol/l
Tested, at 1-20
McCann et al..
1975; La Vole
et al.. 1979.
1981b
Sakal et al.,
1985
Pahlman and
Pelkonen. 1987
Bos et al..
1988
Kaden et al.,
1979
Probst et al.,
1981
McQueen et al.,
1983
Snyder and
Matheson. 1985
Williams
et al.. 1989
Oberly et al.,
1984
Wangenhelm and
Bolcsfoldl,
1988
Oshlro et al.,
1988
-------
TABLE V-24 (cont.)
Test
Organism (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
CHROMOSOME EFFECTS
BACTERIA
Mutation
BACTERIA
ONA damage
Mutation
o
CD
Mouse lymphoma cells (DMA
strand breaks)
Chinese hamster cell line
(chromosomal aberrations)
S. typhlmurlum
(reverse mutation, his)
Rat liver S9 (Aroclor)
Positive
S. typhlmurlum (expression
of lac Z gene under control
of umu C gene)
S. typhlmurlum
(reverse mutation, his)
Liver S9 mix source unknown Positive
INDENOfl.2.3-cd1PYRENE
Rat liver S9 (Aroclor) Positive
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Positive
NAPHTHALENE
Negative
With or without rat liver S9 Negative
(Aroclor)
With or without rat liver S9 Negative
Rat liver S9 Negative
(3-methylcholanthrene)
Rat liver S9 (Aroclor) Negative
With or without rat liver S9 Negative
(Aroclor)
Rat or hamster S9
(Aroclor)
Rat liver S9 (20-100
lit/plate) (Aroclor)
Negative
Negative
At 0.15 MM both with and
without metabolic activation.
25 pg/mi
At 20 |ig/plate for TA100 and
2 pg/plate for TA9B
At -3 pg/plate TA100
Tested at 6.3 pg/ml
Tested up to 1000 pg/plate In
strains TA98. TA100. TA1535
and TA1537
Tested up to 300' pg/plate In
strains TA1535. TA1537. TA1538.
TA9B. TA100
Tested at 3 pinol/plate In
strains TA98 and TA100
Tested up to 1.6 mN 1n
strain TA1537
tested In two DNA-repalr defi-
cient strains, TA100 and TA98.
and two strains which have
full DNA repair capacity:
UTH8414 and UTH8413
Tested up to 10 ing/plate In
strain TA100
Tested up to 1000 pg/plate
1n TA98
Garberg et al..
1988
Hatsuoka
et al., 1987
LaVole et al..
1979; Hermann
et al.. 1980
Rice et al..
1985a
Nakamura
et al.. 1987
McCann et al.
1975
Godek et al.,
1985
Florin et al..
1980
Selxas et al..
1982
Connor et al..
1985
Mortelmans
et al.. 1986
Narbonne
et al.. 1987
-------
TABLE V-24 (cortt.)
Test
Nutation (cont. )
Organism (Assay)
S. typhlnurlum
(reverse nutation, his)
Exogenous Activation*
System
Rat liver S9 (20-100
til/plate) (Aroclor)
Results Reported
Positive
Comments
Tested up to 1000 ng/plate
In TA1S35. Naphthalene gave
Reference
Narbonne
et al.. 1987
MAMMALIAN CELLS
DNA damage
ro
10
S. typhlmuMuni
(reverse mutation, his)
(taped-plate assay)
Chromosomal effects
Rat hepatocytes
(unscheduled ONA synthesis)
Bone marrow of CD-I mice
(mlcronuclel Induction)
Chinese hamster ovary cells
(slster-chromatld exchange)
With or without rat liver
S9 (polychlorlnated
blphenyls)
Rat liver S9 (Aroclor)
With or without rat liver
S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None
None
Rat liver S9
Negative
Weakly positive
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Positive
>2 times the control value of
revertants at 100 pg/plate
with 20 pi S9/plate and at
16 et al.. 1985
pg/ml were extremely cytotoxlc.
Tested at 250 mg/kg (the MTO) Sorg et al.,
In corn oil 1985
Positive with and without rat NTP. 1991b
liver S9 when tested at 15 and
27 tig/ml
-------
TABLE V-24 (cont.)
o
J>
•i* Test
ro
O
Chromosomal effects
(cont.)
Cell transformation
BACTERIA
DMA damage
<
i
-t»
Ul
o
«x
ro
4k
vO
Organism (Assay)
Chinese hanster ovary cells
(chromosomal abberatlons)
Fischer rat embryo cell line
(morphologic changes)
AKR leukemia. virus-Infected
Swiss mouse embryo cell line
(morphologic changes)
B. subtlUs (rec)
E. coll (polA+1
S. typhlnmrlum (expression
of lac Z gene under control
of umu C gene)
S. typhlmurlum
(reverse mutation, his)
Exogenous Activation*
System
Rat liver S9
None
None
Rat liver S9 (Aroclor)
Rat liver S9 (none)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver mlcrosomes
(Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat or hamster S9
(Aroclor)
Results Reported
Positive
Negative
Negative
PHENANTHRENE
Negative
Negative
Negative
Positive
Negative
Negative'
Negative
Negative
Positive
Positive
Positive
Comments
Tested at 45 and 67.5 vq/ml
Tested up to 100 pg/mi
Tested at 5 pg/mi
Tested at 125 Mg/well
Tested up to 250 |ig/mi
•
Tested up to 8.3 |ig/mi
At 12 vg/plate In strain TA100
Tested up to 50 nmol/plate In
strain TA100
Tested up to 50 pg/plate In
strains TA1535. TA1537. TA98
and TA100
Tested up to 200 pg/plate In
strain TA100
Tested at 0.28 mN 1n strain
TA1537
Positively Identified In a
highly mutagenlc fraction of
raw gas condensate
At 5 ng/plate TA97
Tested from 50*200 t>g/plate
In strain TA100
Reference
NTP. 1991b
Freeman et al. ,
1973
Rhlm et al..
1974
McCarroll
et al., 1981
Rosenkrantz and
PoUler. 1979
Nakamura
et al.. 1987
Oesch et al . ,
1981
Mood et al.,
1979
McCann et al . ,
1975
La Vole et al..
1981a
Selxas et al.,
1982
Benson et al. .
1984
Sakal et al..
1985
Carver et al..
1986
-------
TABLE V-24 (cont.)
r\>
o
o
CD
Test
DNA damage (cont.)
FUNGI
Recombination
HAHNALIAN CELLS
ONA damage
Nutation
Chromosomal effects
Cell transformation
Organism (Assay)
»
S. typhlmurlum
(forward mutation. 8AGR)
S. cerevlslae
(mltotlc recombination, 03)
Human foreskin epithelial
cells (unscheduled DNA
synthesis)
Primary rat hepatocytes
(unscheduled ONA synthesis)
F344 rat trachea epithelial
organ culture (unscheduled
ONA synthesis)
House skin (effect of co-
application with benzo[a]-
pyrene on formation of ONA
adducts)
Chinese hamster V79 cells
(forward mutation. 8AGR,
QUA") ,
Human lymphoblastotd TK6
cells (forward mutation,
TFT«)
Chinese hamster ¥79-4 cells
(sister -chromatld exchange,
aberrations)
House prostate C3HG23 cells
(morphologic changes)
Syrian hamster embryo cells
(morphologic changes)
House BALB/3T3 cells
Exogenous Activation*
System
Rat liver S9 (Aroclor or
phenobarbltal)
Rat liver S9 (Aroclor)
None
None
None
None
Syrian hamster embryo cell
feeder layer
Rat liver S9 (Aroclor)
Syrian hamster embryo cell
feeder layer
None
None
None
Results Reported
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Comments
Tested up to 300 nmol/ml In
strain TH677
None
Tested up to 400 pg/ml
Tested up to 100 nmol/ml
Tested at 0.1x10"* to
10x10"* H
Average 17X decrease 1n forma-
tion of benzo[a]pyrene-DNA
adducts upon co-application
with benzo[a]pyrene
Tested at 1 pg/ml
Tested at SO nmol
Tested up to 10 pg/mi
Tested up to 10 pg/mi
Tested up to 40 pg/mi
Tested up to SO pg/mi
Reference
Kaden et al..
1979
Simmon. 1979a
Lake et al..
1978
Probst et al..
1981
Ide et al..
1981
Rice et al..
1984
Huberman and
Sachs. 1976
Barfknecht
et al.. 1981
Popescu et al..
1977
Narquardt and
Heldelberger,
1972
Plenta et al. .
1977
Kakunaga, 1973
(morphologic changes)
-------
TABLE V-24 (cont:)
0
CO
Test
Cell transformation
(cont.)
BACTERIA
DNA damage
Nutation
Organism (Assay) Exogenous Activation*
System
S
Guinea pig fetal cells None
(morphologic changes)
Mouse embryo C3H/10T1/2 None
flbroblast cells
E. coll Various
Tree. eolA*..E°l*. uvrA)
B. subtllls (rec-J Various
None
S. typhlmurlum (expression Rat liver S9 (Aroclor)
of lac Z gene under control
of urau C gene)
S. typhlmurlum *°Co gamma Irradiation
(reverse mutation, his) of pyrene
Rat liver S9 (poly-
chlorinated blphenyl)
Rat liver S9 (Aroclor)
Various
I
Results Reported
Negative
Negative
PYRENE
Negative
Negative
Negative
Negative
Positive
Positive
Negative
Negative
Positive
Comments
Tested a 1 5 pg/mi
Tested at 4 MM
International collaborative
program (consensus view of
participants)
International collaborative
program (consensus view of
participants)
No difference In Inhibition
at 100 pg/plate
Tested up to 167 pg/ml
Tested at 160 pg/plate In
strains TA9B. TA1537 and TA1538
At 100 pg/plate with activa-
tion 1n strain TA1537
At 100 pg/plate with and
without activation In strains
TA98 and TA100
Tested up to 1000 pg/plate In
strains TA1535. TA1537. TA98
and TA100
At 25 pg/plate In strain
TA1537; consensus view of
Reference
DIPaolo. 1975
Peterson
et al.. 1981
Ashby and
K1lby, 1981
Ashby and
Kllby. 1981
K1nae et al.,
1981
Nakamura
et al.. 1987
Gibson et al. ,
1978
Klnae et al..
1981
NcCann et al.,
1975; La Vole
et al.. 1979;
Florin et al..
1980; Ho
et al.. 1981
Bridges et al. .
1981
Rat liver S9 {Aroclor)
Positive
participants In International
collaborative program
Positive at 2 pg/plate.
TA1537 TA97; higher doses
TA100
Hatljasevlc and
Zelger. 1985
-------
TABU V ?4 (cont.)
Test
Organism (Assay)
Exogenous Activation*
System
Results Reported
Comments
Reference
Nutation (cont.)
CD
FUNGI
Nutation
INSECTS
Nutation
NANNALIAN CELLS
ONA damage
oo
10
S. typhlimirlum
(reverse nutation, his)
S. typh1mur1um (reverse
mutation, his) (taped-
plate assay)
S. typhlmurlum
("forward imitation. 8AGR)
S. typhlmurlum
(forward mutation, ARAR)
S. cerevlslae and
Schlzosaccharomyces pombe
(different genetic endpolnts)
0. melanogaster (sex-
linked recessive lethals)
Human foreskin epithelial
cells (unscheduled ONA
synthesis)
Primary rat hepatocytes
(unscheduled DNA synthesis)
HeLa cells (unscheduled
ONA synthesis)
Human flbroblast cell line
W138 (unscheduled DNA
synthesis)
F344 rat trachea epithelial
organ culture (unscheduled
DNA synthesis)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Various
None
None
None
Rat liver S9
(3-methylcholanthrene
Rat liver S9 (Aroclor)
None
Positive
Negative
Negative
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Positive
Negative
At 2 ng/plate TA97. 4
wg/plate TA9B
Tested up to SO pg/plate In
strain TA98
Tested up to 50 ug/plate In
strain TA98
At 140 nmol/ml In strain TN677
The minimum positive dose In
strain 813 was 0.25
Consensus view of participants
In International collabratlve
program
800 dig/kg fed 1n diet for up
to 72 hours
Tested up to 400
Tested up to 500 mol/mt
None
At 7.2
Tested from O.lxlO"6 to
10x10"* N
Sakal et al.,
1985
Bos et al..
19B8
Kaden et al.,
19/y
Dorado and
Pueyo. 1988
de Serres and
Hoffman. 1981
Valencia and
Houtchens, 1981
Lake et al..
1978
Probst et al..
1981
Martin et al..
1978
Robinson and
Mitchell. 1981
Ide et al..
1981
-------
TABLE V-24 (cont.)
0
-*».
j».
o
<
1
U3
n/12/91
Test Organism (Assay)
ONA damage (cont.) Mouse skin (effect of co-
appllcatlon with benzo[a]-
pyrene on DNA adduct formation)
Primary F344 rat hepatocytes
(unscheduled DNA synthesis)
Mutation Mouse lymphoma L5178V cells
•(forward mutation, TFTR)
Mouse lymphoma L517BY cells
Mouse lymphoma L517BY cells
(forward mutation. TK~)
Mouse lymphoma LSI 78V cells
(forward mutation. TK~)
Chinese hamster ovary cells
(forward mutation. HGPRT)
Chromosomal effects Rat liver epithelial ARL18
cells (slster-chromatld
exchange)
Chinese hamster ovary cells
(slster-chromatld exchange)
Chinese hamster V79 cells
(slster-chromatld exchange)
Rat liver RLj cells .
(aberrations)
Chinese hamster liver
epithelial cells (slster-
chromatld exchange)
Human peripheral lymphocytes
(slster-chromatld exchange)
Exogenous Activation*
System
None
None
Rat liver S9 (Aroclor)
None
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
Rat liver S9 (Aroclor)
None
Rat liver S9 (Aroclor)
Syrian hamster embryo cell
feeder layer
None
None
None
Intact rat hepatocytes
Results Reported
Positive
Negative
Positive
Negative
Positive
Positive
Negative
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Positive
Comments
Co-application with benzo[a]-
pyrene resulted 1n average In-
crease In level of DNA adducts
of 56X
Tested up to 1E-4 M
At 10 |ig/ml
Tested up to 128 pg/ml
Tested from 5.9-9 pg/mi
Tested at 1.5xlO"» to
2.02xlO'» M
Tested from 2.5-15 pg/ml
Tested up to 1 pmol/mt
Between 19 and 300 pg/mi
Tested up to 100 pg/ml
Tested at 10 pg/ml
Tested up to 100 pg/ml
Tested at 5-45 ng/ral
Tested at 10'4 to 10'6 M
lested at 10~4 M
Reference
Rice et al..
1984
Williams
et al.. 1989
Jotz and
Mitchell. 1981
Mitchell
et al.. 1988
Mitchell
et al.. 1988
Wangenhelm and
Bolcsfoldl.
1988
Oshlro et al. .
1988
Tong et al. .
1981b
Evans and
Mitchell. 1981
Perry and
Thomson. 1981
Popescu et al .
1977
Dean. 1981
DeSalvIa
et al.. 1988
Llndahl-
Klessllng
et al.. 1989
-------
TABLE V-24 (cont.)
c\>
o
en
O
Test
Chromosomal effects
(cont.)
Cell transformation
•\
Organism (Assay) Exogenous Activation*
System
Mouse lyaphoma cells (DNA Rat liver S9 (Aroclor)
strand breaks)
Syrian hamster embryo cells None
(morphologic changes)
Mouse prostate C3H cells Mouse embryo flbroblast
(morphologic changes) feeder layer
Mouse BALB/C-3T3 cells None
(morphologic changes)
Guinea pig fetal cells None
(morphologic changes)
Syrian hamster embryo cells None
(morphologic changes)
Results Reported
Positive
Negative
Negative
Negative
Negative
Negative
Comments
Tested at 0.5xlO~4 and
5xlO"« M
Tested up to 20 pg/mi
Tested at 1 gg/mi
Tested at 20 jig/ml
Tested at SO pg/mi
Tested at 10 pg/mt
Tested at 1-100 tig/mi
Reference
Garberg et al..
1988
DIPaolo et al.,
1969; Plenta
et al.. 1977;
Casto. 1979
Chen and
Heldelberger,
1969
DtPaolo et al..
1972; Kakunaga.
1973
Evans and
DIPaolo. 1975
Tu et al.. 1986
•Compound In parentheses refers to type of Inducing treatment administered to animal from which the exogenous activation system was prepared.
o
CO
-------
other PAHs 1n an ethylene glycol, trlcaprylln or ethyl laurate vehicle.
The mice were then observed for 18 months to determine the Incidence of
sarcomas. Survival was 90-100X during the critical period of tumor
formation, the 4th to llth month. The authors found that both dose and
Interval between administration of the carcinogen and noncardnogen affected
the degree of observed Inhibitory response. Phenanthrene had substantial
Inhibitory effects on the production of sarcomas by d1benz[a,h]anthracene,
particularly at lower dosages. At 275 yg of the carcinogen, concomitant
administration of a 24:1 molar ratio of phenanthrene to d1benz[a,h]-
anthracene (1n ethyl laurate) yielded ~50X reduction 1n the percentage of
tumor-bearing animals as compared with administration of d1benz[a,h]-
anthracene alone. When the dosage was decreased to 60 yg of the
carcinogen, a similar molar ratio of phenanthrene to d1benz[a,h]anthracene
was shown to yield a comparable reduction In tumor development. It was
found that either concomitant administration of carcinogenic and
noncardnogenVc PAHs or administration of the noncardnogen either 2 days
prior to or after the carcinogen administration resulted 1n maximum
/
reduction 1n the percentage of tumor-bearing animals. When the vehicle was
changed to trlethylene glycol, phenanthrene In combination with
d1benz[a,h]anthracene had a substantial promoting effect, approximately
doubling the percentage of tumor-bearing animals.
In the same study benzo[a]pyrene was also tested for carcinogenic
effects 1n combination with other noncardnogenlc, weakly carcinogenic and
potent carcinogenic hydrocarbons 1n a trlcaprylln vehicle (Falk et al.,
1964).. The chosen ratios of putative antlcardnogen to carcinogen approxi-
mated those that occur 1n the environment. PAHs, Including benzofa]-
fluorene, chrysene, benzo[k]fluoranthene, perylene, and a mixture of
04420 V-151 10/08/91
-------
anthracene, phenanthrene and pyrene, had substantial Inhibiting effects on
the ability of benzo[a]pyrene to produce Injection site sarcomas. Other
PAHs, Including acenaphthylene, fluorene, anthracene, benzo[g,h,1]perylene,
1ndeno[l,2,3-cd]pyrene and coronene, had no such Inhibiting effects.
By contrast, Pfelffer (1973, 1977) found no Inhibitory effects of 10
noncardnogenlc PAHs on benzo[a]pyrene or d1benz[a,h]anthracene cardnogen-
1c1ty. Groups of 100 NRHI female mice were given single subcutaneous Injec-
tions of various dosages of benzo[a]pyrene and d1benz[a,h]anthracene, and 10
noncardnogenlc PAHs, separately and 1n combination. In p.5 mi tMcapry-
I1n. The PAHs, which the authors considered noncardnogenlc, Included
benzo[a]anthracene, phenanthrene, anthracene, pyrene, fluoranthene,
chrysene, benzo[g,h,1]perylene, perylene, benzo[e]pyrene and coronene. The
animals were examined weekly for the development of sarcomas for 114 weeks.
Relationships were found to exist between Increasing dose and tumor
development for benzo[a]pyrene, d1benz[a,h]anthracene, benzo[a]pyrene plus
•
d1benz[a,h]anthracene, and all 12 PAHs administered as mixtures. No
treatment-related Increases In sarcoma development were observed for any of
the 10 noncardnogenlc PAHs when administered separately. Interestingly,
the noted dose-response curves for combinations of PAHs most closely
resembled the curve for
-------
Pott et al. (1977) conducted comparable experiments on the carcinogenic
effects of automobile exhaust condensates (AEC) 1n mice. Female NRHI mice
1n groups of ~88 animals were administered single subcutaneous Injections of
various dosages of AEC and benzo[a]pyrene, separately and 1n combination,
which had been dissolved or suspended In trlcaprylln. AEC contained both
.PAHs and non-PAH substances. When AEC was present with benzo[a]pyrene, the
Incidence of tumors (most of which were sarcomas) was less than when benzo-
[a]pyrene was administered alone. This Inhibitory effect of AEC was par-
ticularly obvious at the higher dosages of benzo[a]pyrene, but a decreasing
relationship between tumor Incidence and level of AEC was observed at all
dosages of benzo[a]pyrene. In a second series of experiments, Pott et al.
(1977) sought to assess the effects of a PAH fraction and non-PAH substances
prepared from the AEC. These were Injected separately and 1n combination
with benzo[a]pyrene. Mice Injected 1n this set of experiments were observed
for a year. The PAH-conta1n1ng fraction of AEC was tumorlgenlc. When
assayed 1n combination with other AEC fractions, tumor1gen1c1ty was reduced.
The relative proportions of PAHs found 1n automobile exhaust gas
condensates were determined 1n relation to benzo[a]pyrene. The PAHs were
divided Into two groups; the first group was composed of benzo[a]pyrene,
d1benz[a,h]anthracene, benz[a]anthracene and benzo[b]fluoranthene and the
second group phenanthrene, anthracene, fluoranthene, pyrene, chrysene,
benzo[e]pyrene and benzo[g,h,1]perylene. The concentrations used 1n each
dose group were determined by the relative concentration of benzo[a]pyrene.
The benzo[a]pyrene concentrations selected for the first dose group
(benzo[a]pyrene, d1benz[a,b]anthracene, benz[a]anthracene and benzo[b]-
fluoranthene) were 1, 1.7 and 3.0 yg benzo[a]pyrene and the benzo[a]pyrene
04420 V-153 10/08/91
-------
concentrations selected for the second dose group (phenanthrene, anthracene,
fluoranthene, pyrene, chrysene, benzo[e]pyrene and benzo[g,h,1]perylene)
were 1, 3, 9 and 27 yg benzo[a]pyrene (benzo[a]pyrene was not Included 1n
the group). A third dose group was created by combining the compounds In
groups one and two; the relative benzo[a]pyrene concentrations selected for
this dose group were 1, 1.7 and 3.0 yg benzq[a]pyrene. All compounds were
dissolved 1n acetone. Four control groups that received only benzo[a]pyrene
at doses of 1, 1.7 or 3.0 yg 1n acetone and a solvent control group were
also employed. The shaved skins of 100 female NHRI mice/group were treated
twice/week until natural death or development of an application site
carcinoma (Schmahl et al., 1977).
The Incidence of application site sarcomas 1n the solvent control group
was 1/81; no other tumors were reported. In the benzo[a]pyrene control
groups the carcinoma Incidences were 10/77, 25/88 and 43/81 In the 1, 1.7
and 3 yg benzo[a]pyrene groups, respectively. The carcinoma Incidences
for the first dose group (benzo[a]pyrene, d1benz[a]anthracene,
benz[a]anthracene and benzo[b]fluoranthene) were 25/81, 53/88 and 63/90 1n
the 1, 1.7 and 3 yg benzo[a]pyrene groups, respectively. The carcinoma
Incidences for the second dose group (phenanthrene, anthracene,
fluoranthene, pyrene, chrysene, benzo[e]pyrene and benzo[g,h,1]pery1ene)
were 1/85, 0/84, 1/88 and 15/86 1n the 1, 3, 9 and 27 yg benzo[a]pyrene
groups, respectively. In the third group (combination of groups one and
two) the carcinoma Incidences were 44/89, 54/93 and 64/93 1n the 1, 1.7 and
3 yg benzo[a]pyrene groups, respectively. The reported papHloma and
sarcoma Incidences of all groups did not exceed 5X (Schmahl et al., 1977).
04420 V-154 10/08/91
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The cocardnogenlc potential of various substances found 1n cigarette
smoke was studied by Van Ouuren et al. (1973) and Van Duuren and Goldschmldt
(1976). In the 1973 study, female ICR/Ha Swiss mice 1n groups of 50 were
given repeated dermal applications of 5 yg benzo[a]pyrene 1n 0.1 ml
acetone 3 times/week either alone or with pyrene, benzo[g,h,1]perylene or
benzo[e]pyrene. Control groups received either no treatment or treatments
of acetone, benzo[g,h,1]perylene, pyrene or benzo[e]pyrene alone. The
experiment ran for 52 weeks. Cocardnogenlc activity was shown by the three
PAHs (Table V-25). No skin tumors were found In the control groups. When
benzo[a]pyrene was applied with the Individual noncarclnogens, the number of
mice with paplllomas Increased 1.5-2.6 times over the number observed when
benzo[a]pyrene was applied alone. Likewise, the total number of paplllomas
was 2-6 times higher when the noncarclnogens were present as compared with
benzo[a]pyrene alone. Moreover, the number of days from first application
to appearance of the first tumor In the multiple treatment groups was
similar to that 1n the benzo[a]pyrene group alone.
In'a second study, Van Duuren and Goldschmldt (1976) assessed both the
cocardnogenlc and tumor-promoting activity of benzo[g,h,1]perylene,
fluoranthene, benzo[e]pyrene and pyrene 1n conjunction with benzo[a]pyrene.
Again, female ICR/Ha Swiss mice In groups of 50 were used. Various dosages
of the above noncardnogenlc PAHs alone and 1n conjunction with 5 yg
benzo[a]pyrene were applied 1n 0.1 ml of acetone or 1n DHSO 3 times/week
for <440 days. Pyrene, fluoranthene and benzo[e]pyrene were found to be
potent cocardnogens for benzo[a]pyrene, while benzo[g,h,1]perylene was
determined to be "moderately cocardnogenlc". Pyrene and f luoranthene also
proved to be weak tumor-promoting agents when an Initiating dose of benzo-
[a]pyrene was used. The results of the study are presented 1n Table V-26.
04420 V-155 10/08/91
-------
TABLE V-25
Cocardnogenlc Activity of Various PAHs with Benzo[a]pyrene 1n Mouse Sk1na
(1 Year Exposure)
Carcinogen0
Cocarclnogen
Dose of
Cocarclnogen0
Np. of Mice with
PapHlomas/No.
of Survivors
at 52 Weeks
Benzo[a]pyrene
Benzo[a]pyrene
Benzo[a]pyrene
Benzo[a]pyrene
—
Benzo[e]pyrene
Benzo[g,h,1]perylene
Pyrene
—
15
21
12.
13/42
34/39
20/37
27/41
aSource: Van Duuren et al.t 1973
DBenzo[a]pyrene (5 yg) and/or cocardnogen was applied as acetone solu-
tions 3 times/week for 52 weeks. There were no tumors In the groups
treated only with acetone, benzo[e]pyrene. benzo[g,h,1]perylene or pyrene.
04420
V-156
10/08/91
-------
TABLE V-26
Cocarc1nogen1c Activity of Various PAHs with Benzo[a]pyrene on Mouse Sk1na
Carcinogen''
Benzo[a]pyrene
—
Benzo[a]pyrene
Benzofajpyrene
—
Benzo[a]pyrene
Benzo[a]pyrene
—
Benzo[a]pyrene
—
—
Benzo[a]pyrene
Benzo[a]pyrene
Benzo[a]pyrene
Cocarclnogen
Acetone
Acetone
Benzo[e]pyrene
Benzo[e]pyrene
Benzo[e]pyrene
Benzo[g,h,1]perylene
Benzo[g,h,1]perylene
Benzo[g,h,1 jperylene
Fluoranthene
Fluoranthene
Pyrene
Pyrene
Pyrene
Pyrene
Pyrene
Dose of
Cocarclnogen
(v9)
._
--
15
15
5
21
21
7
40
40
40
12
40
12
4
Duration
of
Experiment
(days)
368
440
368
368
368
368
368
368
440
440
440
368
440
368'
368
Days to
First
Papllloma
251
210
--
246
249
—
222
238
--
99
—
—
229
186
250
Mice with
Paplllomas/
Total Paplllomas
14/16
16/26
0
33/79
24/33
0
20/39
19/31
0
39/126
0
0
35/66
26/42
12/14
aSource: Van DQuren and Goldschmldt, 1976
&Benzo[a]pyrene (5 pg/0.1 ml acetone or DMSO) was applied 1n the same solution as the Cocarclnogen,
3 times/week.
o>
-------
R1ce et al. (1984) also found fluoranthene and pyrene to have cocardno-
genlc potential when combined with benzo[a]pyrene. When female CD-I mice
were given dermal applications of 150 yi [3H] benzo[a]pyrene and either
fluoranthene or pyrene, the occurrence of DNA adduct formation (after 24
hours) was 66 and 56X, respectively, above the level found for benzofa]-
pyrene alone. Phenanthrene co-application resulted 1n an average 17X
decrease and formation of [3H]benzo[a]pyrene-DNA adducts.
Slaga et al. (1979) examined the effects of several PAHs on skin tumor
Initiation by 7,l2-d1methylbenz[a]anthracene (DMBA) and benzo[a]pyrene.
Either benzo[e]pyrene, pyrene or fluoranthene 1n acetone was applied to the
backs of female CD-I mice (30/group) 5 minutes before Initiation with DMBA
or benzo[a]pyrene. Beginning 1 week after Initiation mice received 10 v9
of 12-0-tetradecanoylphorbol-13-acetate (TPA) 1n acetone twice weekly for 30
weeks. The Incidences of both paplllomas and carcinomas were observed
weekly and removed at random for hlstologlc verification. When applied
before Initiation, pyrene and benzo[e]pyrene slightly enhanced
benzo[a]pyrene Initiation whereas fluoranthene had a marginal effect. These
same PAHs had an Inhibitory effect on 7,l2-d1methylbenz[a]anthracene skin
tumor Initiation. When pyrene and fluoranthene were applied after
benzo[a]pyrene Initiation, they had no tumor promoting effects.
Huang et al. (1986) Investigated the ability of benzo[a]pyrene phenolic
metabolites to Interfere with the mutagenlclty and tumor1gen1c1ty of benzo-
[a]pyrene or the putative ultimate carcinogen, benzo[a]pyrene-7,8-d1ol-9,!0-
epoxlde. Of 12 Isomerlc phenolic metabolites, 3-hydroxy-benzo[a]pyrene was
the most potent antagonist of 7,8-d1ol-9,lO-epox1de mutagenlclty for
04420 V-158 10/08/91
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Salmonella typhlmuMum strain TA100; 3 ymol reduced mutagenlcHy by 50%.
The 3-hydroxy-benzo[a]pyrene also decreased m1crosome-med1ated mutagenlcHy
of benzo[a]pyrene and benzo[a]pyrene-7,8-d1ol for TA100. Likewise, mutagen-
lcHy of the 7,8-d1o1-9,lO-epox1de for mammalian cells (V79) was decreased
by 50% upon addition of 8 yM 3-hydroxybenzo[a]pyrene. A 2500 ymole dose
of 3-hydroxybenzo[a]pyrene was applied topically to the shaved backs of
female CD-I mice 5 minutes before addition of a tumor-Initiating dose of 200
vmole of benzo[a]pyrene-7,8-d1ol-9,!0-epox1de. This was followed by 16-20
weeks of promoting treatment with TPA. At the end of this time a 72-78%
reduction 1n numbers of tumors/mouse and a 42-55% decrease. 1n Incidence was
observed by comparison with animals treated with the 7,8-d1ol-9,lO-epox1de
and TPA alone. A less dramatic reduction (35-41% decrease 1n Incidence,
40-52% Inhibition In number of tumors/animal) was observed when benzo[a]-
pyrene was the Initiating agent.
The enhancing or Inhibiting action of non-PAH materials on PAH cardno-
genlclty has also been a subject of study. El-Bayoumy (1985) showed that
two synthetic organoselenlum compounds, p-methoxybenzeneselenol and benzyl-
selenocyanate, Inhibited forestomach tumors 1n mice treated with benzofa]-
pyrene. As described 1n the oral cardnogenUHy section, 9-week-old female
CD-I mice were fed a standard diet for 1 week. At the beginning of week 2,
25 mice/group were fed diets containing varying levels of the above two
compounds or their nonselen1um-conta1n1ng congeners. A third pair of com-
pounds, phenothlazlne and phenoselenazlne, were also tested. Beginning with
week 3, mice were gavaged twice weekly for 4 weeks with 1 mg benzo[a]pyrene
1n corn oil. The test diets were continued for 1 week post-treatment and
then standard diets were fed until week 28 when the mice were killed. Mice
04420 V-159 10/08/91
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receiving benzo[a]pyrene and standard diet had a forestomach tumor Incidence
of 85X. This was decreased to 25X In the high-dose p-methoxyphenol-treated
group. Benzylselenocyanate and methoxybenzeneselenol produced Insignificant
reductions 1n tumor Incidence but significant reductions 1n the number of
tumors/mouse.
As noted 1n the nonoral carc1nogen1c1ty section, the pulmonary cardno-
genlcHy of benzo[a]pyrene can be greatly enhanced by co-administration of
partlculate material. Pershagen et al. (1984) studied the pulmonary
cardnogenlcHy of benzo[a]pyrene alone and 1n combination with arsenic
trloxlde (ASpO-) 1n male Syrian golden hamsters. The animals were
divided Into four groups: ASpCL, benzo[a]pyrene, Aso°3 Plus benzo-
[a]pyrene. and vehicle controls. At each of 15 weekly Instillations, 3
mg/kg of arsenic and/or 6 mg/kg of benzo[a]pyrene was administered. All
groups received a carrier dust (charcoal carbon), which Increased the lung
retention of arsenic. Carcinomas of the larynx, trachea, bronchi or lungs
were found 1n 3/47, 17/40 and 25/54 animals examined 1n the As.Og,
benzo[a]pyrene and As-O. plus benzo[a]pyrene groups, respectively. No
respiratory tract carcinomas were found 1n the 53 controls. The Incidences
of pulmonary adenomas, paplllomas and adenomatold lesions were markedly
higher In the arsenic group than In the control group (p<0.01). There was
also some evidence of a positive Interaction between arsenic and benzo[a]-
pyrene In relation to adenomatos1s lung tumors. The authors noted that the
presence of the carrier dust prevented rapid lung clearance of the
As90.. They proposed- that the arsenic may have had an adverse effect on
£
-------
Met1v1er et al. (1984) studied the effects of different levels of
/
Plutonium oxide (PuO?) and benzo[a]pyrene on lung cardnogenesls 1n SPF
Wlstar rats In an effort to determine the cocardnogenlc effects of Ionizing
radiation and benzo[a]pyrene. Eight different experimental groups Inhaled
PuO^ at four dose levels with and without benzo[a]pyrene (given In two
doses of 5 mg by Intratracheal Instillation). The results are summarized 1n
Table V-27. They show that the Incidence of lung tumors Increased as a
function of both PuCL dose and benzo[a]pyrene exposure. The authors felt
that a multiplicative relative risk model fH the observed effects.
Jones et al. (1984) Investigated the effect of Ia,25-d1hydroxychole-
calclferol [1,25-{OH)_D3] on the transformation of cells pretreated with
benzo[a]pyrene and benzo[e]pyrene. Treatment of Syrian hamster embryo cells
with benzo[a]pyrene for 3 days followed by treatment with 1,25-(OH)2D3
for 4 days Increased transformation of the cells. Benzo[e]pyrene, not a
complete carcinogen by Itself, Induced cell transformation when followed by
•
1,25-(OH) 0, application.
b 0
/
Rahlmtula et al. (1977) examined the capabilities of nine antloxldants
(ascorbate, butylated hydroxyanlsole, butylated hydroxytoluene, ethoxyquln,
glutathlone, NNN'N'-tetramethyl-p-phenylened1am1ne dlhydrochlorlde, nordlhy-
#
droguarlarotlc add, propyl gallate and pyrogallol) to affect benzo[a]pyrene
hydroxylatlorr by rat Hver mlcrosomal MFCs. All of the antloxldants tested
reduced the bacterial mutagenldty of benzo[a]pyrene 1n the presence of rat
liver mlcrosomes and cofactors. They also found that several synthetic and
naturally occurring flavones, when Incorporated In the diet (3-5 mg/g) or
applied to the skin, caused an Increase 1n benzo[a]pyrene hydroxylase
04420 V-161 10/08/91
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TABLE V-27
Co-administration of Pu02 and Benzo[a]pyrene by Inhalation*
No. of Animals
Pu02 Initial
Lung Burden
(Bq)
0
220
630
6300
0
220
630
6300
Benzo-
[a]-
pyrene
(mg)
0
0
0
0
2x5
2x5
2x5
2x5
No. of
Animals
89
89
30
19
38
29
22
19
Median
Survival Time
(days)
864
820
798
345
675
444
480
330
Median Lifetime With Pulmonary
Dose Malignancies
(By)
0.0
3.3
9.4
76.3
0
2.9
8.5
75.4
Fatal
0
4
6
1
7
15
14
18
Incidental
0
13
8
5
3
2
2
1
With Fatal
Benign Tumors
0
0
0
0
2
10
0
0
'Source: Met1v1er et al., 1984
o
CO
-------
activity In the small Intestine and skin, respectively. In addition,
pulmonary adenoma formation resulting from oral administration of
benzo[a]pyrene was totally prevented, and skin tumors Initiated by
benzo[a]pyrene application to mice were significantly reduced (>50X) by
treatment with the synthetic flavbne, B-naphthoflavone. Pulmonary tumor
formation was also reduced 50% by Incorporation of the naturally occurring
flavone, quercetln pentamethyl ether, Into the diet. Sullivan et al. (1978)
demonstrated that BHA, BHT, phenothrazlne, phenothrazlne methosulfate and
ethoxyquln all can reduce the quantitative yield of benzo[a]pyrene
metabolites In Incubations with rat liver mlcrosomes.
Summary
Studies of the general and specific health effects 1n animals associated
with exposure to PAHs tend to be limited 1n the number of PAHs considered
and the route of administration. A wide variety of general toxic effects
have been linked to exposures. Effects on the hematopoletlc and lymphold
systems seem to predominate, such ,as anemia, hemolysls, leukemia and lympho-
cyte toxlclty. Equally Important to the nature of effects, the dosage
eliciting such effects appears to vary by route of administration; for
example, for compounds like fluoranthene, acute toxlclty (LD5Q) occurs at
a lower dosage for oral versus other routes of exposure.
Some PAHs.are known to be carcinogens for animals. In terms of specific
health effects,- relatively potent carcinogens like benzo[a]pyrene and
d1benz[a,h]anthracene as well as moderate-to-strong cocardnogens like
fluoranthene and pyrene are found among the PAHs. Dose-response relation-
ships for oral exposures have been documented for some of the carcinogenic
04420 V-163 10/08/91
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PAHs. The carcinogenic effect of some PAHs when administered to animals by
other routes such as subcutaneous and IntraperKoneal Injections, Inhalation
and skin-painting has also been documented.
Environmental PAH exposures are thought to occur to mixtures rather than
Individual compounds. Experimental PAH 'mixtures research assessing
carcinogenic activity has focussed on nonoral routes of administration;
these Include subcutaneous Injection, dermal application and Inhalation.
When studied 1n combinations. Inhibition, promotion and cocardnogenlc
effects have been reported for PAHs. PAHs' have also been reported to act as
Initiators of cardnogenesls when followed by repeated treatments with
non-PAHs such as plutonlum oxide. Mixtures, such as automobile exhaust
condensate, that contain a variety of PAHs as well as other compounds, have
also been shown to be carcinogenic 1n animals.
Reproduct1ve/teratogen1c effects have not been evaluated for most PAHs.
However, there 1s some evidence suggesting that Ingestlon of benzo[ajpyrene
reduces fertility and reproductive capacity and has deleterious effects on
the offspring of mice so exposed.
Several PAHs have been extensively studied for genotoxlc effects.
Benzo[a]pyrene seems clearly to be mutagenlc In both prokaryotlc and
mammalian cells 1n the presence of an activation system and causes various
chromosomal effects 1n mammalian cells. Benzo[g,h,1]perylene, chrysene and
d1benz[a,h]anthracene are mutagens In several systems, and the latter also
damages the DNA of both prokaryotlc and eukaryotlc cells. Benz[a]anthra-
cene, normally a weak carcinogen, can be activated to exhibit mutagenlc
04420 V-164 10/08/91
-------
activities comparable to benzo[a]pyrene. For the remainder of the compounds
under review, there are either too few data to draw firm conclusions, or the
results have been mixed. Most of these compounds have exhibited
genotoxldty 1n some systems.
04420 V-165 10/08/91
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VI. HEALTH EFFECTS IN HUMANS
Introduction
Human beings are likely to be exposed on a dally basis to various levels
of PAHs 1n the ambient environment. Primary vehicles of exposure among the
general population Include Inhalation of mainstream and sldestream tobacco
smoke and exhaust products from the combustion of commonly used fuels.
Certain occupational groups have the additional burden of exposure to
extremely high levels of complex mixtures that contain PAHs as well as other
toxic and tumorIgenlc compounds. Those considered at high risk for these
exposures Include steelworkers, coke oven workers, gas workers, foundry
workers, aluminum reduction plant workers, roofers, chimney sweeps, and
possibly auto mechanics. Scrotal cancer among chimney sweeps 1s considered
to be the first and oldest Identified occupational cancer (Pott, 1775).
These occupational exposures occur almost exclusively via Inhalation or
direct dermal contact. It 1s from this body of scientific evidence that a
causal relationship has been clearly established between PAH-conta1n1ng
mixtures and several specific cancer sites (IARC, 1984, 1985; WHO, 1988).
These studies are briefly summarized at the end of this chapter, although
their direct relevance to oral Ingestlon of PAHs 1n drinking water 1s
unclear.
PAHs do occur In Ingested media such as water and foods, but these
exposures have not been well-characterized or evaluated. PAHs have been
Identified In smoked foods, charbrolled meats, raw vegetables, shellfish,
and refined fats and oils. To study ep1dem1olog1cally the relationship
between any specific nutrient and a specific adverse event Is very
difficult, however, and extreme caution must always be exercised In
04430 VI-1 08/09/91
-------
Inferring causality. Howson et al. (1986) point out that, although an early
study Implicated 1ngest1on of PAHs via charcoal broiled and smoked foods
with an Increased risk of gastric cancer, subsequent experimental research
showed that PAHs were not good candidates for carcinogens of the glandular
stomach and therefore probably were only Indirectly associated with gastric
cancer.
Cigarette smoking 1s strongly and consistently associated with cancer at
several sites Including the lung, larynx, oral cavity, esophagus and bladder
(Schottenfeld and Fraumenl, 1982; Maclure and MacMahon, 1980). In addition
to n1trosam1nes and other chemicals, 34 PAHs, some of which are recognized
as carcinogens, have been Identified 1n mainstream and sldestream cigarette
i
smoke (IARC, 1983; Uynder, 1988; Appel et al., 1990). Inhalation of tobacco
smoke 1s also considered to be a major risk factor for cardiovascular
disease. However, the specific relationship of any of the compounds 1n
tobacco smoke to the complex etiology of this disease 1s unclear. Albert et
al. (1977) demonstrated 1n chickens that direct Injection of PAHs [DHBA and
/
B[a]P] Into the aorta could Induce atherosclerotic lesions. In humans, H
1s likely that other constituents of tobacco smoke, primarily carbon
monoxide and nicotine, also are Involved 1n the disease process and may
possibly be the main culprits, not PAHs (Uynder. 1988). This will be
briefly addressed later 1n the review of more recently published
occupational studies (Hansen, 1983, 1989; Gustavsson et al., 1988), but a
complete review of the smoking and human health literature 1s well beyond
the scope of this document.
04430 VI-2 08/09/91
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Clinical Case Studies
Oral. The only available case reports of oral 1ngest1on of Isolated
PAHs are concerned with accidental Ingestlon of naphthalene. Acute
hemolytlc anemia 1s the most frequent manifestation of naphthalene poisoning
1n humans. Case reports have described the appearance of acute hemolytlc
anemia after naphthalene Ingestlon by children (Jacobzlner and Raybln, 1964;
Athreya et al., 1961; Gross et a!., 1958; Zlnkham and Chllds, 1957, 1958;
Haggerty, 1956; Chusld and Fried, 1985; Bregman, 1954; MacGregor, 1954;
Abelson and Henderson, 1951; Mackell et al., 1951; Zuelzer and Apt, 1949)
and adults (Anz1ulew1cz et al., 1959; Zlnkham and Chllds, 1957, 1958; Gldron
and Leurer, 1956).
The reported mechanisms and range of exposure to naphthalene 1n these
case studies were: 1) chewing, sucking, or swallowing of mothballs (one to
numerous) as a single Incident or for periods up to 3 months; 2) Ingestlon
of toilet bowl deodorant cakes (pure naphthalene) by a child over a period
of a year; and 3) Ingestlon of naphthalene-containing deodorant 1n a diaper
pall for an unspecified period. Doses of naphthalene were not generally
reported In these case studies because of the poorly defined nature of the
exposure. Tests to detect naphthalene derivatives 1n the urine of the
anemic Individuals were negative 1n some cases and positive In others.
Symptoms of naphthalene tox1c1ty that frequently precede the diagnosis
of acute hemolytlc anemia 1n persons of all ages Include mild to severe
jaundice, dark urine (red, orange, or port wine colored), pallor, and
lethargy (U.S. EPA, 1987a). Severe Jaundice Is often the reason for
hospUaHzatlon, since the jaundice often develops before severe anemia
04430 VI-3 08/09/91
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becomes manifest. However, It 1s clear that anemia and Jaundice can develop
In parallel as shown by a time-course study of hematologlc changes 1n a
16-year-old girl who had Ingested about 6 g of naphthalene 1n a suicide
attempt (Gldron and Leurer, 1956). Vomiting and tachycardia are occasion-
ally observed as precHnlcal signs of naphthalene poisoning In persons of
all ages. Precllnlcal signs of naphthalene toxldty observed primarily 1n
neonates or children Include anorexia, cyanosis, shallow respiration or
apnea, convulsions, and diarrhea. Precllnlcal symptoms of naphthalene
poisoning reported by children or adults Include fever, confusion, pain In
abdominal or kidney region, pain at urination, nausea, headache, fainting,
and vertigo (U.S. EPA, 1987a).
Frequent laboratory findings Indicative of severe hemolytlc anemia after
naphthalene poisoning 1n persons of all ages Include depressed hemoglobin,
hematocrU, and erythrocyte count; elevated leukocyte and retlculocyte
counts; erythrocyte anlsocytosls, polychromatophHla, fragmentation, sphero-
.cytosls, and mlcrospherocytosls; and occasional hemogloblnuMa (U.S. EPA.
1987a)r Additional laboratory findings Indicative of severe hemolytlc
anemia after exposure of neonates or children to naphthalene Include the
following: erythrocyte polkllocytosls and mlcrocytosls; elevated serum
blllrubin; occasional observation of Heinz bodies, nucleated erythrocytes,
and Howell-Jolly bodies; and occasional . observation of methemogloblnurla
(U.S. EPA, 1987a).
In most studies of persons who have developed severe hemolytlc anemia
after exposure to naphthalene, treatment with blood transfusions, treatment
with blood transfusions plus alkali therapy, or observation without either
04430 VI-4 05/15/91
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of these treatments has led to complete patient recovery with no observed
complications. However, deaths have been observed after naphthalene-Induced
hemolytlc anemia (U.S. EPA, 1987a), but It 1s unknown whether they were
directly related to the exposure.
Other Routes. In addition to the cases of acute naphthalene poisoning
following oral exposure, similar effects have been reported following
exposure to naphthalene by other routes: combined dermal absorption and
Inhalation of naphthalene vapor by neonates (GMgor et a!., 1966; Nalman and
Kosoy, 1964; Valaes et al., 1963; Dawson et al., 1958; Cock, 1957; Schafer,
1951) and adults (Younls et al., 1957); Inhalation of naphthalene vapor by
neonates (Hanssler, 1964; Irle, 1964); Inhalation of naphthalene vapor by a
child and adults (L1n1ck, 1983); and transplacental ^exposure of the fetus to
naphthalene that had been Ingested by the mother (Anzlulewlcz et al., 1959;
Zlnkham and ChUds, 1957, 1958). These exposures occurred via combined
dermal absorption and Inhalation for a few days of naphthalene vapor from
.apparel and bed clothing that had been stored 1n mothballs; Inhalation of
vapor 'from a naphthalene-containing medication; Inhalation of naphthalene
vapor for several years from excessive numbers of mothballs kept throughout
the home; and transplacental exposure, for about 3 months, of fetuses to
naphthalene Ingested by the mother.
Two groups of Individuals have been shown to be especially susceptible
to naphthalene-Induced hemolytlc anemia:
1. Persons whose erythrocytes are deficient 1n glucose 6-phosphate
dehydrogenase (G6PDH) or persons 1n whom erythrocyte GSH Is
rapidly depleted by certain oxldant chemicals (U.S. EPA, 1987a).
The precise mechanism by which GSH 1s depleted or a deficiency
of G6PDH leads to naphthalene-Induced hemolysls In these cases
Is not clear.
04430 VI-5 05/15/91
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2. Neonates (U.S. EPA, 1987a). The sensitivity of neonates to
naphthalene 1s explained In part by the same factors that confer
sensitivity to children and adults; namely, G6PDH deficiency
and/or diminished levels of GSH as described above. Additional
naphthalene sensitivity 1n newborns may be conferred by the
Immaturity of pathways necessary for the conjugation and
excretion of naphthalene metabolites (Valaes et al., 1963).
Evidence for the latter hypothesis 1s suggested by the finding
that glucuronlde excretion by human newborn Infants Increased
gradually during the first week of life and that the Initial
levels and the rate of Increase were lower In the premature
Infant than 1n the full-term Infant (Brown and Burnett, 1957).
Fanburg (1940) described the case of a man who had developed an allergic
reaction to naphthalene from clothing that had been stored In mothballs.
The reaction was an exfollatlve dermatitis resembling mycosis fungoldes.
The elimination of naphthalene from the patient's environment resulted 1n
prompt recovery, which lasted uninterruptedly during a 7-year period of
observation.
Case studies that describe the presence of cataracts 1n persons exposed
to naphthalene by the oral, dermal or Inhalation routes have been summarized
•
-1n an ambient water quality criteria documnt for naphthalene (U.S. EPA,
1980c).' Ghettl and Mar1an1 (1956) associated the occurrence of cataracts In
8/21 workers with naphthalene exposure 1n a manufacturing plant. Other
cases of occupational Instances of cataract formation have been described by
Hollwlch et al. (1975).
Possible preneoplastlc epidermal changes 1n humans have been associated
with dermal exposures to benzo[a]pyrene (Cottlnl and Mazzone, 1939; Klar,
1938; Rhoads et al., 1954). A group of 26 patients was given dally applica-
tions of a 1% solution of benzo[a]pyrene on protected (thigh) or unprotected
(upper extremity) skin. These Individuals were hospitalized for a variety
04430 VI-6 08/09/91
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of conditions Including pemphigus vulgaMs, mycosis fungoldes, prokeratosls,
psoriasis, xeroderma plgmentosum, basal cell cancer, squamous cell cancer,
lupus erythematosls, syphllUs (1n various stages) or ringworm. Treatment
was for ~4 months and resulted in a series of progressive alterations 1n the
skin proceeding from erythema and pigmentation to the development of lesions
described by the authors as verrucae. The skin changes and lesions
regressed within 2-3 months after cessation of treatment. The authors noted
that the Individual with xeroderma plgmentosum did not respond differently
from the other subjects (Cottlnl and Mazzonl, 1939). Klar (1938) and Rhoads
et al. (1954) reported potentially deleterious epidermal changes 1n men who
had experienced accidental dermal exposures to benzo[a]pyrene.
Ep1dem1oloq1c Studies
Oral. There are no studies of oral exposures to Identified PAHs In
the literature. PAHs are known to occur In some foods as by-products of the
preparation process and 1n others as a consequence of environmental
pollution, but their role as dietary causes or contributors to cancer can
only be assessed Indirectly In epldemlologlc studies of nutrition. This
body of data Is not specific for PAHs and beyond the scope of this document.
Other Routes. Epldemlologlc studies Unking PAHs to human health
effects have studied human exposures as they actually occurred. These
studies can, therefore, be evaluated to determine the health effects of
exposure only to mixtures of PAHs by Inhalation and dermal contact. Occupa-
tional exposures to PAH-conta1n1ng complex mixtures and 70 Industrial
processes that generate PAHs have been evaluated by the IARC Working Group.
It 1s not possible to determine from these studies the effect of Individual
04430 VI-7 05/15/91
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PAHs. Toxic effects Include a variety of skin lesions and noncancer lung
diseases such as bronchitis.
The compiled case reports and large cohort studies of exposures to
complex mixtures of PAHs have shown strong cause-effect relationships In
that high rates of cancer can be associated- with exposure, dose-response
gradients can be demonstrated and results are consistent across time and
geographic location. The data Include thousands of case reports spanning
over 200 years and cohort studies of cancer Incidence and mortality from
several countries. Data-summarized In IARC (1984) monographs of polynuclear
aromatic compounds demonstrate cardnogenlclty to humans frpm Industrial
exposures to mixtures of PAHs 1n aluminum production, coal gasification,
coke production and Iron and steel founding. Bitumens, coal tars and
derived products, shale oils and soots have also been evaluated for their
human health effects (IARC, 1985). A detailed evaluation of PAH mixtures
can be found 1n the document, "Carcinogen Assessment of Coke Oven Emissions"
.(U.S. EPA, 1984b). Evidence of human cardnogenlclty based largely on the
IARC review mentioned Is summarized 1n Table VI-1. The extent to which the
data derived from these types of exposure will be applicable to drinking
water exposures 1s unknown and creates the major difficulty for the risk
assessment.
Despite the high levels and large proportion of PAHs, other exposures to
gases and metals are always present In these occupational environments. For
example, coke oven emissions contain other carcinogenic agents Including
arsenic, beryllium, chromium, nickel, 2-naphthylamlne and benzene. Asbestos
1s a possible exposure for chimney sweeps (soot). Cocarclnogens (compounds
04430 VI-8 05/15/91
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TABLE VI-1
IARC Determinations of PAH Carclnogenesls Based on Human Oataa
Agent or Process ' Route''
Sites
Human*
Carcinogenic Factor*1
Data Base
Coal
gasification
Inhalation,'
derMl
lung
bladder
skin*
sufficient
Halted
sufficient
Coal tar fro* the destructive
distillation of coal.
Older coal gasification pro-
cesses.
Coal tar fro* the destructive
distillation of coal.
Analysis of 3753 skin tumors, bladder
cancer deaths by occupational group;
cohort studies of gas Industry.
Coke production Inhalation,
derMl
Aluminum
production
Bltunens
Coal tars and
derived products
Inhalation
Inhalation,
dermal
Inhalation,
derMl
lung
skin
kidney
Intestinal
pancreatic
lung
bladder
pancreas
lung
skin
skin
skin
sufficient
sufficient
Halted
Inadequate
Hatted
Halted
Inadequate
Inadequate
Inadequate
sufficient
Halted
Possible coal tar fume.
Coal tar froa destructive
distillation of coal.
Coal tar froa destructive
distillation of coal.
Coal tar froa destructive
distillation of coal.
Aluminum production Industry.
possibly pitch fume.
Bitumens only.
Coal tars froa destructive
distillation of coal, coal
tar pitches.
Creosotes.
Analysis of 3753 skin tumors, bladder
cancer deaths by occupational group;
cohort studies In coking plant, In steel
Industry and coke oven workers. Cohort
studies In five countries; two case-
control studies.
Cohort studies In aluminum production
plants In USSR. USA. Canada. Norway
(21.829 workers In USA).
Cohort study of roofers. Includes coal
tar exposures.
Numerous case reports of occupational and
pharmaceutical exposures; cohort study of
roofers.
to
-------
TABLE VI-1 (cont.)
o
j*.
-Oi
CO
Agent or Process
Shale oils
Routeb
Inhalation.
dermal
Sites
skin
Humanc
sufficient
Carcinogenic Factor^
Shale oils.
Data Base
Case reports; occupational mortality;
cohort study of shale oil workers; mule-
skinners cancer.
Soots Inhalation. skin sufficient Soot. Historic and contemporary case reports
(chimney origin) dermal from various countries; cohort studies of
sweeps In four countries.
*TMs table Is summarized from IARC (1984. 1985).
"Both Inhalation and dermal exposures are possible In most cases. Skin contact Is most likely for coal tars and derivatives, shale oils and
soots and 1n occupations where these products are used such as textile manufacturing (mule skinners).
cThe preamble to IARC document states "The evidence for carclnogenlclty from studies In humans 1s Judged to fall Into one of four groups.
defined as follows: 1) Sufficient evidence of carclnogenlclty Indicates that there 1s a causal relationship between the exposure and human
cancer; 2) limited evidence of carclnogenlclty Indicates that a causal Interpretation Is credible, but that alternative explanations, such
as chance, bias or confounding, could not adequately be excluded; 3) Intdeqmte evidence, which applies to both positive and negative
evidence. Indicates that one of two conditions prevailed: (a) there are few pertinent data, or (b) the available studies, while showing
evidence of association, do not exclude chance, bias or confounding; 4) Ho evidence applies when several adequate "studies are available
that do not show evidence of carclnogenlclty.
dpAHs have been measured In the occupational setting. All processes listed Included exposure to complex mixtures Including at least seven
of those PAHs of concern In this document (see Chapter II for occurrence).
eCase reports of PAH associated skin cancers Include a large proportion of scrotal cancers.
ro
-------
that enhance tumor production In experimental animals when applied
concurrently with carcinogens) and tumor promoters, both PAH and non-PAH,
may also be components of the mixtures.
Data published since the IARC summary continue to provide evidence of
the cardnogenldty of PAH mixtures from Inhalation and dermal exposures.
Hansen (1983) conducted a 5-year mortality follow-up among Danish chimney
sweeps and noted a 2-fold overall excess compared with males employed In
other occupations. When the excess was examined by age and cause, It was
found that the high mortality of the sweeps 1n the older age group (45-74
years) was exclusively due to an excess number of deaths from cancer and
1schem1c heart disease, whereas the excess among the younger sweeps (15-44
years) was due to other causes. Those sweeps dying of cancer or Ischemlc
heart disease had been occupatlonally exposed for an average of 30 years.
Auto mechanics are considered to be another group at risk of
occupational exposure to PAH mixtures but have not been as thoroughly
studied as chimney sweeps. The recent work of Hansen (1989) reports on the
10-year mortality among a cohort of Danish auto mechanics. Overall, a 21%
Increase 1n total mortality was observed 1n relation to a comparable
population of skilled workers presumed to be unexposed to PAH mixtures. In
addition to the moderate Increase 1n cancer mortality, attributed primarily
to an excess of pancreatic cancer, a statistically significant Increase 1n
Ischemlc heart disease deaths was found.
Gustavsson et al. (1988) conducted a study of cancer Incidence 1n a
cohort that comprised nearly all Swedish chimney sweeps employed any time
04430 VI-11 09/25/91
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between 1918 and 1980. Earlier mortality analyses among the cohort had
shown excess mortality from cancers of the lung, esophagus, liver, and from
leukemia. Increased mortality from Ischemlc heart disease was also found.
This study of Incident tumors found a significant excess of cancers of the
lung, esophagus, and bladder, which contributed to an overall excess
Incidence In the cohort. A nearly significant Increase of hematopoetlc
malignancies that Included both multiple myeloma and leukemia was also
observed. H1stolog1c examination attributed the lung cancer Increase to an
excess of squamous cell and und1fferent1ated/small cell carcinomas. The
excess risk for the solid tumors depended mainly on an excess during
followup for more than 30 years from start of exposure.
S1em1atyck1 et al. (1988) explored potential occupational exposures as
risk factors for about 20 cancer sites In a population-based Incident
case-control study In Montreal. The study was designed to generate
hypotheses for further analytic study. This particular report examined
-associations with occupational exposures to 10 types of exhaust and
combustion products and the cancer sites of Interest. The 10 exposures were
classified as follows: four were exhaust products of Internal combustion
engines, distinguished by the type of engine fuel used (gasoline, dlesel,
jet fuel or propane), and the remaining six were products derived from
"nonenglne" combustion of coal, coke, wood, liquid fuels (heating oil,
kerosene, naphtha and lamp oil), natural gas and propane. Among the many
relationships examined In the study, several are of particular Importance.
A major finding was the association between squamous-cell lung cancer and
both dlesel and gasoline exhaust. Five other relationships were noted and
considered by the Investigators to warrant further study regarding the role
04430 VI-12 08/09/91
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of PAH-contalnlng mixtures 1n the etiology of human cancer. The most
promising leads were the following: 1) the effects of exposure to gasoline
and dlesel exhaust on the occurrence of colorectal cancers; 2) the effects
of exposure to gasoline exhaust on the occurrence of kidney cancer; 3) the
effects of exposure to coal combustion products on the occurrence of
pancreatic cancer and possibly on nonadenoma lung cancer; 4) the effects of
exposure to combustion products of liquid fuels on the occurrence of
prostatlc cancer; and 5) the effects of exposure to natural-gas combustion
products on the occurrence of bladder cancer (S1em1atyck1 et al., 1988).
w
The findings from these four studies provide further Important evidence
that occupational exposure to complex PAH mixtures, which occurs primarily
via Inhalation or dermal contact, may also result 1n cancers at sites other
than those logically expected. I.e., lung and skin. It 1s unclear at this
time as to what the mechanism of exposure to the target organs might
Involve. Gustavsson et al. (1988) propose that, for chimney sweeps, PAHs
*
probably enter the body through direct resorptlon In the bronchi, or are
expectorated, swallowed and absorbed through the gastric and Intestinal
mucosa. It Is likely that both routes of uptake are relevant.
The reported Increase 1n 1schem1c heart disease In three of these
studies (Hansen, 1983, 1989; Gustavsson et al., 1988) 1s quite striking when
viewed 1n light of the normal expectation of a "healthy worker effect". The
usual expectation 1n an occupational study of morbidity and/or mortality 1s
that there will be a depression of the standardized morbidity ratio (SHR)
for cardiovascular diseases. This occurs due to pre-select1on of the
workforce for fitness for physical labor. Further studies .specifically
04430 VI-13 08/09/91
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designed to address the Issue must be carried out to determine 1f this 1s
Indeed a true disease excess and not an artifact of the study methods.
Sensitive Populations
Data to determine what Individuals within the general population may be
more susceptible to PAH-lnduced toxlclty are generally lacking. One factor
that Increases an Individual's risk for lung cancer 1s cigarette smoking,
which 1s suspected to act synerglstlcally with other occupational exposures
such as asbestos and uranium mine atmospheres. Although high exposure to
sunlight and low level of skin pigmentation are well documented risk factors
for skin cancer, no data exist to determine whether Individuals with these
risk factors are more susceptible to PAH-lnduced skin lesions.
Aryl hydrocarbon hydroxylase (AHH) 1s the term given to the enzyme or
enzyme systems Involved In the oxldatlve metabolism of PAHs, especially
benzo[a]pyrene. Genetically determined Individual differences In the
.ability to Induce both Increased levels and Isozymes of AHH have been
proposed as a possible reason for differences among humans In susceptibility
to lung cancer (for further discussion see Chapters III and VII). Some
early studies claimed that persons with lung cancer had higher InduclblHty
of AHH In cultured lymphocytes, but subsequent studies have not all
supported this claim (Perera, 1990). Busb.ee et al. (1980) present evidence
supporting the need to analyze AHH 1n more than one tissue and recommend
measuring 1ndudb1l1ty In alveolar macrophages and lung tissue as well as
lymphocytes. More res.earch will be required to determine the sensitivity
and specificity of this enzyme system as a predictor of human susceptibility
to lung cancer.
04430 VI-14 05/15/91
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Cardnogen-DNA adducts may ultimately prove to be helpful 1n Identifying
those persons at high risk of adverse outcome following exposures to PAH
mixtures. It Is currently possible to detect PAH-DNA adducts In several
types of human tissue and sera In those known to be either occupatlonally or
environmentally exposed (Herbert et al., 1990; Hemm1nk1 et al., 1990;
Perera, 1990). However, the role of adducts- 1n Inducing cancer or other
adverse outcomes Is still a matter of much scientific discussion. Long-term
•'*'"..""' •
validation studies are required 1n appropriate human populations before
wide-scale monitoring for these blomarkers can be considered as a more
refined and specific way of predicting who may or may not be at higher risk
(I.e., more susceptible) of a particular outcome.
Summary
With the exception of naphthalene, which most commonly causes acute
hemolytlc anemia, no data were located directly pertaining to PAH toxldty
to humans by the oral route of exposure. A large body of literature exists
on toxldty and carclnogenldty to humans of a variety of mixtures contain-
ing PAHs. The summarized IARC data clearly demonstrate that Inhalation and
dermal exposures to complex mixtures containing PAHs result In lung, skin
and other cancers. The most recent occupational studies continue to provide
compelling evidence regarding the carclnogenldty of PAH mixtures. The
Increased risk of cardiovascular disease 1s noteworthy and must be studied
further. In the occupational environments studied, some airborne PAHs may
be cleared by mucodllary action and swallowed. This Information may be
useful for determining the potential carclnogenldty to humans of Ingested
PAHs.
04430 VI-15 05/15/91
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One must be cautioned that using these data to predict the effects of
exposure to any single PAH 1s Inappropriate. Furthermore, most 1f not all
PAH exposures occurring to humans are 1n the form of a complex mixture of
the compounds, 1t seems both unrealistic and unnecessary to attempt to
assess the potential role of each Individual compound. Studies to date
clearly Implicate this chemical family as human carcinogens at relatively
Intense, high level exposures. Recent work has also more clearly delineated
the role of PAHs and benzo[a]pyrene 1n particular as causal carcinogenic
constituents In the highly complex mixture known as tobacco smoke (Wynder,
1988). While all the Information summarized 1n this chapter Is Important
for evaluating and clarifying the role of PAH mixtures and their relation-
ship to cancer and cardiovascular disease 1n humans, the direct relevance of
the Information to the assessment of any adverse effect arising from
1ngest1on of PAHs In drinking water remains to be determined.
04430 VI-16 09/25/91
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VII. MECHANISMS OF TOXICITY
The carcinogenic potential of PAHs has been a subject of study for >50
years. They were among the first chemical agents shown to be tumorlgenlc,
and their role 1n the causation of human cancers has been a subject of
•
speculation almost since Pott's (1775) first description of "soot wart" In
chimney sweeps 1n the 18th century. Noncarclnogenlc health effects of PAHs
have not been studied extensively; mechanisms of PAH toxlclty apart from
their ability to Induce neoplasla are, thus, not well elucidated.
Mechanisms Involved 1n PAH Carc1noqen1c1ty
Structure Activity Relationships. Many physico-chemical and enzy-
matic parameters must be considered with respect to PAH carclnogenldty.
Solubility and Intracellular localization proximate to metabolic enzymes are
likely to be Important determinants of the cardnogenlcUy of a particular
PAH.
Following the Identification of the first carcinogenic hydrocarbon from
soot, benzo[a]pyrene, an Intensive effort was mounted to Isolate the various
active components of carcinogenic tars (IARC, 1973). It became apparent
that carcinogenic PAHs are structurally derived from the simple angular
phenanthrene nucleus (Pullman and Pullman, 1955; Arcos and Argus, 1974).
UnsubstUuted PAHs with fewer than four condensed rings that have been
tested have so far not shown tumorlgenlc activity. Furthermore, of the six
possible PAHs with arrangements with four benzene rings, only three of these
compounds are active as carcinogens, namely benzo[c]phenanthrene,
benz[a]anthracene and chrysene. The unsubstUuted penta- and hexacycllc
04440 VII-1 05/07/91
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aromatic hydrocarbons are generally the most potent carcinogens of the
series. These Include benzo[a]pyrene, d1benz[a,h]anthracene, dlbenzo-
[a,h]pyrene, d1benzo[a,1]pyrene, d1benzo[a,l]pyrene, d1benzo[a,e]pyrene.
benzo[b]fluoranthene, and benzo[j]fluoranthene. Less potent carcinogens are
the dlbenzanthracenes and dlbenzophenanthrenes. Only a few heptacycllc
hydrocarbons show carcinogenic activity, Including phenanthro[2',3':3,4']-
pyrene, peropyrene, and d1benzo[h,rst]pentaphene. There are very few known
carcinogenic hydrocarbons with more than seven unsubstltuted aromatic rings
(Santodonato et al., 1981).
Active carcinogens are also found among those PAHs containing a non-
aromatic ring. Examples of this type of compound are cholanthrene,
1,12-ace-benz[a]anthracene, 8,9-cyclopentanobenz[a]anthracene, 6,7-acebenz-
[a]anthracene, acenaphthanthracene, l,2,5,6-tetrahydrobenzo[J]cyclo-
pent[f,g]aceanthrylene, and "angular" steranthrene. All of these compounds
contain an Intact conjugated phenanthrene moiety.
Alfcyl substitution at certain positions In the ring system of a fully
aromatic hydrocarbon has been observed to confer carcinogenic activity or to
enhance carcinogenic potency. Arcos and Argus (1974) noted that monomethyl
substitution of benz[a]anthracene makes these dervatlves potent carcinogens
1n mice. Potency depends on the position of substitution 1n the decreasing
order position 7 > 6 > 8*12 > 9. A further enhancement of carcinogenic
activity 1s produced by appropriate dimethyl substitution of benz[a]anthra-
cene. Carcinogenic compounds are produced by 6,8-d1methyl-, 8,9-dlmethyl-
8.12-dlmethyl-, 7,8-d1methyl-, and 7,12-dlmethyl-substHutlon. The last
04440 VII-2 05/07/91
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compound 1s among the most potent PAH carcinogens Identified 1n
cardnogenesls bloassays. It has not been shown, however, to occur as a
product of fossil fuel pyrolysls or to be a major environmental
contaminant. Carcinogenic trlmethyl- and tetramethyl-benz[a]anthracenes are
known, and their relative potencies are comparable with the parent
7,l2-d1methylbenz[a]anthracene. Methyl substitution 1n the angular ring of
benz[a]anthracene tends to reduce the carcinogenic potential of the
molecule; 4,5-d1methylbenz[a]anthracene may be an exception In this regard.
Alkyl substitution of partially aromatic condensed ring systems may also
enhance the carclnogenlcHy of a compound. 3-Methylcholanthrene, a highly
potent carcinogen, 1s the best example of this type.
Data derived largely from skin painting studies have shown that the
carclnogenlcHy of PAHs tends to decrease with alkyl substHuents longer
than methyl, possibly due to a decrease 1n transport through cell
membranes. Benz[a]anthracene 1s especially sensitive to decreased cardno-
genlclty caused by the addition of bulky substHuents at the 7-pos1t1on.
This observation lent credence to the view for most polycycllcs that high
.reactivity of the mesophenanthrenlc region (called the "((-region*) was a
critical determinant for carclnogenlcHy. Other studies, however, show that
the K-reg1on may not be Involved 1n binding to DNA, a step considered
Important Vn the carcinogenic process (Blobsteln et al., 1976; Welnsteln et
al., 1976; Hoore et al.. 1977).
Partial hydrogenatlon of the polycycllc aromatic skeleton has been
observed to decrease the carcinogenic potency of some PAHs. This was noted
04440 VII-3 10/03/91
-------
for hydrogenated derivatives of benzo[a]pyrene, benz[a]anthracene, and
3-methylcholanthrene. By constrast, the cardnogenldty of d1benz[a,h]anth-
racene, d1benzo[a,1]pyrene, and d1benzo[a,h]pyrene 1s not significantly
altered by mesohydrogenatlon, probably because extensive resonance Is
preserved 1n the molecule. Moreover, 5,6-d1hydrod1benz[a,h]anthracene
actually displayed a 4-fold Increase 1n cardnogenlcHy by comparison with
the parent unsaturated hydrocarbon (Arcos and Argus, 1974), possibly due to
the hydroph1l1c1ty and ease of Intracellular transport of Us dlhydrodlol
derivative.
For many years, Investigators have sought a common molecular feature
among PAH carcinogens that would serve to explain their biologic activity.
The "electronic theory of cardnogenesls" has relied upon an analysis of the
Influence of electron density at specific molecular regions to explain
unique reactivity with cellular constituents. A basic assumption arising
from the work of the Pullmans and others (Pullman and Pullman, 1955) was
that a mesophenanthrenlc region ("K-reg1on") of high ^-electron density
and with a propensity for addition reactions was a critical structural
feature for polycycllc carcinogens. In an expansion of this hypothesis,
further biologic significance was attributed to the concomitant presence of
a rather unreactlve meso-anthracenlc region ("L-reg1on") for high cardno-
genlcHy. In addition, a region of comparatively low reactivity, which
characteristically undergoes metabolic perhydroxylatlon (corresponding to
the 3,4-pos1t1ons of benz[a]anthracene), had been designated the M-reg1on.
According to the theory, only binding of the K-reg1on to critical cellular
sites would Initiate tumor formation. Binding at the L-reg1on could cause
no tumorlgenlc effect, while 1nact1vat1on would be produced by metabolic
04440 VII-4 12/31/90
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perhydroxylatlon In the M-reg1on. The three regions of reactivity are
Illustrated below 1n the benz[a]anthracene skeleton:
H-reg1on of metabolic
perhydroxylatlon
K-rtglon
The electronic K-L theory of carcinogenic reactivity has encountered numer-
ous Inconsistencies, primarily because these relationships were derived from
physico-chemical properties of the parent hydrocarbon and gave no considera-
tion to the biologic effects of metabolites.
That many chemical carcinogens require metabolism to reactive electro-
phlUc forms for their activity 1s generally accepted. PAHs are certainly
1n the class of those carcinogens requiring so-called "metabolic activation"
for binding to critical macromolecules. The realization that this metabolic
activation to reactive Intermediates was a necessary first step has made
possible an understanding of some of the Inconsistencies encountered 1n
structure-activity theories, such as K-reglon binding, that are based solely
on a consideration of the parent compound.
The metabolic processes undertaken by mammals exposed to PAHs are
described 1n Chapter III. In general, they consist of oxidation steps
catalyzed by cytochrome P-450-assodated enzymes. Products Include epoxlde
Intermediates, dlhydrodlols, phenols, qulnones and combinations of these.
These oxidized forms of PAHs have been shown to exhibit various biologic
04440 VII-5 05/07/91
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activities. For example, various benzo[a]pyrene epoxldes (Including the
K-reg1on epoxlde), phenols, qulnones, dlols and dlol-epoxldes have been
shown to be mutagehlc 1n several mammalian cell and bacterial assays
(Gelboln, 1980; IARC, 1973; Schoeny et al., 1985; Chesls et al., 1984).
Research 1n recent years has focused attention on the potential reactiv-
ity of d1ol-epox1de metabolites of PAHs, and their ease of conversion to
trlol carbonlum Ions. The "bay-region" theory proposes that d1ol-epox1des,
which are more readily converted to carbonlum Ions, will be better alkylat-
Ing agents and thus mutagens and Initiators of cardnogenesls (Jerlna and
Lehr, 1977; Wood et al., 1979). Examples of a bay-region In a polycycllc
hydrocarbon are the regions between the 10 and 11 positions of benzo[a]-
pyrene and the 1 and 12 positions of benz[a]anthracene:
Bay-region
9
/ 8 r ,7 u 6
Benzo[a]pyrene Benz[a]anthracene
The theory predicts that dlol-epoxldes 1n which the oxlrane oxygen forms
part of a bay-region (such as benzo[a]pyrene 7,8-d1ol-9,lO-epox1de) will be
more reactive and hence more carcinogenic than d1ol-epox1des 1n which the
oxlrane oxygen 1s not situated 1n a bay-region. The unique structural
feature of the d1ol-epox1des appears to be that the epoxlde Is on a
saturated angular benzo-rlng, which forms part of a bay-region on the PAH.
Perturbatlonal molecular orbital calculations, which predict ^-electron
energy changes. Indicate that the epoxldes on saturated benzo-rlngs (which
form part of the bay-region of a hydrocarbon) undergo ring opening to form a
04440 VII-6 11/13/91
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carbonlum 1on much more easily than do nonbay-reglon epoxldes (Pelkonen and
Nebert, 1982). Synthetic bay-region d1ol-epox1des of benz[a]anthracene,
benzo[a]pyrene, and chrysene have been shown to be more mutagenlc In vitro
and/or tumoMgenlc than other dlol-epoxlde metabolites, their precursor
dlhydrodlols, the parent hydrocarbons, or other oxldatlve metabolites.
Moreover, quantum mechanical calculations provide support for the concept
that reactivity at the bay-region 1s highest for all the dlol epoxldes
derived from polycycllc hydrocarbons.
The bay-region concept has received enough confirmation to lead to
i
suggestions that an analysis of theoretical reactivity 1n this manner may be
useful In screening PAHs as potential carcinogens (Smith et al., 1978).
Among several Indices of theoretical reactivity examined, the presence of a
bay-region for a series of PAHs displayed a high degree of correlation with
positive carcinogenic activity (Table VII-1).
It 1s possible to predict what would be an ultimate carcinogenic form
from the bay-region theory and a consideration of a PAH structure. There
1s, however, no way at the present time to predict whether a particular PAH
will, 1n fact, be metabolized to a bay-region dlol-epoxlde 1n any given
mammalian tissue. For example, benzo[e]pyrene 1s generally considered
noncardnogenlc. Neither the parent compound nor the 9,10-dlhydrodlol, the
presumed bay-region d1ol-epox1de precursor, has significant Initiating
potency 1n mouse skin (IARC, 1983). It has been shown that neither cultured
mammalian cells nor rat liver mlcrosomes metabolize benzo[e]pyrene to the
9,10-dlhydrodlol; furthermore, 9,lO-d1hydrod1ol 1s not metabolized 1n these
systems to the expected dlol-epoxlde. When synthetic benzo[e]pyrene
9,10-dlhydrodlol was Injected 1n newborn mice, hepatic tumors were observed,
04440 VII-7 12/31/90
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suggesting that the neonatal "liver possessed some metabolic capacity not
extant 1n mouse skin or Yn vitro systems (Buenlng et al., 1980). Synthetic
trans and ds 9,lO-d1ol-ll,!2-epox1des produced hepatic and pulmonary
tumors, respectively, 1n newborn mice (Chang et al., 1981). Thus, a
bay-region d1ol-epox1de of benzo[e]pyrene appears to be carcinogenic, but 1s
not formed 1_n vivo under normal circumstances.
Various researchers have attempted to refine and extend the K- or bay-
region theories to allow for predlctlvlty of carcinogenic potential from a
consideration of PAH structure. For example, the reactivity Index used by
Mohammed (1983) established a correlation between the K-reglon reactivity
and the bay-region theory to determine PAH metabolic products. A later
development considered contribution of both slgma and pi-electronic systems
when calculating bond superdelocaHzabllUy (Mohammed, 1985). The potential
of PAHs for one-electron oxidation, as discussed by Caval1er1 and Rogan
(1983) 1s likely to be a factor In cardnogenlcHy and needs Inclusion 1n a
predictive quantitative structure activity scheme.
*
Tissue Specificity of PAH Metabolism. The capacity of mammalian liver
to metabolize PAHs Is well-documented. Other tissues may also be Involved
1n the metabolism of a specific PAH to reactive forms. AHH, the primary
benzo[a]pyrene metabolizing system, has been found 1n human liver, placenta,
lymphocytes, monocytes and lung macrophages (Gelboln, 1980).
Cytochromes P-450 and associated enzymes are known to be present 1n mam-
malian lungs at lower concentrations than are generally found 1n the liver.
Studies using lung mlcrosomal preparations, cultured trachea and alveolar
macrophages and Isolated perfused lungs have shown that benzo[a]pyrene 1s
04440 VII-9 12/31/90
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metabolized to the same oxidized forms as are produced by the "hver. What
differs 1s the proportion of classes of metabolites, the rate of metabolism
and the ultimate tissue distribution of metabolites (Moore and Cohen, 1978;
Santodonato et al., 1981). Co-administered particles or gases may also
Influence the measures (Warshawsky et al., 1981; Schoeny and Warshawsky,
1983).
Intestinal mucosa has been shown to have MFO activity and presumably to
have the capacity to metabolize PAHs. It has also been observed that the
susceptibility of mice to Induction of forestomach tumors can be correlated
with these enzymatic activities. Generally, levels of benzo[a]pyrene
metabolizing enzymes In rodent small Intestine and colon are rather low and
not readily Indudble to higher levels (Santodonato et al., 1981).
Genetic Control of PAH Metabolism. As noted 1n the preceding sections
and In Chapter III, oxldatlve metabolism of PAHs 1s generally accomplished
•
through a series of enzymes associated with cytochrome P-450. Both quanti-
tative and qualitative changes 1n these enzymes can be Induced In response
to exposure to a variety of agents, Including the PAHs themselves. PAHs
can, thus, be responsible for Inducing enzymes for their own metabolism.
Including activation to mutagenlc and carcinogenic forms. The degree of
1nduc1b1l1ty as well as the spectrum of enzymes produced 1n response to a
particular agent are not only tissue-specific, but also species/strain
specific. In particular the enzyme system, loosely called AHH, that 1s
Involved In PAH metabolism (especially benzo[a]pyrene) has been shown to be
under genetic control. The genetics of AHH Induction has been well-charac-
terized In mouse strains observed to be "responders" or "nonresponders" to
04440 VII-10 05/07/91
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Induction by PAH (usually 3-methylcholanthrene) (Nebert et al., 1981). The
expression of the responsive trait Is due to Inheritance of an allele of the
Ah locus, which codes for a cy.tosollc receptor. This receptor regulates
expression of a set of cytochrome P-450 and associated enzymes. When PAH,
2,3,7,8-tetrachlorod1benzo-£-d1ox1n or other compounds are bound to the
receptor, the sets of genes coding for the various metabolic enzymes are
Induced to higher levels of synthesis. Responsive mice have a high-affinity
receptor that readily binds to a number of PAHs and 1s thus more easily
Induced 1n responders than 1n nonresponders, which have a low affinity
receptor (Nebert et al., 1982; Elsen et al., 1983). A number of biologic
effects observed 1n test animals as a consequence of PAH exposure can be
shown to be affected by the responsiveness or nonresponslveness of the
strain. For example, mouse strains C3H/HeJ and C57B1/6J, both responsive to
d1benz[a,h]anthracene Induction of AHH, were more susceptible to
cardnogenesls after s.c. exposure to that compound than were two
nonresponslve strains, AKR/J and DBA/2J. Incidences were 24/30 for C3H/HeJ
and 16/30 for C57B1/63 by comparison with 0/30 and 1/30 for AKR/3 and
DBA/2J, respectively (Lubet et al., 1983a). Similar results were reported
by Kourl et al. (1983), who also showed that among progeny of a heterozygous
responder (B6D2F1) x a homozygous nonresponder (D2) backcross, that
susceptibility to subcutaneous .tumor formation by d1benz[a,h]anthracene
segregated with responsiveness.
Route of exposure plays a part 1n the extent of biologic effects
produced by PAHs In responsive and nonresponslve mice.. When benzo[a]pyrene
1s administered topically, s.c., 1.p. or Intratracheally, responsive mice
are more likely than nonresponders to develop tumors or toxic responses at
the site of application. When benzo[a]pyrene exposure 1s through the oral
04440 VII-11 08/09/91
-------
route, nonresponslve mice are more likely to develop leukemia or bone marrow
toxldty (Nebert, 1981; Legraverend et al.t 1983). This 1s due to the
effects of first pass metabolism and excretion of the PAH. Nebert et al.
(1980) showed, for Instance, that after oral exposure 10-20 times more
[3H] benzo[a]pyrene reaches the bone marrow and spleens of nonresponders
than of responsive mice.
When fetal toxldty Is being assessed there Is the additional complica-
tion of the dam's responsiveness. This will determine to some extent the
dose of maternally administered compound that Is delivered.to the fetuses,
which will themselves be responders or nonresponders. For Instance, when
nonresponder dams received oral benzo[a]pyrene on days 2 and 10 of gesta-
tion, nonresponder fetuses showed a greater toxic response and more
malformations than did responders. When heterozygous responder dams were
•*
similarly exposed, there was no segregation of toxic response with the
nonresponder allele; that 1s, both nonresponder and responder fetuses were
equally affected. In the case of the nonresponder dam, no Induction of
/
benzo[a]pyrene metabolism took place and the fetuses received a larger dose
of the PAH. This allowed the genetic differences In the embryos to be
detected; 1n this 'instance, greater amounts of toxic benzo[a]pyrene metabo-
lites have been Isolated from nonresponder fetuses than from responders.
When the responsive dam was exposed, Intestinal and hepatic metabolism of
benzo[a]pyrene was Increased. Fetuses received less PAH than 1n the above
Instance; thus there was less overall fetal toxldty, and the genetic
differences among Individual fetuses was not seen. The reverse of these
effects was observed when dams were treated Intraperltoneally (Legraverend
et al., 1983).
04440 VII-12 05/07/91
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It has been shown that ovarian AHH 1s Induclble 1n responsive (e.g.,
C57B16/N) but not 1n nonresponder (e.g., OBA/2N) mouse strains. Responders
have likewise been observed to be more sensitive to oocyte destruction by
PAHs than nonresponders. The susceptibility to oocyte killing by PAHs,
however, 1s not Inherited as a simple autosomal dominant trait as Is
responsiveness. This suggests that other factors In addition to ovarian PAH
metabolism are Involved 1n oocyte destruction by PAH (Hattlson et al., 1983).
An area of considerable uncertainty 'with regard to the carcinogenic
hazard of PAHs to humans Involves the relationship between AHH activity and
cancer risk. Genetic variation 1n AHH InduclbllUy has been Implicated as a
determining factor for susceptibility to lung and laryngeal cancer
(Kellerman et al., 1973). It was suggested that the extent of AHH Inducl-
bllUy In lymphocytes was correlated with Increasing susceptibility to lung
cancer formation.
Paigen et al. (1977, 1978a,b) examined the question of genetic suscepti-
bility to cancer and concluded that epldemlologlc evidence -supports this
hypothesis. Moreover, they were able to show that' AHH InduclbllUy 1n
lymphocytes segregates 1n the human population as a genetic trait. However,
their studies failed to find a correlation between this InduclbllUy and
presumed cancer susceptibility, either among healthy progeny of cancer
patients or In patients who had their cancer surgically removed. It 1s
noteworthy that previous Investigations on AHH InduclbllUy were conducted
In persons with active cancer.
04440 VII-13 11/13/91
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In an attempt to elucidate sources of variability 1n human AHH Inducl-
blllty, a study was undertaken using cultured monocytes obtained from 10
sets of monozygotlc twins and 17 sets of dlzygotlc twins. Both benz[a]-
anthracene-lnduced and basal levels of AHH were determined. Genetic factors
were found to account for 50-66% of observed 1nter1nd1v1dual differences 1n
•
AHH 1nduc1b1l1ty. The authors felt that a relatively few number of genes
were Involved 1n regulation of AHH Induction (Okuda et al., 1977). It would
seem that susceptibility to cardnogenesls by PAHs could be a genetically
determined trait at the level of metabolism. The contradictory nature of
studies In this area, however, point to the fact that many factors regarding
PAH activation and subsequent steps In the carcinogenic process need to be
elucidated.
Other Pathways Involved 1n Activation of PAH. PAHs are photoreactlve
compounds. Visible light can be absorbed by several PAHs at sufficient
energy levels to result 1n photoox1dat1pn. Benzp[a]pyrene can be photo-
oxidized to the 1,3-, 6,12- and 3,6-qu1nones as well as to dlhydrodlols and
phenols' (Katz et al., 1979; Gibson and Smith, 1979). It 1s likely that
qulnone formation Is through a phenoxy radical and that phenol and
dlhydrodlol formation may also proceed through radical formation (Inomata
and Nagata, 1972; Jeftlc and Adams, 1970; Greenstock and Wlebe, 1978).
Benz[a]anthracene 1s also known to form qulnones as a consequence of light
exposure 1n aqueous media (Mill et al., 1981).
Caval1er1 et al. (1985) described a 1-electron PAH oxidation pathway as
opposed to the 2-electron mono-oxygenase pathway. This produces radical
cations or radicals depending on the PAH. In addition to cytochrome P-450,
04440 VII-14 . 11/13/91
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hydroperoxldases, peroxldases and prostaglandln synthetase can participate
1n these types of reactions. PAH free radicals {for example, 6-oxy-
benzo[a]pyrene free radical) have been Implicated In PAH binding to DNA
(Santodonato et al., 1981).
Cobalt ^-Irradiation of PAHs, Including benz[a]anthracene, benzo[a]-
pyrene and chrysene, has resulted In formation of mutagenlc compounds.
Likewise UV-1rrad1ated benzo[a]pyrene was shown to be mutagenlc for Salmo-
nella typhlmurlum {Gibson et al., 1978). The potential for visible light
and other electromagnetic radiation to activate PAHs has Implications for
mechanisms Involved In PAH skin cardnogenesls.
PAH Involvement 1n Carcinogenic Processes. A description of the
hypothetical mechanisms purported to be Involved In the carcinogenic
processes 1s beyond the scope of this document. Suffice It to say that PAHs
may participate In many proposed carcinogenic steps. The majority of PAHs
described 1n this document are mutagenlc 1n one or more test systems as
described 1n Chapter V. Many have been shown to be Initiators 1n mouse skin
Initiation-promotion assays. PAHs can, thus, generally be described as
capable of DNA-b1nd1ng, or of causing DNA damage leading to mutations, which
could be Involved 1n the Initiation phase of cardnogenesls.
Consideration must also be given to the fact that, 1n addition to the
Initiation of resting cells by a chemical carcinogen, a promotion phase
Involving cell proliferation Is also Involved In skin cardnogenesls (Yuspa
et al., 1976). Promotion 1s likely to be a phase common In the carcino-
genic process for most tissues. Certain PAHs may function only as
Initiators and have no promoting ability. It would appear, however, that
04440 VII-15 05/07/91
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the more potent complete carcinogens also serve as promoters by Interacting
with cellular membranes, altering- genetic expression, or 1n some other
fashion causing cell proliferation. It should be noted, also, that as PAHs
have been shown to have various 1mmunosuppress1ve effects, they may Impair
the body's capacity for Immune surveillance of neoplastlc growths. PAHs
may, thus, play a part In all steps of a carcinogenic process.
Mechanisms Involved In Noncarclnogenlc Endpolnts
The preferred target sites of PAHs appear to be rapidly proliferating
tissues such as Intestinal epithelium, bone marrow, lymphold tissue and
gonads. This has led Investigators to the hypothesis that the toxic effects
of PAHs are due to a specific attack on DNA of cells 1n the DNA synthesis or
S phase of the cell cycle. Alterations 1n enzyme activity resulting from
the direct attack on DNA may also have Important significance to the adverse
effects resulting from PAH exposure. Information on certain PAHs not
considered In the rest of the document 1s presented here for Illustrative
purposes.
/
Hemolymphatic System. 7,l2-D1methylbenz[a]anthracene Is well known
for Us effects on the hematopoletlc system. Female Sprague-Dawley rats fed
112 or 133 mg/kg 7,12-d1methylbenz[a]anthracene developed pancytopenla due
to a severe depression of hematopoletlc and lymphold precursors (Caweln and
Sydnor, 1968). Maturation arrest occurred at the proerythroblast, promylo-
blast and promegakaryocytoblast levels; no Injury occurred to stem cells or
circulating formed elements. In another study, rats receiving 300 mg/kg
(orally) and 50 mg/kg (1.v.) of 7,l2-d1methylbenz[a]anthracene displayed
extreme atrophy of hematopoletlc elements, shrinkage of lymphold organs,
04440 VII-16 12/31/90
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agranulocytosls, lymphopenla and progressive anemia (Philips et al., 1973).
Similar results have been noted 1n mice and rats given repeated Injections
of d1benz[a,h]anthracene. D1benz[a,h]anthracene administered to mice 1n
weekly subcutaneous Injections for 40 weeks caused an Increase In the number
of lymph gland stem cells, an overall decrease 1n lymphold cells, and
dilation of lymphold sinuses. The weights of the spleens of mice treated
with d1benz[a]anthracene were also significantly less than those of both
controls and animals treated with anthracene or benz[a]anthracene (Hoch-
L1get1, 1941).
In a similar study, rats given subcutaneous Injections 5 times weekly
for several weeks underwent changes 1n lymphold tissue characterized by the
presence of extravascular red blood cells 1n the lymph spaces and the
presence of abnormal large plgmented cells (Lasn1tsk1 and Woodhouse, 1944).
The noncardnogen, anthracene, did not produce as dramatic a change In
lymphold tissue. These studies led Investigators to believe that Inhibi-
tion of DNA replication may be Involved since only very rapidly proliferat-
ing hematopoletlc elements were affected.
Acute hemolytlc anemia 1s the most frequent manifestation of naphthalene
poisoning 1n humans and has been described 1n newborn Infants, children and
adults (U.S. EPA, 1980c, 1987a). Pertinent Information regarding the
mechanism of naphthalene-Induced hemotoxldty has been obtained by examining
two groups of Individuals that have been shown to be especially susceptible
to naphthalene-Induced hemolytlc anemia.
04440 VII-17 12/31/90
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The first group of naphthalene sensitive Individuals comprises persons
whose erythrocytes are deficient 1n glucose 6-phosphate dehydrogenase
(G6PDH) or persons 1n whom erythrocyte GSH 1s rapidly depleted by certain
oxldant chemicals (GMgor et al., 1966; Nalman and Kosoy, 1964; Valaes et
al., 1963; Athreya et al., 1961; Dawson et al., 1958; Gross et al., 1958;
Zlnkham and Chllds, 1958). The precise mechanism by which GSH 1s depleted
or a deficiency of G6PDH leads to naphthalene-Induced hemolysls 1n these
cases 1s not clear. A deficiency of G6PDH will decrease the rate of conver-
sion of nlcotlnamlde adenlne dlnucleotlde phosphate from Us oxidized (NADP)
to Us reduced form (NADPH). One hypothesis for Increased naphthalene
sensitivity 1n G6POH-defIdent Individuals 1s that the decreased avail-
ability of NADPH will decrease the conversion of oxidized glutathlone to
GSH, reduce the rate of conjugation and excretion of naphthalene metabolites
and Increase the accumulation of naphthalene metabolites 1n the body. A
similar hypothesis may explain Increased naphthalene sensitivity In Indi-
viduals 1n which erythrocyte GSH can be rapidly depleted by certain oxldant
chemicals (Nalman and Kosoy, 1964; Kellermeyer et al., 1962; Dawson et al.,
1958; 'Gross et al., 1958; Zlnkham and Chllds, 1958). Gross et al. (1958)
demonstrated a quantitative correlation between G6PDH deficiency and dimin-
ished levels of GSH 1n Infants beyond 55 hours of age; however, diminished
levels of erythrocyte GSH were observed 1n Infants of less than 55 hours of
age despite high levels of G6PDH activity. A second hypothesis for
Increased naphthalene sensitivity In G6PDH-def1c1ent Individuals 1s that the
decreased availability of NADPH will, 1n the presence of oxldant metabolites
of naphthalene, allow the accumulation of methemoglobln and products of Us
further Irreversible oxidation (Kellermeyer et al., 1962).
04440 VII-18 12/31/90
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The second group of naphthalene-sensitive Individuals comprises neonates
(Grlgor et al., 1966; Nalman and Kosoy, 1964; Valaes et al., 1963; Dawson et
al.. 1958; Gross et al., 1958; Zlnkham and Chllds, 1958). The sensitivity
of neonates to naphthalene 1s explained 1n part by the same factors that
confer sensitivity to children and adults; namely, G6PDH deficiency and/or
diminished levels of GSH as described above. Additional naphthalene sensi-
tivity 1n newborns may be conferred by the Immaturity of pathways necessary
for the conjugation and excretion of naphthalene metabolites (Valaes et al.,
1963). Evidence of the latter hypothesis 1s suggested by the finding that
glucuronlde excretion by human newborn Infants Increased gradually during
the first week of life and that the Initial levels and the rate of Increase
were lower 1n the premature Infant than 1n the full-term Infant (Brown and
Burnett, 1957).
Cardiovascular System. Smoking 1s a known risk factor 1n athero-
sclerosis, and PAHs are a major component of cigarette smoke (McGIll, 1977;
Wald et al., 1973). Injections (1.m.) of pure PAHs Into chickens has
resulted 1n development of prollferaUve lesions bearing a close resemblance
to human atherosclerotic plaques (Albert et al., 1977; Bond et al., 1981).
It has been proposed that human atherosclerotic plaques are In fact benign
hyperplastlc lesions of mutagenlc origin (Hartman, 1983). Majesky et al.
(1983) undertook a. study of PAH metabolism 1n two pigeon strains: athero-
sclerosis susceptible White Careneau (WC-2) and atherosclerosis resistant
Show Racer (SR-39). After treatment with an enzyme Inducer (3-methyl-
cholanthrene) hepatic homogenates from the susceptible strain were more
capable of benzo[a]pyrene metabolism than those from SR-39; furthermore
7,8-dlol, a mutagen1c/cardnogen1c precursor, was Increased. Assays of
aortic homogenates showed that this tissue had an even greater capacity for
04440 VII-19 12/31/90
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benzo[a]pyrene metabolism 1nduc1b1lKy as a function of strain. It would
appear that the responslve/nonresponslve concept may be applicable to
nonrodent species and to noncancer endpolnts.
Pulmonary System. Various Investigators have observed that l.p.
administration of naphthalene to rodents results In selective pulmonary
bronchlolar epithelial cell (Clara) necrosis, but not hepatic or renal
necrosis (long et al.. 1981c, 1982; Warren et al.. 1982; Mahvl et al.r 1977;
Reid et al., 1973). In an effort to determine the mechanism of action,
numerous studies have focused on the biochemistry of naphthalene and the
covalent binding characteristic of Us metabolites.
Shank et al. (1980) found that mice pretreated with dlethyl maleate
prior to 1.p. Injection of naphthalene had three times the level of
covalently-bound naphthalene metabolites In lung, liver, kidney and spleen.
Studies with 14C-naphthalene Injected Into mice revealed a similar binding
pattern: binding was highest 1n the lung but low 1n the spleen. Increased
binding corresponded to rapid and significant depletion of GSH 1n lung and
liver, and to a lesser extent 1n the kidney. Covalent binding was dose-
dependent and exhibited a threshold at dosages between 200 and 400 mg/kg.
Warren et al. (1982) suggested that lung damage may be mediated by P-450
dependent metabolism and GSH depletion.
Buckpltt and Warren (1983) extended these studies, utilizing a variety
of metabolic Inhibitors. The results suggested that some of the metabolites
Involved In GSH depletion and covalent binding 1n extrahepatlc tissues
originated In the liver. Buckpltt et al. (1985, 1987) suggested that the
difference 1n the rates of formation of specific metabolites between target
04440 VII-20 12/31/90
-------
and nontarget tissues, and 1n different species, may reflect the stereo-
chemistry of epoxldatlon by the tissue-specific P-450 Isozymes. This may,
1n turn, relate to the selective pulmonary necrosis observed 1n mice.
Confirmation that P-450 was Involved 1n pulmonary necrosis was obtained
In the studies of BuckpHt et al. (1986). Liver mlcrosomes from
phenobarb1tol-1nduced mice administered 300 mg naphthalene/kg 1.p. exhibited
73X less covalent binding In the presence of plperonyl butoxlde, a P-450
Inhibitor, than controls. A similar degree of Inhibition also was observed
with SKF 525A. It was-reported that plperonyl butoxlde also blocked the
pulmonary Injury exhibited by naphthalene In controls.
Gastrointestinal System. Male and female rats were observed with
Injury to their Intestinal epithelium after oral and Intravenous administra-
tion of 7,l2-d1methylbenz[a]anthracene (Philips et al., 1973). Further
analysis demonstrated that the Incorporation of 14C-labeled thymldlne Into
DNA of small and large Intestine, spleen, cervical lymph nodes and other
lymphatic structures was Inhibited as much as 90% within 6 hours after
d1methylbenz[a]anthracene administration. This finding Indicates a strong
Inhibition of DNA synthesis and led the authors to believe that DNA 1n S
phase cells 1s particularly susceptible to 7,l2-d1methylbenz[a]anthracene
and presumably to other PAH compounds as well. It has been proposed that
somatic cell mutations, such as can be Induced by PAH metabolites, may play
a role In the formation of gastric polyps and chronic gastric ulcers
(Hartman, 1983).
Gonads. Testicles and ovaries contain rapidly proliferating cells and
thus, may be especially susceptible to damage by PAHs. Severe testlcular
04440 VII-21 12/31/90
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damage was Induced 1n adolescent rats by a single Intravenous Injection of
7,l2-d1methylbenz[a]anthracene (0.5-2.0 mg). Similar effects were produced
1n adult rats given 7,l2-d1methylbenz[a]anthracene orally (20 mg) and
Intravenously (5 mg). Lesions Involved destruction of spermatogonla and
resting spermatocytes, both of which are the only testlcular cells actively
synthesizing DNA. The remaining germinal cells and Leydlg cells were not
damaged by 7,l2-d1methylbenz[a]anthracene. No testlcular damage was pro-
duced by a single feeding of 100 mg benzo[a]pyrene (Ford and Hugglns, 1963).
In female mice, 7,l2-d1methylbenz[a]anthracene was shown to cause
destruction of small oocytes and a reduction In numbers of growing and large
oocytes after oral administration. Mattlson and Thorgelrsson (1977) have
shown that destruction of primordial oocytes In mice following treatment
with 3-methylcholanthrene was correlated with the genetic capability for
PAH-lnduced Increases In ovarian AHH activity. This Indicates an apparent
link between ovarian metabolism of PAH and ovatoxldty. This link 1s
strengthened by the observation that the effective dose to kill 50% of small
oocytes (ED,-n) was less for responsive C57B1/6N mice (3.38 tig/ovary)
than for nonresponders DBA/2N (36.14 yg/ovary). An F, generation from
mating of these two strains had an Intermediate Intraovarlan Injection
ED5Q of 8.27 yg/ovary.
Endocrine System. Few data are available concerning the mechanism of
toxlclty and the effect of PAHs on the endocrine system. Selective destruc-
tion of the adrenal cortex and Induction of adrenal apoplexy by 7,12-dl-
methylbenz[a]anthracene has been demonstrated In rats after a single 1ntra-
gastrlc dose of 30 mg. The same amount of adrenal damage could be produced
by a 5 mg dose of 7-hydroxymethol-12-methylbenz[a]anthracene, the principal
04440 VII-22 12/31/90
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7,l2-d1methylbenz[a]anthracene oxldatlve metabolite. No adrenal damage was
noted with other 7,l2-d1methylbenz[a]anthracene metabolites, thus Indicating
that a specific reactive Intermediate may be responsible for this phenomenon
(Santodonato et al., 1981).
Integumentary System. The Integument 1s highly susceptible to agents
that Inhibit DNA synthesis as Is evident 1n cancer patients receiving radia-
tion treatment or chemotherapy. Such Individuals show signs of alopecia,
dermatitis and skin sloughing. Workers exposed to PAH-conta1n1ng materials
such as coal tar, mineral oil and petroleum waxes are known to develop
chronic dermatitis and hyperkeratosls (Heuper, 1963; NAS, 1972a). It Is
well documented that the application of carcinogenic PAHs to mouse skin
leads to destruction of sebaceous glands, skin ulceratlons, hyperplasla and
hyperkeratosls (Bock, 1964). Sebaceous glands undergo rapid cell turnover
and are the most sensitive structures of the skin to PAH-lnduced toxldty.
Visual System. Ocular toxldty, particularly cataract formation, has
long been associated with naphthalene administration 1n rodents and other
laboratory animals as well as 1n humans (U.S. EPA, 1980c, 1987a).
Oral administration of naphthalene 1s believed to result 1n Us metabo-
lism 1n the liver and the metabolites then travel through the bloodstream to
the eye where further metabolism takes place (van Heynlngen, 1979).
Evidence In rats and rabbits suggest that l,2-d1hydroxy naphthalene 1s
enzymatlcally converted to l,2-naphthoqu1none which then reacts with eye
proteins, resulting 1n damage (P1r1e and van Heynlngen, 1966; Rees and
Plrle, 1967).
04440 VII-23 12/31/90
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van Heynlngen (1979), 1n her review of the literature, hypothesized that
susceptibility to naphthalene-Induced cataracts Is more pronounced In rat
and rabbit strains with lightly plgmented or dark eyes, due to the presence
of. polyphenol oxldase. This nonspecific enzyme, found only 1n plgmented
tissues, catalyzes the formation of melanin from tyroslne.
•
van Heynlngen and Plrle (1967) suggested that the toxic metabolite Is
1,2-d1hydroxy naphthalene. In gavage studies 1n which naphthalene was
administered dally to 39 rabbits at 1 g/kg, they detected 1,2-d1hydroxy
naphthalene and 1,2-naphthoqu1none 1n the eyes and three metabolites 1n
blood that could be converted by different enzymes 1n the eye to
l,2-d1hydroxynaphthalene. In more than half the rabbits, lens opacities and
degeneration of the retina were observed. In addition, 1,2-naphthoqu1none
can oxidize ascorbic add present 1n the aqueous and vitreous humors,
resulting 1n oxalic add formation as the ascorbic add concentration
decreases (van Heynlngen, 1970a,b). Although ascorbic add decreases In
aqueous and vitreous humors, the level 1s maintained or Increases 1n the eye
lens Itself (van Heynlgen, 1970a). Presumably dehydroascorblc acid, formed
by oxidation by naphthoqulnones, penetrates the lens and 1s reduced to
ascorbic add. Ascorbic add diffuses only slowly from the lens. Excessive
depletion of ascorbic add may account for the appearance of calcium oxalate
crystals (Plrle and van Heynlngen, 1966). GSH appears to be maintained at
high levels 1n eye lens 1n spHe of extensive oxldatlve reactions (van
Heynlngen, 1970a).
van Heynlngen (1970b) found that the albino Wlstar rat has only about 3X
of the concentration of catechol reductase (an enzyme that catalyzes the
1nterconvers1on of qulnones and dlols) found In the rabbit lens. The rat
04440 VII-24 12/31/90
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also has less ascorbic add In aqueous humor than the rabbit (van Heynlngen,
1979). This would result In a higher level of 1,2-naphthoqu1none. Thus,
polyphenol oxldase may be the most Important factor 1n the rat eye while
catechol reductase may play a crucial role In ocular toxldty 1n the rabbit.
Rao and Pandya (1981) reported Increased I1p1d peroxldatlon In the eyes
of male albino rats administered 1 g napthalene dally for 10 days. Alkaline
phosphatase showed a slight Increase and aniline hydroxylase activity was
not detected. Liver peroxide levels were elevated but serum I1p1d peroxides
were not measured.
L1p1d peroxides have been suggested as a causal factor 1n cataract
formation. Yamauchl et al. (1986) Investigated this aspect 1n relation to
naphthalene. Naphthalene (1 g/kg) In acacia oil was administered to male
Mlstar rats dally for up to 18 days. GSH content 1n lens and serum and
liver I1p1d peroxide levels were measured during Interim sacrifice. Serum
peroxide levels Increased significantly on the fourth day and reached a
maximum on the seventh day. Liver peroxide levels had a similar pattern.
GSH content 1n lenses decreased to about 64X on the fourth day and remained
depressed. The authors suggested that I1p1d peroxides are stable enough to
reach the lens and cause ocular damage. Microscopic observation Indicated
slight cataractous changes 1n some rats .on the 14th day when serum Upld
peroxide levels were elevated (Yamauchl et al., 1986). It was suggested
that peroxides may play a role 1n cataract formation, 1n addition to the
role played by 1,2-n.aphthaqulnone. A decrease In nonproteln sulfhydryl
content 1n lens has previously been associated with naphthalene-Induced
cataracts 1n rabbits (Ikemoto and Iwata, 1978).
04440 VII-25 12/31/90
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PAH-Induced Immunotoxlclty
Numerous Investigators have demonstrated that carcinogenic PAHs can
produce an Immunosuppresslve effect. This effect was first observed by
Malmgren et al. (1952) using high doses of 3-methylcholanthrene and
d1benz[a,h]anthracene In mice. Subsequent studies established that single
carcinogenic doses of 3-methylcholanthrene, 7,l2-d1methylbenz[a]anthracene,
and benzo[a]pyrene caused a prolonged depression of the Immune response to
sheep red blood cells (Stjernsward, 1966, 1969). Noncarclnogenlc hydro-
carbons such as benzo[e]pyrene and anthracene reportedly had no Immuno-
suppresslve activity. In a review on Immunosuppresslon and chemical
cardnogenesls, substantial evidence was presented to Indicate that the
degree of Immunosuppresslon was correlated with carcinogenic potency for
PAHs (Baldwin, 1973). Both cell-mediated and humoral Immune reactions are
affected by PAHs.
The effects of three PAHs, 3-methylcholanthrene, d1benzo[a,h]anthracene
and d1benzo[a,c]anthracene on the Immune response was Investigated 1n mice
1n relation to tumorIgenesls (Lubet et al., 1984). The subcutaneous and
oral routes were used 1n two strains of mice, C57B1/6 (B6N) and DBA/2N
(D2N). The B6N strain Is an AHH Indudble species, and was more susceptible
to tumor formation than was the nonlnduclble D2N strain. D1benz[a,h]anthra-
cene at doses of 25 or 50 mg/kg produced an Immunosuppresslon effect as mea-
sured by a modification of the Jerne plaque assay. This effect was more
pronounced 1n the AHH Indudble B6N mice than In the 02N mice. In contrast,
d1benz[a,c]anthracene caused minimal effects 1n either strain. In general,
1t was noted that the more potent carcinogens produced greater Immunosup-
presslon than the noncardnogens. The route of administration also Influ-
i
04440 VII-26 12/31/90
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enced the Immunosuppresslve effects. Following 1.p. administration of high
concentrations of PAHs striking 1mmunosuppress1on was observed 1n both
strains of mice. When the oral route was employed, the nonlndudble AHH
mice (D2N) showed greater susceptibility than did the B6N mice. The authors
suggested that In AHH 1nduc1ble mice, orally administered PAHs are rapidly
metabolized and are rapidly excreted. In the nonlndudble mice the llpo-
ph1l1c PAHs are absorbed and distributed to target organs. They concluded
that AHH 1nduc1b1l1ty 1s an Important factor In the Immunosuppresslve
activity of PAHs.
A study by White et al. (1985) showed that suppression of an IgM
response to SRBC In mice exposed to PAH was a function of the strain respon-
siveness. Responsive B6C3F1 mice did not experience the degree of Immuno-
suppresslon following subcutaneous exposure as did DBA/2 nonresponslve mice.
This study further showed a correlation between a PAH's capacity for Immuno-
suppresslon and Us cardnogenldty. Anthracene, chrysene, benzo[e]pyrene
and perylene had no significant effect on Immune response while benz[a]'-
anthracene, benzo[a]pyrene, d1benz[a,c]anthracene and d1benz[a,h]anthracene
exposure resulted 1n 55-91% suppression. Even greater 1mmunosuppress1on was
caused by 3-methylcholanthrene and 7,l2-d1methylbenz[a]anthracene.
These results were contradicted to some extent 1n 1n vitro studies using
the weak or noncardnogen/cardnogen pair benzo[e]pyrene and benzo[a]pyrene
(Blanton et al., 1986). These workers found that benzo[e]pyrene did
suppress certain Immune function but required higher doses than did benzo-
[a]pyrene. By contrast to the White et al. (1985) study, anthracene In this
Instance caused j_n vitro Immunosuppresslve effects. These studies also
demonstrated that the 1mmunosuppress1on by benzopyrenes 1s due to effects on
04440 VII-27 12/31/90
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various cell types and 1s not attributable only to cytotoxlclty. It has
been shown that benzo[a]pyrene has potent effects on production of
Interleukln 1 by macrophages In vitro (Lyte and B1ck, 1986).
Summary
The cardnogenes1s-1n1t1at1ng potential of a PAH 1s dependent upon a
number of factors: Us I1p1d solubility and distribution to target organs,
the presence of potentially reactive areas of Us structure and Us poten-
tial for metabolism to reactive electrophlUc forms. This latter factor 1s
tissue and species dependent and 1s related to some extent to the 1nduc1b1l-
Uy of higher levels and particular Isozymes of cytochrome P-450-assodated
enzymes. Carcinogenic PAHs are generally mutagenlc and can damage DNA.
These activities are very likely to be Involved 1n the compound's activity
as an Initiator of carcinogenic processes. Many PAHs are complete skin
carcinogens, serving as their own promoters. Some are active as promoters
or cocarclnogens for other Initiating agents.
Target tissues for PAH-med1ated toxlclty other than cardnogenesls are
generally actively engaged In ONA synthesis. These tissues Include hemato-
poletlc and Immune systems, gonadal tissues and the lens of the eye. Mech-
anisms Involving suppression of DNA synthesis or DNA damage have been pro-
posed for PAH-lnduced toxldty to hematopp1et1c elements, the GI system and
the cardiovascular system. In addition to the organ systems covered,
PAH-1nduced lesions have been demonstrated In the lungs, liver and kidney.
No evidence concerning precise mechanisms of toxlclty has been found, but
many toxic endpolnts are linked to the species or strains capability for
ready Induction of PAH metabolizing enzymes.
04440 VII-28 12/31/90
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Naphthalene appears to be metabolized In a different manner than other
PAHs. Studies Indicate that naphthalene 1s metabolized 1n the liver to
1,2-naphthaqulnone. Administration of naphthalene causes an elevation 1n
serum Upld peroxide and liver peroxide levels. It has been suggested that
l,2-naphthaqu1none and elevated serum llpld peroxide levels may play a role
1n naphthalene Induced cataract formation 1n r-odents. Naphthalene Injection
has also caused bronchlcal epithelial cell necrosis In rodents and has
caused acute hemolytlc anemia 1n Instances of human poisoning.
04440 VII-29 05/10/91
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VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS
Introduction
The quantification of toxlcologlc effects of a chemical consists of
separate assessments of noncarclnogenlc and carcinogenic health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarclnogenlc health effects occur,
while carcinogens are assumed to act without a threshold.
In the quantification of noncarclnogenlc effects, a Reference Dose
(RfD), [formerly termed the Acceptable Dally Intake (ADI)] 1s calculated.
The RfD 1s an estimate (with uncertainty spanning perhaps an order magni-
tude) of a dally exposure to the human population (Including sensitive
subgroups) that 1s likely to be without an appreciable risk of deleterious
health effects during a lifetime. The RfD 1s derived from a no-observed-
adverse-effect level (NOAEL), or lowest-observed-adverse-effect level
*
(LOAEL), Identified from a subchronlc or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfD Is calculated as
follows:
RfD = (NOAEL or LOAEL) =
[Uncertainty Factor(s) x Modifying Factor]
Selection of the uncertainty factor to be employed 1n the calculation of
the RfD 1s based upon professional Judgment, while considering the entire
data base of toxlcologlc effects for the chemical. In order to ensure that
uncertainty factors are selected and applied 1n a consistent manner,
04450 VIII-1 12/31/90
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the U.S. EPA (1991a) employs a modification to the guidelines proposed by
the National Academy of Sciences (NAS, 1977, 1980) as follows:
Standard Uncertainty Factors (UFs)
Use a 10-fold factor when extrapolating from valid experimental
results from studies using prolonged exposure to average healthy
humans. This factor 1s Intended to account for the variation
1n sensitivity among the members of the human population. [10H]
Use an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
Inadequate. This factor Is Intended to account for the uncer-
tainty In extrapolating animal data to the case of humans.
[10A]
Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there 1s no
useful long-term human data. This factor 1s Intended to
account for the uncertainty 1n extrapolating from less than
chronic NOAELs to chronic NOAELs. [IDS]
Use an additional 10-fold factor when deriving an RfD from a
LOAEL Instead of a NOAEL. This factor 1s Intended to account
for the uncertainty In extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (MF)
• Use professional judgment to determine another uncertainty
factor (MF) that 1s greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of
the overall data base and the number of species tested. The
default value for the MF 1s 1.
The uncertainty factor used for a specific risk assessment Is based
principally upon scientific Judgment rather than scientific fact and
accounts for possible 1ntra- and Interspedes differences. Additional
considerations not Incorporated 1n the NAS/ODW guidelines for selection of
an -uncertainty factor Include the use of a less than lifetime study for
deriving an RfD, the significance of the adverse health effects and the
counterbalancing of beneficial effects.
04450 VIII-2 11/15/91
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From the RfD, a Drinking Water Equivalent Level (DUEL) can be calcu-
lated. The DUEL represents a medium specific (I.e., drinking water)
lifetime exposure at which adverse, noncardnogenlc health effects are not
anticipated to occur. The DHEL assumes 100X exposure from drinking water.
The DHEL provides the noncardnogenlc health effects basis for establishing
a drinking water standard. For Ingestlon data, the DUEL Is derived as
follows:
DHEL _ (RfD) x (Body weight 1n kg) _ ^
Drinking Water Volume 1n l/day =
where:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 i/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for expo-
sures of shorter duration (1-day, 10-day and longer-term) are determined.
The HA values are used as Informal guidance to municipalities and other
organizations when emergency spills or contamination situations occur. The
HAs are calculated using an equation similar to the RfD and DWEL; however,
the NOAELs or LOAELs are Identified from acute or subchronlc studies. The
HAs are derived as follows:
(NOAEL or LOAEL) x (bw)
(UF)x(_ l/day)
Using the above equation, the following drinking water HAs are developed
for noncardnogenlc effects:
1. 1-day HA for a 10 kg child Ingesting 1 i .water per day.
2. 10-day HA for a 10 kg child Ingesting 1 l water per day.
3. Longer-term HA for a 10 kg child Ingesting 1 I water per day.
4. Longer-term HA for a 70 kg adult Ingesting 2 i water per day.
04450 VIII-3 08/28/87
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The 1-day HA calculated for a 10 kg child assumes a single acute
exposure to the chemical and 1s generally derived from a study of <7 days
duration. The 10-day HA assumes a limited exposure period of 1-2 weeks and
1s generally derived from a study of <30 days duration. The longer-term HA
1s derived for both the 10 kg child and a 70 kg adult and assumes an
exposure period of ~7 years (or 10% of an Individual's lifetime). The
longer-term HA 1s generally derived from a study of subchronlo duration
(exposure for 10X of animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chemical, based
on the overall we1ght-of-evidence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of
carc1nogen1c1ty 1n animals with limited (Group B1) or Inade-
quate (Group 62) evidence In humans.
Group C: Possible Human Carcinogen. Limited evidence of
cardnogenldty 1n animals 1n the absence of human data.
Group D: Not Classified as to Human Cardnogenlclty. Inade-
quate human and animal evidence of carclnogenlcUy or for which
no data are available.
Group E: Evidence of Noncarc1nogen1c1ty for Humans. No
evidence of carclnogenlcUy In at least two adequate animal
tests In different species or 1n both adequate ep1dem1olog1c
and animal studies.
If toxlcologlc evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the
Ingestlon of the contaminant 1n drinking water. The data used In these
estimates usually come from lifetime exposure studies using animals.
04450 VIII-4 12/31/90
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In order to predict the risk for humans from animal data, animal doses must
be converted to equivalent human doses. This conversion Includes correction
for noncontlnuous exposure, less than lifetime studies and for differences
1n.s1ze. The factor that compensates for the size difference 1s the cube
root of the ratio of the animal and human body weights. It 1s assumed that
ttie average adult human body weight 1s 70 kg and that the average water
consumption of an adult human 1s 2 a of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure from
1ngest1on of water. The cancer unit risk Is usually derived from a.linear-
ized multistage model with a 95% upper confidence limit providing a low dose
estimate; that 1s, the true risk to humans, while not Identifiable, 1s not
likely to exceed the upper limit estimate and, 1n fact, may be lower.
Excess cancer risk estimates may also be calculated using other models such
as the one-hit, Welbull, loglt and probU. There 1s little basis 1n the
current understanding of the biological mechanisms Involved 1n cancer to
suggest that any one of these models 1s able to predict risk more accurately
than any other. Because each model 1s based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an Inherent uncertainty that 1s due to the
systematic and random errors 1n scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there 1s
uncertainty when the data are extrapolated to humans. When developing
04450 VIII-5 12/31/90
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cancer risk rate levels, several other areas of uncertainty exist, such as
the Incomplete knowledge concerning the health effects of contaminants 1n
drinking water, the Impact of the experimental animal's age, sex and
species, the nature of the target organ system(s) examined and the actual
rate of exposure of the Internal targets 1n experimental animals or humans.
Dose-response data usually are available only for high levels of exposure
and not for the lower levels of exposure closer to where a standard may be
set. When there 1s exposure to more than one contaminant, additional
uncertainty results from a lack of Information about possible synerglstlc or
^
antagonistic effects. Since PAH occur only as mixtures In.the environment,
no epidemlologic data are found for Individual PAH.
Noncardnoqenlc Effects
For many of the PAHs experimental data on noncardnogenlc effects are
either nonexistent or provide Insufficient Information on which to base
criteria for drinking water exposure. In general, existing data are not
suitable for criteria derivation for one or more of these reasons:
f
Studies were designed to assess only carcinogenic potential
Studies were designed to determine only lethal dose (1059)
Studies do not give dose/response data
• Studies contain only one dose level, at which severe health
effects occurred
• Studies do not measure chronic exposure
• Studies were by other than oral exposure
Measure of dose Is not known
Sample size 1s too small
• Test animals used do not provide relevant models for human
health assessment.
04450 VII1-6 09/23/91
-------
Recently, the U.S. EPA (1988, 1989a,b,c) and Hazelton Laboratories
America, Inc. (1989c, 1989d) conducted 90-day subchronlc bloassays for
acenaphthylene, anthracene, fluoranthene, fluorene and pyrene. These
studies examined a variety of toxlcologlc endpolnts and were of adequate
design, thereby providing sufficient Information on which to base criteria
for drinking water exposure. These studies were Insufficient to evaluate
potential carclnogenldty of the above PAHs.
Short-term Studies 1n Animals
There were short-term studies wherein animals were treated by the oral
route reported for only acenaphthylene, -anthracene, fluoranthene and
fluorene. For acenaphthylene there 1s an oral LD5Q for rats and mice
reported 1n abstract form (Knobloch et al., 1969). For anthracene there 1s
a report In abstract form stating that a single oral dose of 17 g/kg of
anthracene Is not lethal to mice (Nagornyl, 1969). For fluoranthene the
only data are from a range-finding study on more than 300 compounds; the
.study reported the oral LD5Q for rats as 2000 mg/kg/day (Smyth et al.,
1962).' For fluorene, K1zer et al. (1985) reported that 1n rats fed 10.5
mg/kg/day 1n the diet for 3 weeks, no Increases In either SGOT or hepatic
mlcrosomal epoxlde hydrolase activity were observed. Thus, there are no
data suitable for derivation of 1- or 10-day HAs for any PAH discussed In
this document.
Longer-term Studies 1n Animals
No-longer-term exposures of animals by the oral route were reported for
the following: benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,1]-
perylene, chrysene, 1ndeno[l,2,3-cd]pyrene, phenanthrene and pyrene.
04450 VIII-7 11/15/91
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Acenaphthylene. Acenaphthylene was administered to groups of seven
rats 1n doses of 600 mg/kg bw for 40 days or 2000 mg/kg bw for 32 days.
Effects were reported on body weight, peripheral blood, renal function,
kidney and liver morphology, and bronchitis was Induced (Knobloch et al.,
1969). The report Is In abstract form; data are, thus, Insufficient for
derivation of criteria.
In a subchronlc toxldty bloassay conducted by Hazelton Laboratories
America, Inc. (1989c) acenaphthylene was administered to groups of 20 male
and female CD-I mice by gavage. Dose levels were 0, 100, 200 and 400
mg/kg/day. Criteria evaluated for compound-related effects were mortality,
clinical signs, body weight, food consumption, opthalmology, hematology,
clinical chemistry, organ weights, gross pathology and hlstopatholgy. Based
on liver and kidney changes and deaths 1n females the LOAEL determined from
this study 1s 100 mg/kg/day; no NOAEL was determined. Due to the high
mortality observed 1n females receiving 100 mg/kg/day the data provided are
»
considered Insufficient for derivation of criteria.
t
Anthracene. Two bloassays for carclnogenlclty of Ingested anthracene
have been conducted (Druckry and SchmShl, 1955; SchmShl and Relter, n.d.).
Neither study mentions noncancer health effects, nor were any tumors
reported. The latter study has not been published. In a subchronlc
toxldty study, the U.S. EPA (1989e) administered anthracene to groups of 20
male and female CD-I (ICR)BR mice by gavage. Dose levels were 0, 250, 500
and 1000 mg/kg/day. Criteria evaluated for compound-related effects were
mortality, clinical signs, body weights, food consumption, opthalmology,
hematology, clinical chemistry, organ weights, organ-to-body weight ratios,
04450 VIII-8 11/15/91
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gross pathology and Mstopathology. No .treatment-related effects were
noted; therefore, the NOAEL determined from this study 1s 1000 mg/kg/day.
BenzTalanthracene. Klein (1963) published a report of benz[a]anthra-
cene carclnogenlclty for mice treated by gavage. Only one concentration was
administered, and noncancer endpolnts were not described. Thus, data are
Insufficient to derive criteria for benz[a]anthracene.
Benzo[a]pyrene. Benzo[a]pyrene was administered to mice at multiple
concentrations 1n the diet 1n> order to assess Us carcinogenic potential
(Rlgdon and Neal, 1966, 1969; Neal and Rlgdon, 1967). Treatment-related
Incidences of tumors of the forestomach and lung, and leukemlas were
observed. No noncancer health effects were reported, however. Rlgdon and
Neal (1965) also conducted a series of assays to determine 1f dietary benzo-
[ajpyrene produced deleterious reproductive effects. Oral benzo[a]pyrene
concentrations of 250, 500 or 1000 ppm over various time periods up to
lifetime showed no treatment-related effects, except lack of weight gain
/
related to feed unpalatabllUy.
NacKenzle and Angevlne (1981) dosed groups of 30 or 60 pregnant CD-I
mice with gavage preparations of benzo[a]pyrene to deliver 0, 10, 40 or 160
mg/kg bw/day. This was done only on days 7-16 of gestation. No maternal
toxldty was noted, nor were there signs of fetal toxldty. Pups were
culled to 8/l1tter and used 1n an F, mating study. By days 20 and 42,
F. animals exposed to benzo[a]pyrene Ijn utero were observed to have
decreased body weights 1n comparison with controls. Gonadal weights of
treated animals were also significantly reduced. The testes from animals
04450 VIII-9 07/26/91
-------
exposed In utero to the low benzo[a]pyrene dose weighed ~60X those of
controls; testes from the 40 mg (middle dose) group weighed ~18X of
controls. As most P.. ]j± utero exposed females had no ovaries or only
remnants of ovarian tissues, weights were generally not recorded. Fertility
was reduced among F, treated animals. Of control males 100X were fertile,
20/25 from the group exposed to 10 mg, 3/45 from the 40 mg group, and none
of the males treated In utero with 160 mg benzo[a]pyrene/kg/day were
fertile. Of treated females, 34X of the low-dose group produced a Utter;
none of the middle- and high-dose females produced a litter. This study,
<•
thus, demonstrates a LOAEL for gonadal weight decrease and loss of fertility
of 10 mg/kg/day for benzo[a]pyrene.
DlbenzTa.hlanthracene. D1benz[a,h]anthracene was carcinogenic to mice
receiving the compound 1n an olive o1l/dr1nk1ng water emulsion (Lorenz and
Stewart, 1947; Snell and Stewart, 1962a). Only one concentration of
treatment suspension was administered. The only noncancer health effect
reported was dehydration and emaciation of animals due to poor tolerance of
the vehicle. There are, thus, no data suitable for derivation of criteria.
Fluoranthene. In a 13-week bloassay 1n mice, the U.S. EPA (1988)
administered either 0, 125, 250 or 500 mg/kg/day of fluoranthene to groups
of 20 male or female CD-I mice by gavage. Body weights, food consumption
and clinical signs of toxldty were monitored at regular Intervals during
the experimental period. At the end of the study period the animals were
sacrificed and submitted for autopsy and hematologlc and serum chemistry
evaluations. A LOAEL of 250 mg/kg/day based on statistically significant
(p<0.05) changes 1n SGPT and absolute and relative liver weights, as well as
04450 VIII-10 11/15/91
-------
decreases 1n packed cell volume and red blood cell numbers (females only)
and albumin/globulin ratios, was Identified by the U.S. EPA (1991a); the
corresponding NOAEL Is 125 mg/kg/day.
Fluorene. Neither of two reports of bloassays for oral carcinogenic
potential of fluorene gave Indication of an Increased tumor Incidence
(Morris et al., 1960; Wilson et al., 1947). The former study reported no
effects related to treatment. In the latter study one set of rats was
exposed to dietary concentrations of 0.062 or l.OX fluorene for 104 days and
a second set to 0.125, 0.25 or 0.5X fluorene for 453 days.. The animals of
the short-term group appeared normal 1n all respects except for treatment-
related decreases In growth rate. The longer-term treated animals were
observed to have significantly Increased liver weights (two highest dose
groups) and decreased spleen weights (all treated animals). While these
changes were described as "significant", no numerical data were presented.
This study 1s, therefore, unsuitable for criteria derivation.
•
In' a 13-week subchronlc bloassay 1n mice, the U.S. EPA (1989a) admin-
istered either 0, 125, 250 or 500 mg/kg/day fluorene, suspended 1n corn oil,
to groups of 25 male and female CD-I mice by gavage. Measures used to
assess toxldty Included food Intake, body weight, clinical observations,
hematology and serum chemistry, and gross and hlstopathologlc changes.
Using the data from U.S. EPA (1989a), the U.S. EPA (1991a) Identified a
LOAEL of 250 mg/kg/day for hematologlc effects; the NOAEL 1s 125 mg/kg/day.
Pyrene. In a 13-week subchronlc bloassay 1n mice, the U.S. EPA
(1989b) administered either 0, 75, 125 or 250 mg/kg/day of pyrene to groups
04450 VIII-11 11/15/91
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of 20 male and 20 female CD-I mice by gavage. Criteria used to assess
toxldty Included body and organ weights, food consumption, mortality,
hematology amd serum chemistry, and gross and hlstopathology. Based on
nephropathy, accompanied by statistically significant (p<0.01) changes 1n
absolute and relative kidney weights, the U.S. EPA (1991a) Identified 125
mg/kg/day as the LOAEL; the NOAEL is 75 mg/kg/day.
Quantification of Noncardnoqenlc Effects
Derivation of 1- and 10-Day Health Advisories. There were no data
suitable for calculation of 1-day or 10-day HAs for children for any PAH
covered 1n this document.
A number of short-term exposure studies have been reported wherein
various Immunologlc endpolnts have been measured. These are summarized 1n
Tables VIII-1 and VIII-2. While It Is Important to consider Immune deficits
as Indicators of early toxldty, 1t Is not appropriate to base health
advisories on the data presented 1n these two tables. First, none of the
studies employed an oral route of administration, thereby decreasing the
relevance of the data to drinking water health advisories. Secondly, 1t 1s
not clear whether the Immune deficits observed are, 1n fact. Indicators of
toxldty or are normal adaptive responses to stress.
For benzo[a]pyrene the data of MacKenzle and Anglvlne (1981) could also
be considered as an Indicator of noncancer toxic effects resulting from
short-term exposure in utero. In view of the fact that this exposure
encompassed an entire critical phase of the animal's Hfespan, namely the
fetal developmental phase, the data may be more suitable for derivation of a
lifetime health advisory.
04450 VIII-12 11/15/91
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TABLE VIII-1
Immunotoxldty of Benzo[a]pyrene 1n Mice after Short-Term Exposure
Days
Exposure
1
1
1
1
1
1
10
14
Strain Route
CBA l.m.
C3H/ANF in utero
B6C3F1 s.c.
C57 1.p.
C3H l.p.
C3H l.p.
C57B1/6 l.p.
DBA/2 l.p.
B6C3F1 s.c.
B6C3F1 s.c.
LOAEL/NOEL
(mg/kg)
33.3/ND
150/ND
252/ND
5.0/0.5
5.0/0.5
18.0/1.8
25/-
50/25
5.0/-
40.4/ND
Effect*
PFC
PFC
GvH
mixed lymph
number of
spleen cells
target cell
killing
INF
PFC
PFC
AbT1
CPLPS
spleen
weight
PFU
Reference
Stjernsward,
1966
Urso and
Gengozlan,
1984
White
et al., 1985
Wodjanl
et al., 1984
Griffin
et al., 1986
Lubet
et al., 1984
Dean et al.,
1983
White
et al., 1985
*A11 effects noted were decreases by comparison with controls In the
measurements Indicated. PFC = plaque-forming spleen cells; PFU =
plaque-forming units; GvH = graft vs. host response; mixed lymph = mixed
lymphocyte response; INF = Interferon production; AbT1 = antibody
production to a "[-Independent antigen; CPLPS = cell proliferation 1n
response to Upopoly-sacchaMde mHogen.
ND = Not determined as only one dose was tested
- = No NOEL reported
04450
VIII-13
11/15/91
-------
TABLE VIII-2
Comparative Effects of PAH on Immune Functions In B6C3F1 M1cea
Compound
Doseb
(mg/kg/day)
Effect
Anthracene
Benz[a]anthracene
Benzo[a]pyrene
Chrysene
D1benz[a,h]anthracene
28.5
36.5
40.4
36.5
42.7
None
Decreased antibody
forming cells
Decreased antibody
forming cells;
decreased spleen
weight
None
Decreased antibody
forming cells;
decreased spleen
weight
aData from White et al. (1985)
bAnimals were treated s.c. with 160 ymole/kg/day
04450
VIII-14
07/27/90
-------
Derivation of Longer-Term HA. There were no data suitable for calcu-
lation of longer-term HAs for children or adults for any PAH covered 1n this
document.
Assessment of Lifetime Exposure and Derivation of DWELs. There were
no data suitable for calculation of RfDs for the following: benz[a]anthra-
cene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,1]perylene,
chrysene, d1benz[a,h]anthracene, 1ndeno[l,2,3-cd]pyrene or phenanthrene.
Acenaphthylene — Quantitative data on the subchronlc oral toxlclty of
acenaphthylene has been provided by Hazelton Laboratories America, Inc.
(1989c). Acenaphthylene .was administered to CD-I mice (20/sex/group) at
dosage levels of 0, 100, 200 or 400 mg/kg/day. for at least 90 days. Effects
examined Included mortality, clinical signs, body weights, food consumption,
opthalmology, clinical chemistry, organ weights, gross pathology and hlsto-
pathology. Treatment-related effects were observed 1n all dosage groups.
Due to the high mortality observed 1n all groups of treated females, the low
dose Is considered an PEL. Therefore, this study 1s Insufficient for
deriving criteria since the choice of mortality as the critical effect 1s
not appropriate as the basis for an RfD.
Additionally, four subchronlc studies on the toxldty of acenaphthylene
were located In the literature: two oral studies (Rotenberg and Mashblts,
1965; Knobloch et al., 1969) and two Inhalation studies (Rotenberg and
Mashblts, 1965; Reshetyuk et al., 1970). However, deficiencies (no experi-
mental controls, short duration and Incomplete reporting of study design
and/or results) preclude the use of any of these studies as a basis for an
oral RfD.
04450 VIII-15 11/15/91
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Anthracene ~ Quantitative data on the oral exposure to anthracene
reported 1n U.S. EPA (1989e) failed to demonstrate treatment-related effects
to male or female CD-I (ICR)BR mice at doses up to 1000 mg/kg/day for at
least 90 days. From these results, a NOAEL of 1000 mg/kg/day was Identi-
fied. Using this NOAEL, the DUEL 1s derived as follows.
Step 1 - RfD Derivation
RfD m (1000 mq/kq/day) = ^^ mg/kg/day
3000 (rounded to SxlO'1 mg/kg/day)
where:
1000 mg/kg/day = NOAEL reflecting no treatment-related effects 1n
mice (U.S. EPA, 1989e)
3000 = combined uncertainty factors: 100 to account for
1ntra- and Interspedes extrapolation, 10 for the
use of a subchronlc study for RfD derivation, and
3 for the lack of reproductive/developmental and
supporting chronic toxlclty data
Step 2 - DUEL Derivation
nun OxIO"1 mq/kq/day) (70 kg) in ,. mn
DWEL = (2 I/day) = 10'5 mg/l
where:
/
3x10'* mg/kg/day = RfD
70 kg = assumed body weight of an adult
2 i/day = assumed volume of water consumed by an adult
Benzol"aIpyrene — For benzo[a]pyrene an Indicator of a noncancer toxic
effect was the decrease 1n fertility and gonadal weight 1n mice exposed In
utero. Dams were gavaged with 10 mg/kg/day benzo[a]pyrene In corn oil on
days 7-16 of gestation (MacKenzle and Angevlne, 1981). While this does not
constitute a subchronlc or chronic exposure 1t does cover the entire period
of development most sensitive to Insult of the test population. The study
could, thus, be considered for use In estimating a longer-term risk.
04450 VIII-16 11/15/91
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For benzo[a]pyrene, available data show that the appearance of
neoplastlc effects occurs at lower doses than do Indicators of systemic
toxldty. For example a LOAEL based on the MacKenzle and Angevlne (1981)
data Is 10 mg/kg/day, while significant Increases 1n tumor Incidence have
been observed In mice receiving 6.5 mg/kg/day benzo[a]pyrene 1n the diet
(Neal and Rlgdon, 1967). It, thus, seems Inadvisable at this time to
propose any health advisories for benzo[a]pyrene based on noncancer effects.
Fluoranthene — In a 13-week subchronlc bloassay 1n mice the U.S. EPA
(1988) Identified a LOAEL of 125 mg/kg/day of fluoranthene, This LOAEL was
based on statistically nonsignificant Increases 1n clinical signs, serum
chemistry and changes 1n liver and kidney pathology as well as significant
Increases In relative liver weights of male mice. In a revaluation of U.S.
EPA (1988), the U.S. EPA (1991a) Identified the 125 mg/kg/day dose level as
the NOAEL and the 250 mg/kg/day dose level as the LOAEL. The low dose was
considered the NOAEL as clinical signs (I.e., salivation) were not dose-
related effects, and changes 1n serum enzymes and kidney and liver hlsto-
pathology were not considered adverse at 125 mg/kg/day. The 250 mg/kg/day
dose 1s considered the LOAEL based on significant reductions 1n packed cell
volume and red blood cell numbers 1n females, and albumin/globulin ratios
and significant Increases 1n SGPT and absolute and relative liver weights In
both sexes. Based on a NOAEL of 125 mg/kg/day, the DWEL 1s derived as
follows:
Step 1 - RfD Derivation
»«- • -4.17x10-- mg/kg/day
(rounded to 4x1 0'* mg/kg/day)
04450 VIII-17 11/15/91
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where:
125 mg/kg/day = NOAEL reflecting the absence of dose-related
effects In mice (U.S. EPA, 1988)
3000 = combined uncertainty factors: 100 to account for
1ntra- and Interspedes extrapolation, 10 for the
use of a subchronlc study for RfD derivation, and 3
for the lack of reproductive/developmental and
supporting chronic toxldty data
Step 2 - DUEL Derivation
DUEL . (4x10-' mq/kq/day) (70 kq) =
(2 l/day)
where:
4xlO~2 mg/kg/day = RfD
70 kg = assumed body weight of an adult
2 l/day = assumed volume of water consumed by an adult
Fluorene — In a 13-week subchronlc bloassay 1n CD-I mice (U.S. EPA,
1989a), quantitative data were reported on the adverse health effects
associated with oral exposure to fluorene. From these results, the U.S. EPA
/
(1991a) Identified a LOAEL of 250 mg/kg/day for hematologtc effects; the
corresponding NOAEL 1s 125 mg/kg/day. Using this NOAEL, the OWEI 1s derived
as follows.
Step 1 - RfD Derivation
RfD m (125 mq/kq/day) = 4
-------
3000 = combined uncertainty factors: 100 to account for
1ntra- and Interspedes variability, 10 for the use
of a subchronlc study for RfD derivation, and 3 for
the lack of reproductive/developmental and
supporting chronic toxldty data
Step 2 - DUEL Derivation
nuci (4x10"* mq/kq/dav) (70 kg)
DWEL = (2 i/day) = K4 m9/l
where:
4x10~2 mg/kg/day = RfD
70 kg = assumed body weight of an adult
2 a/day = assumed volume of water consumed by an adult
Naphthalene ~ Quantitative results on the chronic oral toxldty of
naphthalene have been provided by Schmahl (1955). Groups of 28 BD I and
BD II rats received naphthalene (estimated dally dose 10-20 mg) 1n the diet,
starting when the rats were -100 days old; the experiment was terminated on
the 700th experimental day when a total dose of 10 g/rat was achieved. No
treatment-related effects were observed. The use of this study to derive an
/
oral RfD has been questioned by the U.S. EPA (1989c) because this study was
designed to assess the cardnogenlcHy of naphthalene, small numbers of
animals were used, and there 1s uncertainty about the actual dose admin-
istered.
Subchronlc studies with mice and rats by NTP (1980a,b) and Shopp et al.
(1984) support a lower chronic NOEL determined by the Schmahl (1955) study.
In the NTP (1980a) study, mice were treated by gavage with 0, 12.5, 25, 50,
100 or 200 mg/kg/day naphthalene, 5 days/week for 13 weeks. Comprehensive
hlstologlc examination of the high-dose and control groups revealed no
04450 VIII-19 11/15/91
-------
treatment-related lesions. Clinical signs of toxlclty occurred at or above
the 100 mg/kg/day dose, but no effects were observed at or below the 50
mg/kg/day dose. No adverse effects on mortality, weight gain, 1mmunolog1c
effects, clinical chemistry and hematologlc parameters or organ weights were
observed 1n mice given a 53 mg/kg/day dose by gavage for 90 days (Shopp et
al., 1984). H1stolog1c examinations were not'performed In this study. Sub-
chronic NOELs from these studies were 35.7 mg/kg/day (NTP, 1980a) and 53
mg/kg/day (Shopp et al., 1984).
Derivation of quantitative data on the oral toxldty of naphthalene are
not derived because concurrence on the most appropriate study on which to
base the calculation has not been reached (U.S. EPA, 1989c).
Pyrene — In a 13-week subchronlc bloassay 1n CD-I mice (U.S. EPA,
1989b), quantitative data were reported on the adverse health effects
associated with oral exposure to pyrene. From these results, the U.S. EPA
(1991a) Identified a LOAEL of 125 mg/kg/day for nephropathy accompanied by
changes 1n absolute and relative kidney weights; the corresponding NOAEL 1s
75 mg/kg/day. Using this NOAEL, the DWEL 1s derived as follows.
Step 1 - RfD Derivation
(75 mq/kq/day) = ? ^Q_2 mg/kg/(jay
3000 (rounded to 3xlO~* mg/kg/day)
where:
75 mg/kg/day = NOAEL reflecting the absence of nephropathy In mice
(U.S. EPA, 1989b)
3000 = combined uncertainty factors: 100 to account for
Intra- and Interspedes variability, 10 for the use
of a subchronlc study for RfD derivation, and 3 for
the lack of reproductive/developmental and support-
Ing chronic toxldty data.
04450 VIII-20 11/15/91
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Step 2 - DUEL Derivation
nun (3x10*2 mq/kg/day) (70 kg) ,
UWtL = ,~ = '•
(rounded to 1 mg/a)
where:
0.025 mg/kg/day = RfD
70 kg = assumed body weight of an adult
2 I/day = assumed volume of water consumed by an adult
Carcinogenic Effects
The majority of health effects data for Individual PAHs concerns their
potential as carcinogens. The data base for these compounds consists
entirely of animal studies. There are numerous case reports and epldemlo-
loglc Investigations on human health effects of PAH-contalnlng materials.
These, however, have been reports on exposures to environmental or to
occupational ly generated mixtures containing PAHs and other compounds.
There are no reports of exposures to Individual PAHs with the exception of -a
case study of skin painting of benzo[a]pyrene. The exposed subjects
/
developed verrucae, wart-Uke benign lesions that regressed upon cessation
of treatment (Cott1n1 and Mazzone, 1939). This observation Is significant
1n that H 1s not unlike the process observed 1n animals skin painted with
PAHs. In response to a carcinogenic PAH, animals generally develop non-
malignant lesions (paplllomas) that may regress upon cessation of treatment
or may progress to carcinomas 1f treatment Is continued or followed by a
promoting treatment.
As reviewed 1n Chapter V, much of the data on PAH cardnogen1c1ty comes
from skin-painting bloassays, subcutaneous Injection studies and to a lesser
04450 VIII-21 09/23/91
-------
extent from experiments wherein PAHs were administered Intratracheally or by
Inhalation. There are comparatively few oral bloassays. The relevance of
data from skin painting bloassays to evaluate the potential for Induction of
nonskln cancer 1n humans has been a subject of discussion. The example of
benzo[a]pyrene may be Instructive 1n this context. This PAH causes
•
Injection site sarcomas upon subcutaneous administration, skin tumors when
applied topically, lung neoplasms when delivered Intratracheally or by
Inhalation and forestomach tumors In rats when given orally. It appears
that benzo[a]pyrene produces neoplastlc growth at the site of delivery when
at least two conditions are met: that the tissue Is capable of metabolism
of benzo[a]pyrene to reactive forms, and that the tissue 1s of a type that
normally undergoes some degree of proliferation. The requirement for
metabolism of benzo[a]pyrene 1s not a limiting factor for formation of
distant site tumors; many metabolites can be transported 1n the blood and
the majority of tissues assayed show some capacity for PAH metabolism.
The other PAHs In this document have been studied to a lesser extent and
by fewer exposure routes than has benzo[a]pyrene. There are a few examples,
however, to Indicate that when a PAH 1s positive 1n skin painting bloassays,
H will produce tumors when administered by other routes. Benz[a]anthracene
and d1benz[a,h]anthracene, which produce skin tumors when applied topically,
also produce neoplasms when delivered orally (Klein, 1963; Bock and King,
1959; LaMnov and Soboleva, 1938; Lorenz and Stewart, 1947, 1948; Snell and
Stewart, 1962a, 1962b). It appears justified to say that evidence of
cardno- genlclty from a PAH skin-painting bloassay Is a cause for concern
and should not be Ignored when evaluating a PAH as a potential human
carcinogen.
04450 VIII-22 11/15/91
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Classification of PAHs as to Potential for Human Carc1noqen1c1ty
Assignment of compounds to a classification based on their likelihood of
producing carcinogenic effects 1n humans was done according to the U.S. EPA
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986). Using these
criteria the PAHs described 1n this document may be classified 1n the
following two groups.
Group D. Not classifiable as to human cardnogenldty. This 1s due to
a lack of specific human evidence and Inadequate animal data for cardno-
genldty.
Acenaphthylene. One skin painting blassay 1n mice showed no Increase
1n tumor Incidence. One assay for mutagenldty 1n Salmonella was not
positive.
Anthracene. Studies wherein rats were administered anthracene orally
did not result 1n tumor Induction. Lung Implantation and skin painting
/
bloassays, and subcutaneous, Intraperltoneal and Intracerebral Injection
likewise have not shown a tumorlgenlc effect. Mutagenldty for Salmonella
strain TA97 has been reported.
Benzorq.n.llpervlene. A bloassay by Intrapulmonary Injection of rats
was considered Inadequate for evaluation of this PAH due to the presence of
Impurities; Two skin-painting studies for complete cardnogenldty and
three Initiation-promotion assays In mice were negative. There are data to
suggest that this compound may act as a co-cardnogen for benzo[a]pyrene
applied to mouse skin. Benzo[g,h,1]perylene was mutagenlc for S.
typhlmurlum.
04450 VIII-23 09/23/91
-------
Fluoranthene. Skin painting bloassays for complete carclnogenlcHy
have not shown positive results. Initiation/promotion protocols wherein
fluoranthene was used as the Initiator, as well as a subcutaneous Injection
study, were negative. There 1s some evidence that fluoranthene served as a
cocardnogen for benzo[a]pyrene. Fluoranthene produced an Increase 1n
Incidence 1n lung adenomas for males and females combined In a short-term jm
vivo bloassay generally considered not adequate for evaluation of carclno-
genlcHy. Evidence for genetic toxldty of fluoranthene Is equivocal.
Fluorene. Two oral bloassays reported no Increase ,1n tumors as a
consequence of fluorene treatment. Both assays had deficiencies limiting
their usefulness. IARC considered two apparently negative skin-painting
bloassays to be Inadequate for-evaluation; a more recent Initiation/promo-
tion assay was not positive. Two subcutaneous administration studies were
negative. Genetic toxicology data are limited but negative.
Naphthalene. Bloassays of naphthalene by the oral route and as a skin
tumorlgen were negative or Inadequate for evaluation. An Inhalation and an
1ntraper1toneal Injection study were negative, and a subcutaneous Injection
study was Inadequate for evaluation. Supporting data for genetic toxldty
are negative but limited.
Phenanthrene. An assay 1n which rats received a single oral treatment
showed no Increase In tumor Incidence. Treatment of mice topically,
subcutaneously and 1ntraper1toneally has not resulted 1n tumor Induction.
One study reported phenanthrene to be an Initiator of skin tumorlgenesls 1n
CD-I mice when followed by high concentrations of TPA. This was countered
04450 VIII-24 09/23/91
-------
by negative studies 1n three other mouse strains. Phenanthrene and a dlol-
epoxlde derivative were mutagenlc for Salmonella typhlmurlum and human
lymphoblast cells. Other genetic toxicology testing has not shown positive
results.
Pyrene. Intratracheal Instillation of pyrene and Fe.03 particles
did not Induce tumors 1n hamsters. Skin painting assays 1n mice for
complete cardnogenesls or Initiating capacity have been negative or Incon-
clusive. Mice Injected either subcutaneously or Intraperltoneally did not
develop tumors, but there 1s evidence that pyrene enhances tumor1gen1dty of
topically applied benzo[a]pyrene 1n mice. Both positive and negative
results have been reported for assays of mutagenlc effect; pyrene was not
shown to transform mammalian cells.
Group B2. Probable human carcinogen. These Judgments were based on
sufficient animal evidence In the absence of human data for Individual PAHs.
/
BenzTalanthracene. Benz[a]anthracene produced tumors 1n mice treated
orally, and 1n various mouse strains treated Intraperltoneally, Intrave-
nously, topically, subcutaneously and Intramuscularly. It was mutagenlc for
Salmonella. DrosophUa and mammalian cells and produced DNA damage, SCE and
morphologic transformation 1n cultured cell. Benz[a]anthracene was positive
1n a mouse lung adenoma assay.
BenzoTalpyrene. Orally administered benzo[a]pyrene was carcinogenic
to three mouse strains. It produced tumors when administered Intratrache-
ally to rats and hamsters. Benzo[a]pyrene delivered Intraperltoneally has
04450 VIII-25 11/15/91
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Induced tumors 1n mice and rats. It 1s the best documented experimental
skin carcinogen producing tumors 1n mice, rats, rabbits and guinea pigs. It
was carcinogenic when administered subcutaneously to mice, rats, hamsters,
guinea pigs and some primates. Benzo[a]pyrene has produced positive
responses 1n a number of genetic toxlology assays In bacterial and mammalian
cells.
Benzo[b]f1uoranthene. Exposure of rats by lung Implantation resulted
1n tumor formation as did 1ntraper1toneal exposure of newborn mice. A total
of three skin painting and Initiation/promotion studies 1n. mice were posi-
tive as was an assay by subcutaneous Injection of mice. Benzo[b]fluoran-
thene was mutagenlc In a forward assay 1n Salmonella.
Benzo[k]f1uoranthene. Lung Implantation produced tumors In rats, and
Initiation/promotion protocols 1n two mouse strains resulted 1n Increased
tumor Incidence. IntrapeMtoneal Injection 1n newborn mice produced
equivocal results. Benzo[k]fluoranthene was mutagenlc 1n a forward assay 1n
Salmonella.
Chrysene. Chrysene produced tumors In several mouse strains when
applied topically 1n assays for complete skin cardnogenldty or In Initia-
tion/promotion protocols. Several early studies employing Intramuscular or
subcutaneous Injection of mice and rats produced negative or equivocal
results. Three studies wherein neonatal mice of two strains were exposed
Intraperltoneally reported Increased tumor Incidence 1n liver and other
sites. Chrysene produced mutations In Salmonella and chromosome aberrations
and morphologic transformation 1n mammalian cells.
04450 VIII-26 09/23/91
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D1benz[a.h]anthracene. Mice of various strains have been shown to
develop tumors as a consequence of oral exposure to d1benz[a,h]anthracene.
Mice were also reported to develop tumors after pulmonary, Intratracheal,
Intravenous or topical treatment. Injection site tumors (Intramuscular or
subcutaneous) have been observed In mice, rats, guinea pigs, pigeons and
unspecified fowl. Results of DNA damage, mutation and morphologic trans-
formation assays have been positive.
IndenoH .2.3.-cdlpyrene. Lung Implantation of 1ndeno[l,2,3-cd]pyrene
produced tumors 1n rats. Skin painting assays for complete carclnogenldty
and Initiating ability were positive 1n two mouse strains, and Injection
site tumors were reported after subcutaneous exposure of a third strain.
Limited data Indicate mutagenldty for Salmonella.
Quantification of Carcinogenic Effects
Compounds classified as Group A, B and C carcinogens are generally
regarded as suitable for quantitative risk assessment. There are studies
sufficient to classify seven PAHs In this document 1n group B2. Many of
these studies, however, do not provide a suitable basis for dose-response
assessment for the following reasons:
• Studies do not give dose/response data.
Studies contain only one dose level.
Studies use exposure other than oral exposure (such as skin
painting).
Sample size 1s too small.
No carclnogenldty bloassays using the oral route of exposure have been
reported for benzo[b]fluoranthene, benzo(k)fluoranthene, chrysene and
1ndeno[l,2,3-cd]pyrene.
04450 VIII-27 09/23/91
-------
BenzTalanthracene. In a study by Klein (1963), male B6AF1 mice were
gavaged with a 3X solution of benz[a]anthracene 3 times/week for 5 weeks and
observed for either 437 or 547 days. Incidences of both lung adenomas and
liver tumors were Increased at both observations. As there was only one
dose of compound administered, and exposure was of short duration, deriva-
tion of a quantitative risk estimate based on this study Is Inappropriate.
Benzofalpyrene. Several studies have reported Increased Incidence of
alimentary tract tumors 1n rodents as a consequence of oral benzo[a]pyrene
exposure. In the majority of these, there was a single, gavage exposure
(e.g., Muggins and Yang, 1962; HcCormlck et al., 1981), or only one gavage
or dietary dose was employed (Berenblum and Haran, 1955; G1bel, 1964; Chu
and Halmgren 1965; B1andf1or1 et al., 1967; Wattenberg, 1972, 1974;
El-Bayoumy, 1985). Trlolo et al. (1977) observed forestomach tumor Induc-
tion 1n female mice (9/group) fed 200 and 300 ppm benzo[a]pyrene 1n the
diet. Treatment was for a relatively small percentage of the animals' usual
Hfespan; that 1s, 12 weeks. Effective numbers of animals were not
reported. In part of the Wattenberg (1972) study, mice receiving two
dietary doses of benzo[a]pyrene were observed to develop forestomach tumors.
Treatment was for <1 year, and no concurrent controls were reported.
A quantitative assessment for oral exposure to benzo[a]pyrene regarding
Its carcinogenic effects can be based on the experiments reported by Neal
and Rlgdon (1967), In which benzo[a]pyrene at doses ranging between 1 and
250 ppm 1n the diet was fed to strain CFW mice for <197 days. No tumors
were found 1n the control group nor 1n the groups treated with 1, 10 or 30
ppm benzo[a]pyrene. The Incidence of tumors, however. Increased between the
04450 VIII-28 09/23/91
-------
40 and 250 ppm benzo[a]pyrene dosages. Stomach tumors, which were mostly
squamous cell paplllomas but also some carcinomas, appeared with an Inci-
dence significantly higher than controls (p<0.001. Fisher Exact Test) at
several doses. Quantitative risk estimates for human cancer Incidence were
developed by U.S. EPA (1980d) using the linearized multistage procedure and
more recently by Clement Associates (1988, 1990a) using a two-stage model.
Krewskl and Murdock (1990) also applied the ArmHage-Doll and a two-stage
model to these data. These approaches appear below.
U.S. EPA (1980d) Approach. The Nedl and Rlgdon (1967) data, with
adjustments to approximate dally lifetime doses (Table VIII-3), were used 1n
calculation of a quantitative risk estimate for human lifetime cancer
Incidence by use of the linearized multistage procedure. Tumor Incidence
data at the highest three doses were not used 1n the extrapolation due to
lack of fH to the multistage model. [A discussion of the fit of data to
the multistage model appears In Human Health Methodology Appendix to the
•
October 1980 Federal Register (45 FR 79379).]
/
A carcinogenic potency factor for humans (q,*) was determined to be
11.53 (mg/kg/day)"1. An estimate of the cancer risk from consuming 1 yg
benzo[a]pyrene/l water (unit risk) could be calculated as follows:
Unit risk - "•53 (mfl/kq/daY)-ix 2 I/day x 0.001 mq/vq . 3.29xl
-------
TABLE VIII-3
Incidence of Tumors 1n Mice Treated Intragastrlcally with Benzo[a]pyrenea»b
Experimental Dose
(ppm diet)
0
1
10
20
30
40
45
50
100
' 250
Transformed Dose
(mg/kg/day)
0.0
0.1
1.3
2.6
3.9
5.2
5.9
6.5
13.0
32.5
Incidence0
No. Responding/No. Tested
0/289
0/25
0/24
1/23
0/37
1/40
4/40
24/34
19/23
66/73
aSource: Neal and Rlgdon, 1967
^Length of exposure = 110 days; length of the experiment = 183 days;
Hfesrpan of mouse = 630 days; average weight of mouse = 0.034 kg
cThe Incidences at the highest three doses were not used 1n the extrapo-
lation due to lack of fit of the multistage model.
04450 VIII-30 09/23/91
-------
There are substantial difficulties with the procedure used to derive a
risk estimate from the Neal and Rlgdon (1967) data as given In Table VIII-3.
Among the factors that render this data set problematic are the following:
1. Exposure time was variable 1n the higher dose groups (Table
VIII-4). The exposure time was reported as a range, and
Individual animal data are now unavailable.
2. Animals were started on test at differing ages.
3. A very sharp Increase 1n tumor Incidence was noted 1n those
animals fed 50 ppm by comparison with those fed 45 ppm.
4. There was an apparent leveling off of tumor Incidence among the
highest dose groups.
In the U.S. EPA (1980d) use of the multistage model the peculiarities of
the data set were handled by using one estimate of exposure time (110 days)
and by discarding data at the highest three doses as they did not fit the
model. This precluded use of the highest Incidence data and resulted In the
odd situation of eliminating a dose group (50 ppm) essentially equivalent to
one (45 ppm) that was used In the modeling procedure.
Clement Associates (1988). An alternate approach was proposed by
Clement Associates (1988) 1n which the Neal and Rlgdon (1967) data were fit
to a two-stage dose-response model. This two-stage model 1s a special case
of the Moolgavkar and Knudson (1981) and Hoolgavkar (1986) cancer risk model
as adapted by Thorslund et al. (1987) to account for exposure to known
levels of carcinogens. According to the two-stage model, the population of
cells at risk for Induction of cancer consists of stem cells. These cells
can divide, undergo differentiation to terminal, nond1v1d1ng cells, die, or
undergo changes that result 1n a preneoplastlc state. It 1s assumed that
this last option Includes a mutation or heritable change at a critical site.
04450 VIII-31 11/15/91
-------
trt
C3
OJ
TABLE VIII-4
Forestomach Tumors 1n Mice Fed Benzo[a]pyrene*
First Subgroup
Exposure
B[a]P ppm
X
0
1
10
20
30
40
45
50
100
250
250
250
250
250
250
250
100
100
5000
Age First
Exposed
toi
300
30
30
116
33
33
31
17
20
18
49
56
49
62
49
91
74
48
98
Age Last
Exposed
tfl
300
140
140
226
143
143
141
124
118
88
50
58
53
67
56
121
81
78
99
Age Last
Observed
tl
300
140
140
226
143
143
143
124 .
118
88
155
162
155 .
168
155
198
182
156
209
Second Subgroup
Age First
Exposed
102
67
101
71
22
24
20
180
Age Last
Exposed
tf2
177
211
181
219
146
185
•
181
Age Last
Observed
t2
177
211
183
219
146
185
294
No. with
Fores tomach
Tumors/No.
of Mice
r/n
0/289
0/25
0/24
1/23
0/37
1/40
4/40
24/34
19/23
66/73
0/10
1/9
1/10
4/9
3/10
26/26
0/10
12/18
17/33
*Source: Adapted from Neal and Rlgdon (1967) by Clement Associates (1990a)
VO
-------
The preneoplastlc cell then has the options of division, differentiation,
death, or further change to a fully transformed or cancer cell. This cancer
cell, 1f permitted to proliferate, will form the basis of a tumor. In the
Moolgavkar and Knudson (1981) model, these processes are described mathe-
matically by postulating specific exposure-dependent rates for cell changes.
A simplified version of the model can be expressed 1n the following manner:
•
Kt) = H0H-\f C0{v) [exp(B-D)(t-v)]dv (8-1)
where
I(t) = age-specific cancer Incidence at age t
MO = transition rate from stem to preneoplastlc cell
M-j = transition rate from preneoplastlc to cancerous cell
C0(v) = number of susceptible stem cells per Individual target
organ at age v
B = birth rate or rate of cell proliferation of preneoplastlc
cells
0 = death rate of preneoplastlc cells
It Is likely that at least some of the Increased Incidence Is attributable
to the fact that the 50 ppm dosed animals were younger when exposure was
begun. It 1s, nevertheless, appropriate to use as much of the data as
feasible In calculating the quantitative estimate. It Is reasonable to
assume that the biologic processes described by the above equation can be
affected by exposure to carcinogens and that the likelihood of their
occurrence 1s a function of exposure time. Thorslund et al. (1987) thus
developed a version of the model that Incorporated time and exposure
dependence.
04450 VIII-33 11/15/91
-------
In applying the two-stage model to the Neal and Rlgdon (1967) data 1n
Table VIII-5, some modifications were made by Clement Associates (1988) and
some parameters were estimated. In the absence of experimental Information
about cell stages and differences 1n exposure over time, 1t was not possible
to estimate the Individual background and exposure-Induced mutation rates
for preneoplastlc and transformed cells using tumor rate data. Nor could
the relative transition rates that correspond to each stage be Identified.
Two exposure-Induced relative transition rates and a background transition
rate were estimated from bloassay data. If these transition rates are
linear functions of dose (which 1s likely at low doses), they may be
expressed as Mn = an f &nx and Mi ~ ai f &ix where a 1s
the background transition rate and 6 1s the PAH-lnduced transition rate per
unit of exposure for each stage. It was assumed that the factors
Influencing the background transition rates and the PAH-lnduced transition
rates have the same relative effectiveness for each stage. Under this
assumption, BO/<*O = B-i/a, = s» tne relative transition rate, so
that MQ^ = OQO^l+Sx)2 = H(l+Sx)*, where M = o0or
In this application of the model H was assumed that promotional effects
result from multiple molecular Interactions. Thus, the growth rate of
preneoplastlc cells G at low doses 1s virtually Independent of exposure
level so that G = 8 - D. If this assumption 1s violated at higher doses,
the shape of the curve will have greater curvature than quadratic, and the
quadratic model will be rejected.
Finally, as a first approximation H was assumed that the number of stem
cells post-maturity, CQ(v), Is relatively constant and may be taken to be
unity [I.e..' CQ(v) * CQ * 1).
04450 VIII-34 11/15/91
-------
TABLE VIII-5
Variable Exposure Data Used to Estimate Parameters In the Ingestlon
Dose-Response Model for Benzo[a]pyrenea
Dose Age First
(ppm) Exposed (days)
x ts
0
1
10
20
30
40
45
50
100
250
—
30
30
116
33
67
33
101
31
71
17
22
20
24
18
20
Age Sacrificed
(last exposed)
(days)
t
300
140
140
226
143
177
143
211
141
181
124
219
118
146
88
185
Number of Number of
Animals Forestomach Tumors
Exposed
n Observed Expected**
289
25
24
23
37
40
40
34
23
73
0
0
0
1
0
1
4
24
19
66
i
0.000
0.002
0.232
0.875
3.091
5.746
7.560
19.692
14.220
64.259
aSource: Neal and Rldgon (1967) as adapted by Clement Associates (1988)
blt was assumed that one-half the animals 1n each group were exposed
during each of the Intervals reported, ts to t. x2 = 17.12, d.f. = 8,
pssO.03
04450
VIII-35
12/31/90
-------
Substituting these expressions [G, M, S, CQ(v)] Into equation 8-1 and
Integrating yields a cumulative hazard function H(x,t), so that the prob-
ability that a tumor will develop by time t as a result of exposure to a
level of genotoxlc agent x can be expressed as:
P(x.t) = 1 - exp [-H(x.t)] = 1 - exp [-M(USx)2][exp (Gt) - 1 - Gt]/G2
(8-2)
where
M = background tumor rate parameter
S = fractional Increase 1n the transition rate between cell
stages per unit dose, assumed to be the same for each stage
G = B - D and 1s the exposure-Independent growth rate of preneo-
plastlc cells
t = the time (or age) at which risk 1s evaluated
The level of agent at the target tissue was assumed to be directly
related to the administered dose. This was based on observations of experi-
ments discussed earlier In which the rate of formation of the major benzo-
[a]pyrene dlol epoxIde-DNA adduct was found to be linearly related with
respect to dose 1n the forestomach, lung, and skin (Perelra et al., 1979;
/
Adrlaenssens et al., 1983).
The specific dose-response model derived for benzo[a]pyrene (equation
8-2) was thus a restricted form of the model developed by ArmUage and Doll
(1957), Moolgavkar and Knudson (1981), and Thorslund et al. (1987). It was
restricted by assuming that G Is Independent of treatment (x) and that the
two transition rates are linear functions of x with proportional coeffi-
cients. The consequence of these assumptions Is that at constant t the
dose-response function has only two parameters:
P(x) = 1 - exp-A(l + Sx)2 (8-3)
2
where A = M [exp(Gt)-l-Gt]/G and t = age at last observation.
04450 VIII-36 09/23/91
-------
Use of this modification of the two-stage model for quantitative risk
estimation for benzo[a]pyrene has several advantages.
1. At low doses, the model converges to a linear, .nonthreshold
form.
2. Only two parameters need to be estimated.
3. A stable point estimate of risk could be obtained directly.
4. The mathematical form of the two-stage model 1s based on an
acceptable theory of cancer Induction.
5. The model Is consistent with what 1s known regarding the
mechanism of tumor Induction by PAHs, Including benzo[a]pyrene
(e.g., exposure to cigarette smoke).
6. Data from all dose groups reported 1n Neal and Rlgdon (1967)
could be used In the risk estimation.
As Indicated earlier, the animals 1n the Neal and Rlgdon (1967) study
were exposed at various ages and for varying times. In order to describe
the dose-response relationship for the 1ngest1on of benzo[a]pyrene and tumor
Incidence for each exposure group, estimates for each of the exposure
variables were made as Indicated 1n Table VIII-5. As a first approximation,
each mouse was assumed to be exposed to one of two exposure patterns:
(1) those that were youngest at first exposure and were sacrificed
at the earliest age (e.g., for the group exposed to 0.04 mg
benzo[a]pyrene 1n Table VIII-4, were first exposed at age 33
days and last exposed at age 143 days), and
(2) those that were oldest at first exposure and were sacrificed
at the oldest age (e.g., for the 0.04 mg/group, were first
exposed at 67 days and last exposed at 177 days).
One-half of the mice In each exposure group were assumed to have been
exposed to each exposure pattern. Using the maximum likelihood method, the
data 1n Table VIII-5, and the assumption that the tumors are Incidental, the
parameters 1n the hypothesized risk equation were estimated as outlined 1n
04450 VIII-37 09/23/91
-------
Gart et al. (1986). The resulting best-fitting model has the parameters
estimates y<* -»0, G = 0.194, and YB2 = 0.2142. This mathematical
model gives a statistically adequate fH (I.e., the resulting x2 has a p
value >0.01, which 1s the criterion used for the multistage model by the
U.S. EPA). This fH 1s shown 1n Table VIII-5.
The resulting model 1s a quadratic function of dietary concentration at
high exposure levels. This model 1s assumed to be valid over the longest
exposure duration employed for a group of animals; that 1s, t-t =(219-22)/
7=28.143 weeks. The parameter value for G' (0.194), however, 1s Incompatible
with Increases 1n age-specific cancer death rates that have been observed.
This value for G Implies that the rates Increased at about the 15th to 16th
powers of age (since h(t)=exp Gt-1 -at ). This 1s greater than the
highest reported rate, which 1s that for human prostate cancer (k=13).
Values for k are usually between 4 and 8. This large value of G suggested
to the authors of Clement Associates (1988) that benzo[a]pyrene exerts a
•
promotional effect, which was not Incorporated Into the model.
t
To calculate a lifetime risk estimate for humans H was assumed that a
mouse consumes 13X of Us weight/day as food, a standard surface area
adjustment was made for differences between species and 70 kg was assumed
for human body weight. The low dose linear term for humans was calculated
to be 3.22 per(mg/kg)/day.
Krewskl and Murdoch (1990). Krewskl and Murdoch (1990) also made an
attempt to make greater use of the available data 1n Neal and Rlgdon (1967)
shown In Table VIII-4. Appropriate adjustments were made for start and
04450 VIII-38 11/15/91
-------
duration of exposure and age at sacrifice. Both the Arm1tage-Doll and a
two-stage model adapted from Moolgavkar (1986) with exponential expansion of
preneoplastlc cells, were fH to the data. As Is shown 1n Table VIII-6,
however, neither model was very successful 1n predicting the observed tumor
Incidences. The authors suggested that this failure to fH the data may be
due to an underlying deviation of the theoretical basis of the models from
reality, but may also represent Inadequate experimental technique 1n the
generation of data or the use of the approximate mathematical forms of the
model. Alternative explanations for the failure of the model to fit the
data are the following:
use of an Inappropriate method of adjusting for differences 1n
the exposure Interval within an exposure group;
• lack of an appropriate exposure-dependent growth rate of
preneoplastlc cell growth rate expression;
• background tumor rates poorly defined by the data;
• lack of data on whether observed tumors are paplllomas or
carcinomas.
Clement Associates (1990a). Hore recently a model was developed
Incorporating a different assumption as to how to treat the exposure groups
containing animals with different durations of exposure. • This model differs
from the earlier Clement Associates (1988) report 1n that both transition
rates and the growth rate of preneoplastlc cells were considered to be
exposure-dependent. To this end a simple saturation assumption was used to
define the functional form for the dose-dependence of the preneoplastlc
growth rate.
The cumulative hazard function for the two-stage model for exposure
constant at level x over the Interval tfl to t. for t=t, 1s discussed
by Thorslund et al. (1987).
04450 VIII-39 11/15/91
-------
o
4k
£»
O
o
-J
•V
ro
TABLE VIII-6
Forestomach Tumors 1n Mice Subjected to Variable Exposures of Benzo[a]pyrenea
Dose
Group
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Dose d
(ppm)
0
1
10
20
30
40
45°
50
100
250
250
250
250
250
250
250
100
100
5000
Exposure
Number
Period Time at Sacrifice Number of
t-|-t2 days
20-140
30-140
116-226
50-160
67-177
51-161
20-172
22-132
19-137
49-50
56-58
49-53
62-67
49-56
91-121
74-81
48-78
139-140
T days
300
140
140
226
160
177
163
172
132
137
155
162
155
168
155
198
182
156
252
Animals Exposed
289
25
24
23
37
40
40
34
23
73
10
9
10
9
10
26
10
18
33
Observed
0
0
0
1
0
1
4
24
19
66
0
1
1
4
3
26
0
12
17
X2
d.f.
P
of Animals with Tumors
Predicted
A-D Model
0.0
0.2
1.9
3.6
8.3
11.4
13.0
18.8
13.3
67.0
0.4
0.6
1.3
1.5
2.2
16.1
1.0
5.8
17.9
50.9
16
2xlO~s
M-V-K Model
0.0
0.2
1.4
2.7
6.3
8.8
10.4
23.8
11.1
66.6
0.4
0.8
1.6
1.7
2.5
15.5
1.1
5.6
21.9
44.7
15
9xlO~s
aSource: Adapted from Krewskl and Murdoch (1990)
^Corrected from 0.05 (rounded value) given by authors
-------
To fH the model H was assumed that the exposure groups are made up of
two subpopulatlons with different exposure durations of one of two ranges as
shown 1n Table VIII-4. It was also assumed that the first and second sub-
populations 1n any exposure group j are a proportion of the total of Y*
J
and 1-Y«; Y* may be considered as an unknown parameter. The propor-
tion of animals responding In exposure group j*1s:
P(x,t) = Yj[l-exp-H(x.t01,tfl,t1)]+(l-Yj)[l-exp-H(to2.tf2,t2)]
When Y=! (animals exposed under uniform conditions),
P(x,t)=l-exp-H(x,tortfl.t1).
The data 1n Table VIII-4 were fH to the model with the addition of two
zero dose groups. The control groups reported by Neal and Rlgdon (1967)
showed no Incidence of forestomach tumors. Using zero Incidence data 1n the
model may result 1n some cases 1n unstable parameter estimates; 1t was thus
/
decided to Include some nonzero historical .control data. Mice used by Neal
and Rlgdon (1967) were described as "an Inbred CFW strain kept In this
laboratory for 8 years". The CFW mouse 1s, 1n fact, an outbred strain from
Carworth Farm (CF) and was derived from Swiss Webster. The outbred stock 1s
still maintained by Charles River Breeding Laboratories (Crl). It 1s
believed that the Inbred mice from the testing laboratory are not likely to
be genetically Identical to other Inbred CFW colonies or to other Swiss
Webster mice. However, forestomach tumor Incidences for untreated animals
considered to be similar to the test population are given 1n Table VII1-7.
04450 VIII-41 09/23/91
-------
TABLE VIII-7
Historical Control Incidence Data for Forestomach Tumors 1n
Strains of Swiss (Webster) Bred Mice
Strain
Swiss
random-bred
CPU
SPF Swiss
(Webster)
SWR/J Swill
/
CFW
Texas colony
Sex
M
F
H
F
H
F
M
F
unknown
Age
(days)
_ _
—
--
700
608
300
Tumor Type
papllloma
--
—
'squamous cell
carcinoma
squamous cell
carcinoma
—
Incidence
1/99
0/99
0/100
0/203
0/28
0/38
2/268
1/402
0/289
Reference
Toth et al.,
1976
Sher, 1974
Prejean
et al., 1973
Rabsteln
et al., 1973
Neal and
Rlgdon, 1967
04450
VIII-42
09/23/91
-------
These data Indicate that forestomach tumor Incidence was quite low 1n
Swiss-derived mice In the 1970s. The data chosen for Inclusion were those
for SWR/J mice reported by Rabsteln et al. (1973). These data have the
advantages of being from mice of a known average age and of having a nonzero
Incidence of squamous cell carcinomas 1n both males and females. The SWR/J
Incidence data are consistent wlth'those reported for the test population at
300 days of age.
Two forms of the model were used. The first used the simplifying
assumption that each of the two subpopulatlons of the exposure groups
contained half the animals (y^l/2). The second assumption made was
that the preneoplastlc cell growth rate reverts to background after
exposure; 1n other words, G*(x)=G(0). The growth rate was defined to be of
a general functional form:
bx
E
*
where m 1s a given Integer, here taken to be 1 as a conservative assumption;
b and C are unknown parameters to be estimated from the data.
/
To obtain maximum likelihood estimates of the unknown parameters, the
general approach discussed by Gart et al. (1986) was used (Table VIII-8).
No adequate fit to the full data set was obtained. If, however, the data
were restricted to the lower dose, longer exposure groups (presumably those
most relevant for low-dose continuous human exposure estimates) a reasonable
fH was obtained. Table VIII-9 presents comparisons of estimates derived by
Clement Associates (1988, 1990a), and Krewskl and Murdoch (1990).
04450 VIII-43 11/15/91
-------
TABLE VIII-8
Maximum Likelihood Parameter Estimates for Two-Stage Model with
Saturation of Growth Rate Function
Parameter
Physical Meaning
Estimated Numerical
Values for All
Continuous Exposure
Data
A
6(0)
S
u(x,t)=P(x,t)-P(o,t)
- U730
x=lx!0~s
product of background
transition rates
background growth rate of
preneoplastlc cells
relative cell transition rates
per unit of exposure (mg/g 1n
food)
relative Increase of preneo-
plastlc cell growth rate per
unit of target dose equiva-
lent exposure from mg/g 1n
food
saturation coefficient
exposure (mg/g food)
low-dose risk
2.4569xlO~10
1.1995xlO'2
845.56
159.32
16.303
3.00x10"*
2.97xlO~s
Model
p(x,t)=l-exp-H(x,t)
H(x,t) =
fa(X)
{exp[G(x)t]-G(x)t-l}
G(x) = G(0)
—1
UCxJ
04450
VIII-44
09/23/91
-------
in
o
en
—
vo
TABLE VIII-9
Forestomach Tumors 1n Mice Subjected to Exposures of Benzo[a]pyrene for at least 70 Days3
Number of Animals with Tumors
Dose
Group
1
2
3
4
5
6
7
8
9
10
11
12
Dose d
(ppm)
0
1
10
20
30
40
45
50
100
250
0
0
No. of
Animals
Exposed
289
25
24
23
37
40
40
34
23
73
268
402
Observed
Krewskl and Murdoch
Predicted
Clement Associates
A-0 Modelb M-V-K Modelc . M-V-K:In1t1at1on
0
0
0
1
0
1
4
24
19
66
2
1
0.0
0.2
1.9
3.6
8.3
11.4
13.0
18.8
13.3
67.0
—
—
X2
degrees of
P
0.0
0.2
1.4
2.7
6.3
8.8
10.4
23.8
11.1
66.6
—
—
freedom
1988
0.0
0.0
0.2
0.9
3.1
5.7
7.6
19.7
14.2
64.3
—
—
9.94
7
0.192
1990
0.0
0.0
0.2
0.9
3.1
5.8
7.2
19.4
14.2
64.9
2.6
0.4
18.08
9
0.034
M-V-K:
Initiation
and Promotion
0.0
0.0
0.0
0.2
0.7
2.4
3.7
18.8
18.9
66.1
2.0
1.0
Source: Clement AssocHes (1990a)
Arm1tage-0o1l model
"Moolgavkar-Venson-Knudson model
-------
A difficulty with the previous estimate by Clement Associates (1988) was
that the value of 6 was unreaHstlcally large. This difficulty was
addressed by Clement Associates (1990a) by deriving a G,Q. for humans. It
was assumed that the human G_ 1s equal to the mouse GQ times a constant
equal to the mouse Hfespan of 2 years divided by a human Hfespan of 76
years. The result 1s GQ=0.1152. To relate this value to a power of age,
they solved for the value of k (the number of stages In a multistage model)
that will cause the same relative Increase In the age-specific risk for an
Individual at one-half Hfespan compared with full Hfespan. For 6=0.1152
and Hfespan =76, Clement Associates (1990) found that k-7.?3. As Indicated
In an earlier section, most human values for k fall between 4 and 8; for the
U.S. population the value for k for stomach cancer 1s ~7. They concluded
that the value of G._. for humans of 0.1152 1s 1n a reasonable range.
A maximum likelihood estimate for humans was obtained. Under an addi-
tive risk model the risks become a linear function of dose as x approaches
0. The additional risk over background was expressed as u(x,t)=P(x,t)-
P(o,t), where P(x,t) 1s the probability of cancer by age t given an exposure
level of x. As the exposure x becomes small, one can derive the approxi-
mation u(x,)~L(t).x, where L(t) 1s the low-dose linear term. Extensive
application of the chain rule of derivatives for the model 1n Table VIII-8
resulted 1n the following equation:
L(t) = exp[G(0)t] . [2(S-b)+bG(0)t][1-P(o,t)]
Substituting the maximum likelihood parameter estimates In Table VIII-8
Into this formula provides a value of L(t)=29.63 for t=730 days. To change
the estimate Into units of mg/kg/day, the assumption was made that a mouse
consumes 13X of Us body weight per day.
04450 VIII-46 11/15/91
-------
Thus, the low-dose linear term for mice
= 29.63 x 0.001/0-.13
= 0.2278 per(mg/kg)/day
To obtain the low-dose linear term for humans a standard assumption of
surface area equivalence between mice and humans was made and the resulting
maximum-likelihood estimate of the low dose linear was found:
°-2278 ^ « 2-90 Per(mg/kg)/day
V 0.034 kg
An approximate conditional upper bound was then calculated by Clement
Associates (1990a). The low-dose linear term was considered to be a linear
combination of exposure-related parameters b and S. By assuming that the
age specific rate function for controls was known (that 1s, the parameters A
and G(0) were fixed at their maximum likelihood estimates), the low-dose
linear term could be expressed as
L(t)=H(o,t) . [l-P(o.t)] {2S+b}[G(0)t-2]}
where P(o,t) = l-exp-H(o.t)
/
A
and H(o,t) = {exp[G(0)t]-G(0)t-l}
G(0)2
The equation was solved using t=730, A=2.4599xlO~» and G(0)=0. 11992 . from
Table VIII-8. An upper 95X bound on L(t) was obtained by substituting
values for S (relative transition rate) and b (promotional effect) that
maximized L(t), subject to certain constraints. In this procedure the upper
bound on the promotion parameter, b, was reduced from Us maximum likelihood
value by about 2-fold. The human upper bound of the low-dose linear term
thus obtained was 5.88 per(mg/kg)/day.
04450 VIII-47 11/15/91
-------
In U.S. EPA (1991b) Issue was taken with the manner In which the linear
slope and the upper bound were calculated. The authors' Interpretation of
the above slope factor 1s that 1t represents an lower bound rather than an
upper bound; 1t Is the slope or tangent at dose 0. They further objected to
the following: (1) not all parameters were Included 1n the process of
obtaining the upper bound, but rather only two of the dose-dependent
parameters; (2) 1t was not considered biologically reasonable to require a
more than SOX reduction 1n the cell growth rate In order to obtain an upper
bound; and (3) the historical background tumor Incidence used was from a
mouse strain not used 1n the bloassay.
To these authors the Inability to derive an upper bound by starting from
the parameter values associated with the point estimate of dose response
suggests a peculiarity of the model. It can be shown that a SOX decrease 1n
the cell growth rate value can significantly alter the shape of the dose
response model.
•
As an alternative, they provided an upper bound by extrapolating
linearly from the 10X response point to the background of an empirically
fitted dose-response curve. It was noted In cases wherein the fitted
dose-response curve 1s not linear at low doses that the potency slope or
upper bound can be defined as a secant from a point on the dose-response
curve to the zero dose point. Similar concepts and approaches have been
proposed by Krewskl et al. (1986, 1991) and Gaylor and Kodell (1980). Their
results Indicate that potency slopes thus calculated are comparable to those
obtained from a linearized multistage procedure for the majority of
compounds Investigated.
04450 VIII-48 11/15/91
-------
The upper bound calculated 1n U.S. EPA (1991b) on the Neal and Rlgdon
(1967) data as modeled by Clement Associates (1990) 1s 9.0 per(mg/kg)/day.
U.S. EPA (1991b). The authors of this report chose a model to reflect
the exposure pattern In the Neal and Rlgdon (1967) study; that 1s, partial
lifetime exposure over different (presumably developmentally varied)
portions of their lifetime. To this end they used a We1bull-type dose-
response model that could accomodate partial lifetime exposure. To derive
this model H was asssumed that the hazard rate for tumor occurrence Induced
by benzo[a]pyrene can be given by
hi(t) =/(t-s)k-2D(s)ds
where D(s) = f(d)
= a-jd^d^*. ..+amdm for some positive Integer m
If tfl < s < t1 and
D(s) = 0 otherwise
and d = dose given to animal at time s.
The dose-related hazard function h,(t) Implies that the early benzo[a]-
pyrene exposure contributes more to the hazard rate than does the later
exposure. This assumption 1s consistent with the observation that a single
dose of benzo[a]pyrene of a certain magnitude Is sufficient to Induce
papHlomas after a sufficiently lengthy observation period.
04450 VIII-49 12/10/91
-------
The cumulative hazard by time T for background can be given by
T>
Ho(T) «/atk-1dt
= (l/k)aTk
The cumulative hazard for T>t for the hazard Induced by benzo[a]pyrene can
then be expressed as
Hi(T) =-T [(T-t0)k-(T-tT)k].
v K- i *K
Thus, the probability of tumor observation' by time t (given that animals are
exposed only to benzo[a]pyrene during the time Interval [tQ, t,]) has
the form
P(d,t) = l-exp{-(q0+q1d+...fqmdm)[(t-t0)k-(t-t1)k]},
This model was considered to be equivalent to the multistage model that
would be used when animals are exposed to benzo[a]pyrene for their entire
/
lifetimes. A quadratic model with m=2 was found to be adequate to fit the
Neal and Rlgdon (1967) data. The results of the modeling are given In Table
VIII-10. U.S. .EPA (1991b) reported that data fit was adequate for all
points except those from the 50 and 100 ppro treatment groups. They point
out that the poor fit may be attributable to biologic factors rather than to
statlsltlcal reasons; they note that the animals 1n these dose groups were
started on test at a younger age (<20 days) than were animals 1n other
groups.
04450 VIII-50 11/15/91
-------
TABLE VIII-10
Predicted vs. Observed Tumor Incidence and Data Used
to Calculate the Dose-Response Modela»b
Dose
(mg/g diet)
0.000
0.001
0.01
0.02
0.03
0.04
0.045
0.05
0.10
0.25
to
(day)
0
30
30
116
50
67
50
20
22
19
tl
(day)
300
140
140
226
160
177
160
172
132
137
t
(day)
300
140
140
226
160
177
162
172
132
137
Observed0 Predicted
Incidence Rate
0/289
0/25
0/24
1/23
0/37
1/40
4/40
24/34
19/23
66/73
(0.00)
(0.00)
(0.00)
(0.05)
(0.00)
(0.03)
(0.10)
(0.70)
(0.82)
(0.90)
0.00
0.00
0.00
0.02
0.04
0.08
0.11
0.30d
0.40d
0.98
aSourc«: U.S. EPA, 19915
bto = age of mice (1n days) when exposure began, t] = age of mice (1n
days) when exposure was terminated, t = age of mice (In days) when the
study was terminated. Data on the control and the first nine low-dose
groups were taken from Table 1 of Nell and Rlgdon (1967). Data from other
groups were not Included because animals 1n these groups were exposed to
high doses of B[a]P for only very short periods.
C0bserved number of animals with tumors (Incidence rate)
dThe predicted value lies outside the 95X confidence Interval calculated
from the observed response. This crude test 1s used to determine whether
or not the model reasonably predicts the observed response.
04450 VIII-51 11/15/91
-------
Parameters were estimated by the maximum likelihood method as follows:
q2 = 2.1xlO"5
k = 3.13.
• •
A slope factor at the 10X point (d= 2.4 ppm or 0.3 mg/kg/day) was
calculated by setting tQ = 0, t. = 730, and t = 730. The human upper
bound thus obtained was 4.5 per(mg/kg)/day.
V
Brune et al. (1981). The study by Brune et al (1981) Is the only
reported benzo[a]pyrene Ingestlon study of 2 years duration. Benzo[a]pyrene
was administered to Sprague-Dawley rats 1n either the diet or by gavage In a
solution of 1.5X caffeine. Effective numbers were not reported; 1t was
assumed that the number of animals started on test (32 males and 32 females)
constituted the group examined for tumors. Incidence rates for males and
females combined are given In Table VIII-11.
A linearized multistage procedure was used to calculate an upper bound
slope factor (ql*) .for benzo[a]pyrene from these data. A body surface
equivalence assumption was used for Interpedes conversion; that 1s, the
animal slope was multiplied by a factor equal to
where
U. = 70 kg (assumed adult human weight)
W = 0.4 kg (assumed rat weight)
a
and dose 1s expressed 1n mg/kg/day
04450 VIII-52 11/15/91
-------
TABLE VIII-11
Incidence to Forestomach Only and Total Contact SHe Tumors 1n
Sprague-Dawley Rats Exposed to Benzo[a]pyrene by Gavage
or In the Diet3
Dose
(mg/year)
Gavage
0
6
18
39
Diet
0
6
39
Median Survival Time
(days)
102
112 .
113
87
129
128
131
Tumor
Forestomach Only
3/64
11/64
25/64
14/64C
• 2/64
1/64
9/64
Incidence
Total Contact S1teb
6/64
13/64
26/64
14/64C
3/64d
3/64d
10/64d
aSource: Adapted from Brune et al., 1981
^Includes forestomach, larynx and esophagus
cData from this group were not used 1n calculation due to the shorter
survival time. No adjustment was done as time-to-death data were not
available.
dThe Incidence of total tumors 1n males for the control, low- and high-dose
groups was 3/32, 3/32 and 8/32, respectively.
04450
VIII-53
11/15/91
-------
Higher slope factors were obtained by U.S. EPA (1991b) from the gavage
data (27.1 per(mg/kg)/day based on total contact-site tumors and 27.5
per(mg/kg)/day for forestomach tumors only) than were calculated from the
dietary Intake study [4.7 per(mg/kg)/day based on total tumors and 3.8
per(mg/kg)/day for forestomach tumors]. It 1s not possible to determine
from the Brune et al. (1981) paper whether the enhanced tumor response 1s
due to the effects of gavage (potential Irritation), to the co-carcinogenic
activity of caffeine, or to some combination of both factors.
In the dietary, but not 1n the gavage component of the study, more total
contact-site tumors (larynx, esophagus and forestomach) were observed 1n the
males than 1n the females. Although 1t was possible to separate total
contact-site tumors Into Incidences for males and females, Information 1n
the paper was not adequate for the same adjustment for forestomach tumor
Incidence. In order to derive a potency estimate based on male rat data
only, the slope factor calculated by U.S. EPA (1991b) on the combined male
and female data was multiplied by a factor of 1.5. The SOX Increase was
/
used to adjust for the risk 1n cancer risk for male-only total tumors.
Slope factors based on male rat total contact-site tumors or forestomach
tumors only were 7.1 and 5.7 per(mg/kg)/day respectively.
Chouroullnkov et al. (1967). Chouroullnkov et al. (1967) was another
relatively long-term study (14 months) wherein albino mice of unspecified
strain were administered a total estimated dose of 8 mg of benzo[a]pyrene
mixed 'with olive oil In the diet. The Incidence of forestomach tumors was
0/81 for the controls and 5/81 for the treated animals. As there was only
04450 VIII-54 11/15/91
-------
one non-zero dose the slope factor was calculated 1n U.S. EPA (1991b) by
direct extrapolation from the observed response of 0.062 at 0.63 mg/kg/day
to the background rate of 0:
3
(0.062/063) x (70 kg/0.03 kg) x (24 months/14 months)
= 6.5 per(mg/kg)/day
where (24/14)3 1s a factor to adjust for less-than-Hfetlme observation.
Choice of a Quantitative Estimate for Benzofalpyrene
Slope factors described In the foregoing text are presented for
comparison In Table VIII-12. Note that the slope factors obtained from the
gavage portions of the Brune et al. (1981) study are not Included. This 1s
because of the uncertainty as to magnitude of co-carcinogenic effect
presented by the caffeine vehicle and the potential Irritation component of
gavage. Only results of dietary studies are considered 1n this Instance for
basis of the quantitative risk estimate. Also eliminated from consideration
at thfs point 1s the slope factor at dose 0 (maximum likelihood estimate)
derived 1n Clement Associates (1988), In order to facilitate comparison
among like estimates (upper bounds).
As Indicated 1n Table VIII-12, these estimates span less than an order
of magnitude. Each 1s based on a Iess-than-opt1mal but acceptable data set
from studies 1n two species of outbred rodents. Each estimate 1s based on a
low (tose extrapolation procedure that entails the use of multiple
assumptions and default procedures.
04450 VIII-55 11/15/91
-------
TABLE VIII-12
Slope Factors for Humans Based on Benzo[a]pyrene Feeding Studies
Study
Nell and
Rlgdon, 1967
Brune et al.,
1981
Chouroullnkov
et al., 1967
Slope Factor
per(mg/kg)/day
11.5
5.9
9.0
4.5
4.7
7.1
3.8
5.7
6.5
Comments
Linearized multistage, highest
points dropped (U.S. EPA, 1980d)
Two-stage, conditional upper bound
(Clement Associates, 1990)
Clement two-stage, slope from 10%
response (U.S. EPA, 1991b)
We1bu11-type model (U.S. EPA,
1991b)
Larynx, esophagus, forestomach
tumors, male and female rats
Larynx, esophagus, forestomach
tumors, males only
Forestomach tumors only, male and
female rats
Forestomach tumors only, male rats
only
. Extrapolated from the observed
response.
04450
VIII-56
11/15/91
-------
The least acceptable of the estimates Is that presented by U.S. EPA
(1980d) of 11.53 per(mg/kg)/day based on the Neal and Rlgdon (1967) data.
This application of the linearized multistage model does not account for the
variable times of exposure or the varying ages of exposure of the dose
groups. In fitting the model all data from exposures >45 ppm were dropped.
In 1987 an Agency work group, the Carcinogen Risk Assessment Verification
Endeavor (CRAVE), found sufficient reason to revise this estimate 1n the
light of Improved risk quantUatlon procedures. It was decided at this time
not to Include the 1980 quantitative estimate on the Integrated Risk
Information System (IRIS).
The use of simplified two-stage Moolgavkar-Venson-Knudsen models, has
permitted the use of more of the data reported by Neal and Rlgdon (1967).
The two-stage model 1s described as being biologically based. It 1s
consistent with current thinking that PAHs act as carcinogens by a
multistage process with a small number of stages and allows for
Incorporation of terms that may model other than Initiating activity. In
/
the form of the model presented by Clement Associates (1990a), a term to
permit modeling data for benzo[a]pyrene acting as Us own promoter was
Included. This 1s not Inconsistent with the observation of benzo[a]pyrene's
activity as a complete carcinogen 1n skin. The modified two-stage model 1s
relatively simple requiring the estimation of a limited number of
parameters. As applied 1t 1s linear at low doses as long as the background
Incidence 1s not zero. This application has, however, necessitated the use
of control data of -a historical nature from related but not Identical
strains of mice.
04450 VIII-57 11/15/91
-------
The simplified two-stage model does not accurately predict tumor
Incidence from high-dose short-term exposures. It may be that exposure of
this sort results In tumor Induction by mechanisms sufficiently different
from chronic low-dose exposure that similar models could not be expected to
apply. A potential problem discussed by Clement Associates (1990a) 1s that
the approximation for the exact hazard function used to generate
mathematical expressions may not be sufficiently accurate. They suggested
that Improved accuracy would be possible using the exact model 1f both birth
and death rates of pre-neoplast1c cells were available.
The use of available forms of the two-stage model for generating risk
assessments has come under some criticism. It would appear that this type
of model 1s best defined as biologically based when the several parameters
given biologic meaning can be estimated from specific data. In the case of
the Neal and Rlgdon (1967) study, the data are Inadequate to this
application. A proposed future direction 1s to Investigate the use of human
•
ep1dem1olog1c data for the calculation of estimates of parameters dealing
with background transition rates and background growth rates of
preneoplastlc cells.
For the Neal and Rlgdon (1967) data 1t can only be said that the
two-stage model provides a convenient curve fitting tool, and that Us use
1s not contralndlcated by Information on potential carcinogenic mechanisms
for benzo[a]pyrene.
04450 VIII-58 11/15/91
-------
The use of the Welbull type model 1n a curve-fitting procedure also
allows for the consideration of the variable exposure time of the animals 1n
Neal and Rlgdon (1967). A difficulty Is the lack of fH to the 50 and 100
ppm dose group data. In a test of this model to several other benzo[a]-
pyrene data sets 1t was shown that 1t accurately predicted the tumor
response 1n mice 1n many dietary studies but not 1n those wherein adminis-
tration was by gavage. There are two plausible explanations for this
discrepancy: (1) benzo[a]pyrene given by gavage Induces greater Incidences
of gastric tumors than does 1ngest1on; (2) or the effect of PAH continues
after exposure. The latter hypothesis 1s supported by observation of tumor
Induction as a consequence of a single dose of benzo[a]pyrene.
The Brune et al. (1981) dietary exposure data provide a more limited
dose range than do those of Neal and Rlgdon (1967). For the lower dose of
the two treated groups the Incidence of both total contact-site and
forestomach tumors are equal to or less than the controls. The data are not
•
reported as single sex Incidences for forestomach tumors. This adds some
/
additional uncertainty, as 1t appears from Inspection of the report that
there was essentially no effect 1n the females. It should be noted that
this study was done In rats.
The Welbull model, with parameters that fit the Neal and Rlgdon (1967)
dietary exposure data, tends to underestimate the tumor Incidence observed
1n the gavage studies.
The Chouroullnkov (1967) study offers the twin disadvantages of only one
non-zero dose and less-than-Hfetlme exposure and observation.
04450 VIII-59 11/15/91
-------
There 1s little basis on which to make a recommendation of a single
slope factor from those listed 1n Table VIII-12. One way to present a
quantitative risk estimate for benzo[a]pyrene Is as a range, excluding those
estimates considered Inappropriate. As discussed previously, use of the
11.5 per(mg/kg)/day estimate based on Neal and Rlgdon (1967) 1s not recom-
mended as the higher doses (and tumor Incidences) were excluded from the
modeling procedure nor were duration differences included. The three upper
bound estimates from two different modeling procedures applied to these data
all have both pros and cons, and represent reasonable estimates from
problematic data.
It 1s reasonable to exclude the slope factor of 6.5 per(mg/kg)/day as
this was obtained from direct extrapolation from the observed data
(Chouroullnkov et al., 1967). This Is a rather unusual approach and 1s not
directly comparable with the other calculations. Furthermore, there are no
special circumstances that dictate the desirability of choosing this unusual
approach. There 1s considerable uncertainty 1n applying a linearized multi-
stage procedure to this data set: there 1s only one non-zero dose point and
there 1s uncertainty regarding the duration extrapolation factor. The Brune
et al. (1981) and other data Indicate that for benzo[a]pyrene gavage and
dietary exposure may not be strictly comparable.
For the Brune et al. (1981) data, the most appropriate approach Is to
use the majority of the data. Total contact site tumors. Including
esophagus, pharynx, etc., are a reasonable basis for comparison with the
data obtained for mice, which developed only forestomach tumors. The
estimate derived by applying the linearized multistage procedure to the data
04450 VIII-60 11/15/91
-------
for total contact site tumor for both males and females 1s 4.7
per(mg/kg)/day.
The range of the most acceptable data sets 1s, thus, 4.5-9.0 per(mg/kg)/
day. The median Is 6.25 per(mg/kg)/day.
There Is also precedent for recommending a combined risk estimate by
using a geometric mean (or other combination technique) to calculate a
single number from several slope factors of equal merit. As discussed by
Stlteler and Schoeny (1991) the use of a geometric mean offers some
advantages over other central tendency calculations for values derived from
linearized multistage models. They further recommend that, when possible,
data sets be combined before a modeling procedure when a combined estimate
1s desired. For the data sets 1n question this latter approach seems
Impractical 1n view of the very dissimilar experimental protocols employed.
Vater and Schoeny (1991) have set out a series of biologic considerations to
be weighed when evaluating data sets for combination. These criteria
Include Judgments as to study quality, common mechanisms of action,
similarity of tumor types and sites. Among commonalities of the data sets
for benzo[a]pyrene are the following: mode of administration, tumor site,
tumor types, and presumed mechanisms of action.
There are precedents for using multiple data sets from different studies
using more than one sex, strain and species; for example, quantitative risk
estimates for DDT (U.S. EPA, 1985) and carbon tetrachloMde (U.S. EPA,
1984d).
04450 VIII-61 11/15/91
-------
There 1s less precedent for use of combined risk estimates based on a
mean of slope factors obtained by differing modeling procedures. In the
Interest of using more of the available data, the slope factor of 5.79
per(mg/kg)/day, which Is the geometric mean of the four estimates chosen for
the range, Is recommended. A unit risk for drinking water 1s calculated as
follows:
5.79 per(mq/kq)/day x 2 a/day x 0.001 mq/Pq m K65xlo-4 per
where:
70 kg = assumed weight of adult human and
2 a/day - assumed water consumption of adult human
Concentrations of benzo[a]pyrene corespondlng to lifetime risks of
10~s, 10~6 and 10~7 calculated from the above unit risk are
6.0xlO~2, 6.0xlO~» and 6.0xlO~4 yg/l, respectively. At a 12/04/91
discussion of the Carcinogen Risk Assessment Verification Endeavor (CRAVE)
Work Group, the range, median and geometric means were found to be
acceptable.
DlbenzCa.hlanthracene. Two studies Involving oral exposure to dlbenz-
[a,h]anthracene have been reported (Lorenz and Stewart, 1947; Snell and
Stewart, 1962a). The former study was designed to compare tumor
susceptlbll- 1ty of various mouse strains and to test the suitability of the
olive oil-water emulsions as a vehicle.
The Snell and Stewart (1962a) study reports four sets of tumor Inci-
dences; alveolar carcinoma 1n male and female mice, hemangloendothelloma for
04450 VIII-62 12/10/91
-------
males and mammary carcinoma for females. Exposure duration was reported
only as a range for each group, presenting a difficulty In the use of these
data for calculation of quantitative risk estimates.
In the Errata: PAH Ambient Water Quality Criterion for the Protection of
Human Health (U.S. EPA, 1982), a midpoint of the exposure duration range was
used to calculate quantitative risk estimates using the linearized multi-
stage procedure. These are presented In Table VIII-13. It should be
emphasized that the estimates 1n this table were prepared for purposes of
comparison with risk estimates for other PAHs and were not considered
suitable for calculation of criteria.
Special Considerations
Toxlclty Equivalence Factor Approach for PAHs 1n Group B2. It would
be very useful In the hazard assessment of a diverse but related group of
compounds such as PAHs to be able to prepare quantitative estimates of
biologic effects by comparison with a well-studied type compound.. Various
/
attempts have been made, for example, to compare carcinogenic activity of
Individual PAHs with that of benzo[a]pyrene. If such comparisons were
proven valid then 1t would become feasible, for example, to estimate
carcinogenic potency of a mixture of Identified PAHs by applying a suitable
model 1f all activities were additive. Another potential use would be 1n
regulating less well-studied compounds by comparison with the standard. The
authors of the Errata: PAH Ambient Water Quality Criterion for the
Protection of Human Health (U.S. EPA, 1982) used data sets for several PAHs
administered 1n skin bloassays to generate comparative potency estimates.
Results of these calculations based on skin painting bloassays are shown In
Table VIII-14.
04450 VIII-63 12/10/91
-------
TABLE VIII-13
Cancer Potency Estimates for Excess Risk of 10~5 from Lifetime Exposure
Based on Oral Exposure Data for D1benz[a,h]anthracene*
Spedes/Sex
House/M
Mouse/M
Nouse/F
Mouse/F
Slope Factor
per(mg/kg)/day
0.57
0.75
1.24
2.36
Tumor Type
hemangloendothelloma
alveolar carcinoma
alveolar carcinoma
mammary carcinoma
'Source: U.S. EPA, 1982
04450
VIII-64
09/23/91
-------
T/ VIII-14
Potency Indices for the Carcinogenic PAH Compounds Based on Skin Painting Data9
o
en
o
H- 1
1— (
1— 1
1
cr>
en
Compound
Benzo[a]pyrene '
Benzo[a]pyrene
Benzo[a]pyrene
D1benz[a.h]-
anthracene
Benzo[ b]-
f luoranthene
Benzo[k]-
f luoranthene
Chrysene''
Benz[a]anthracene
Indeno[l,2,3,cd]-
pyrene
Ql*
470
435 .
NA
299.62
35.64
NA
0.53
NA
NA
EOio (and 95X C.L.)
2.98x10-*
(8.62x10"*, 5.98xlO"»)
1.43xlO"»
(3.68x10"*, 2.49xlO"»)
NA
6.34x10"*
(3.24x10"*, 9.44x10"*)
5.0x10"»
(2.75xlO"«, 7.25xlO"»)
NA
0.35
(0.23, 0.47)
NA
NA
qi*
152.49
67.62
20.83
292.81
11.57
0.30
0.88
0.28
1.16
edio (and 95X C.L.)
9.33xlD"«
(6.54x10"*. 1.21x10"*)
(sisixlO"*. 2.57x10-*)
1.43xlO-»
(4.24xlO"». 2.44xlO"»)
6.16x10"*
(3.28x10"*. 9.04x10-*)
1.29x10-»
(8.54xlO"». 1.73xlO"»)
No point estimate
(0.35. none)
0.21
(0.34, 1.12)
0.73
(0.34, 1.12)
0.15
(0.08, 0.22)
Reference
Wynder et al., 1957
Wynder and Hofmann. 1959b
Blngham and Falk, 1969
Wynder and Hofmann, 1959a
Wynder and Hofmann, '1959b
Wynder and Hof roan, 1959b
Wynder and Hofmann, 1959a
Blngham and Falk. 1969
Wynder and Hofmann. 1959a
o
IO
CO
aSource: U.S. EPA. 1982; Chu and Chen. 1984
''Since there Is only one dose group for chrysene, Ql* Is calculated by using Kap1an-He1er survival analysis and the assumption that the
control group has a zero response.
NA - Not available
Ql* « Estimate of carcinogenic potency based on the Incidence data combined with the t1rae-to-tumor data. It Is the 95X confidence upper
bound for the linear coefficient In the multistage model.
qi* « Estimate of carcinogenic potency based on the Incidence data alone. It 1s the 95X confidence upper bound for the linear coefficient In
the multistage model. .
EOjp, " Estimate of carcinogenic potency based on the Incidence data combined with the t1me*to-tumor data as the effective dose associated with
a risk of 10X.
edjQ = Estimate of carcinogenic potency based on the Incidence data alone as the effective dose associated with a risk of 10X.
NOTE: For Ql* and qi*. higher numbers Indicate Increased potency whereas for ED)Q and ed]g. lower numbers Indicate Increased
potencies.
-------
For a complete discussion of the caveats and assumptions Involved 1n the
generation of these estimates, refer to U.S. EPA (1982). The authors note
that the potency estimates 1n Table VIII-14 are not directly comparable.
The q.j* value 1s the slope of 95X upper limit on the linear coefficient 1n
the multistage model (as revised by Howe and Crump, 1982) when tumor Inci-
dence data are used. By contrast the Q * 1s the 95X confidence upper-bound
for the linear coefficient 1n the multistage model developed by Daffer et
al. (1980) and evaluated at t=12 months. This model Incorporates time-to-
tumor data for Individual animals dying In the course of the study. The
ed,Q 1s the dose level 'corresponding to the 10X Incremental tumor response
when Incidence data are used; ED,Q 1s similarly defined for use with
time-to-tumor data. The Q * and q,* reflect estimates of carcinogenic
potency at low doses outside the experimental range. By contrast, the
ED,Q and ed,Q reflect carcinogenic potency within the experimental
range, and presumably are not as model-dependent as the other two Indices.
The authors of U.S. EPA (1982) ranked the PAHs based on the potencies
computed by the four methods as given 1n Table VIII-15.
It 1s Interesting to compare these rankings with a set of rankings based
on unmodeled data. The data 1n Table VIII-16 were prepared by Santodonato
(1986) from published data of Conney (1982). As can be seen from both
tables, the rank assigned to a carcinogenic PAH depends on the type of
model-based procedure applied, the carcinogenic endpolnt measured, and
whether nonmodeled data are used. What 1s not apparent, but Is discussed 1n
U.S. EPA (1982), 1s that variations 1n study protocol for a given route and
endpolnt change the overall outcome of the test and, thus, potency estimates
04450 VIII-66 09/23/91
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171
TABLE VIII-15
Ranking9 of PAHs Based on Estimates of Potency 1n Skin Painting B1oassayb
Ql1
Ql*
"10
ed10
Benzo[a]pyrene
01benz[a,h]anthracene
Benzo[b]fluoranthene
Chrysene
01benz[a,h]anthracene
Benzo[a]pyrene
Benzo[b]fluoranthene
Indeno[1,2,3-cd]pyrenec
Chrysene0
Benz[a]anthracenec
D1benz[a,h]anthracenec
Benzo[a]pyrenec
Benzo[b]fluoranthene
Chrysene
01benz[a,h]anthracenec
Benzo[a]pyrenec
Benzo[b]fluoranthene
Benz[a]anthracened
Indeno[l,2,3-cd]pyrened
Chrysene^
aRank1ng 1s most potent first to least potent last.
bSource: U.S. EPA, 1982
c»Compounds with the same superscript are Indistinguishable 1n potency. '
r\i
CO
-------
TABLE VIII-16
The Carcinogenic Potency of Various PAHs Measured 1n Two Different
Animal Bloassay Systems3
Benzo[a]pyrene
Benz[a]anthracene
Chrysene
D1benz[a,h]anthracene
Phenanthrene
Skin Study5
( tumor s/ymol)
22.5
0.15
0.36
13.4
0.03
Rank
1
4
3
2
5
Newborn Studyc
( tumor s/iimol)
4.5
0.57
0.26
111.7
0.14
Rank
2
3
4
1
5
aSource: Santodonato, 1986
bM1ce received a single topical application of compound followed 7 days
later by twice weekly applications of TPA for 16-25 weeks (Conney, 1982).
cM1ce were given 1.p. Injections of 1/7, 2/7 and 4/7 of the total dose of
compound on the first, 8th and 15th days of life, respectively. The animals
were killed at 22-42 weeks of age (Conney, 1982)
04450 VIII-68 09/23/91
-------
and ranks. Variations 1n use of solvents, animals of varying susceptibil-
ity, and both length of exposure and overall study duration further compli-
cate attempts to compare carcinogenic potency.
While the absolute rank will vary as a function of the considerations
outlined above, certain commonalities can be seen. In general, three groups
of PAHs can be distinguished. The most potent carcinogens are Invariably
benzo[a]pyrene and d1benz[a,h]anthracene. A second group of carcinogens
having Intermediate potency consists of benzo[b]fluoranthene and generally
(but not Invariably) benz[a]anthracene, chrysene and 1ndeno[l,2,3-cd]-
pyrene. The third group consists of weak carcinogens or PAHs that have not
shown significant . cardnogenlcHy Including phenanthrene, pyrene and
benzo[k]- fluoranthene.
An alternative, more quantitative approach to risk estimation for PAHs
has been proposed by Clement Associates (1988, 1990b) and other authors.
This entails deriving a numerical estimate of the carcinogenic potency of a
particular PAH by comparison with that of benzo[a]pyrene. This method 1s
not unlike the generation of toxldty equivalence factors proposed for risk
estimation of chlorinated dlbenzodloxlns and dlbenzofurans proposed by U.S.
EPA (1987c) and recently revised (U.S. EPA, 1989d).
£&fx$" ' '
.•7-WC •?.,.;••, .
What i\i^done* .In this approach 1s to calculate a benzo[a]pyrene-
equlvalent^dose* using data from studies (such as skin painting bloassays)
generally not considered suitable for quantitative risk estimation. In the
Clement Associates (1988, 1990b) reports only those data sets were Included
wherein benzo[a]pyrene was tested concurrently with one or more PAH. This
i,
04450 VIII-69 11/15/91
-------
was done In order to account for Inter-laboratory variations, varying
susceptibility to carcinogenic activity of the different test animals, vary-
ing metabolic capacity of these animals, and differences 1n protocols and
endpolnts measured. For each report considered, the comparison was made
between benzo[a]pyrene activity and the activity of a particular PAH 1n that
same report.
In the Clement Associates (1988) report all risk estimates were gene-
rated using the two-stage model. The low-dose terms or maximum likelihood
estimates are amenable to comparison whereas It 1s felt that comparison of
upper bounds has less meaning. In calculating the estimated transition
rates from the two-stage model on which comparisons were based, the assump-
tion was made that paplllomas and other benign tumors are observable clones
of first stage cells. It was further assumed that carcinomas can develop
from paplllomas, and thus the observation of either a papllloma or carcinoma
serves as evidence of at least one transformation or stage. In those
Instances wherein benign and malignant tumors were combined, the simple form
of the model equation was used:
P(x) = 1 - exp - A(HSx)
The form of the model employing two stages was used for data 1n which
malignant tumors were reported separately:
P(x) = 1 - exp - A(USx)2
04450 VIII-70 09/23/91
-------
To derive the potency for each PAH relative to benzo[a]pyrene, It was
assumed that the PAH and benzo[a]pyrene have similar dose-response curves,
but that H takes a proportionately larger concentration of
nonbenzo[a]pyrene materials to Induce the same response. The relative
potency, Rj, 1s simply the ratio of estimated transition rates, with that of
benzo[a]pyrene taken as 1.0.
The result of a series of calculations was a range of relative potency
estimates. For example, for benzo[b]fluoranthene Individual relative
potencies from the three data sets were 1.067, 0.874 a^d 0.232. In a
similar fashion the ranges of relative potencies presented 1n Table VIII-17
were generated using data from studies described 1n Chapter V. Using benzo-
[b]fluoranthene as an Illustration, a range of relative potencies spanning
an order of magnitude was obtained.
Clement Associates (1988) proposed that selection of the most appro-
priate relative potency be based on a consideration of the qualitative
/
differences among the studies. Criteria used 1n selection of the most
suitable studies Included the following:
1. Relevance of route of administration 1n the bloassay to
presumed human exposure
2. Duration of exposure approximating natural Hfespan
3. Sample size
4. Inclusion of a concurrent vehicle control
'••^- --':«
5. Observance of a dose-response relationship consistent with the
model (linear-quadratic)
6. Extent to which observed responses cover the possible response
range
7. Absence of complicating variables such as administration of
promoting agents
04450 VIII-71 11/15/91
-------
TABLE VIII-17
Summary of Relative Potency Estimates for Indicator PAHsa
1NJ
CO
Compound
Benzo[ a ]pyrene
Benz[a]anthracene
Benzo[b]f luoranthene
8enzo[k]f luoranthene
Benzo[gh1]perylene
Chrysene
01benz[ah]anthracene
Indeno[ 1 . 2, 3-cd Jpyrene
Pyrene
House Skin Subcutaneous
Carclnogenesls Injection
Into Mice
1.0 1.0
0.145C
0.1678
0.0206
0.0151
0.00441
l.lll 2.82k. 4.501
0.021*. 0.0891
i
Test System
Intrapulmonary Initiation-
Administration Promotion
to Rats0 on Mouse Skin
1.0 1.0
0.140 0.258f. 0.1259
0.066 - 0.022f
0.022 0.0051
0.0409
0.232 ' 0.0741
IntraperHoneal
Injection In
Newborn Mice
1.0
0.057,
0.232.
0.040.
0.125.
0.013^
0.081.
0.524. 0.496
-------
Based on application of these criteria single relative potency estimates
were proposed as given 1n Table VIII-18. Only those PAHs classified as B2,
probable human carcinogen, are Included 1n this listing as 1t 1s Inappro-
priate to calculate a quantitative risk estimate for those 1n group D, not
classifiable.
Other approaches for obtaining a single estimate are also feasible. A
weighted average or some other numerical combination procedure could be used
In those Instances wherein a range of relative potencies 1s available.
Other criteria than those described above (or different emphasis on certain
criteria) could result 1n choices other than those 1n Table VIII-18. It
should be noted 1n this context that the Deutsch-Wenzel et al. (1983) study
was thought to be less reliable than more standard skin painting studies
because of the unusual route of exposure (lung Implantation of wax pellets).
This experimental protocol also may entail confounding variables such as
trauma or Irritation (which could have promoting effects) or changes 1n
metabolism of the Implanted PAH. It Is worthy to note that this 1s the only
assay wherein 1ndeno[l,2,3-cd]pyrene had an elevated response by comparison
/
with the generally more carcinogenic PAH benzo[b]fluoranthene. A more
appropriate method of study and relative potency selection may be to employ
an objective method such as that described by DuMouchel and Harris (1983).
In 1990 a modified TEF approach was proposed (Clement Associates,
1990b). In order to Increase the precision of the relative potency
estimates, the analysis Incorporated time-to-tumor Information, vehicle
controls from multiple experiments and historical controls. A form of the
two-stage model was used that was extended to accomodate possible saturation
04450 VIII-73 11/15/91
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TABLE VIII-18
Summary of PAHs Relative Potencies
Compound
Benzo[a]pyrene
Benzo[e]pyrene
Benz[a]anthracene
Benzo[b]f1uoranthene
Benzo[J ]f luoranthene
Benzo[k]fluoranthene
Chrysene
D1benz[a,h]anthracene
Indeno[l ,2,3-cd]pyrene
Relative
Potency
1.0
0.145
0.140
0.061
0.066
0.0044
1.11
0.232
Reference
Deutsch-Wenzel et
Blngham and Falk,
Deutsch-Wenzel et
Deutsch-Wenzel et
Wynder and Hoffman
Wynder and Hoffman
Deutsch-Wenzel et
al., 1983
1969
al., 1983
al., 1983
, 1959a
, 1959a
al., 1983
04450
VIII-74
09/23/91
-------
of processes for converting PAH to reactive metabolites. Data recommended
for this approach were exclusively from a series of experiments on Induction
of lung epldermold carcinomas by Implantation of PAH 1n the lungs of female
Qsborne-Mendel rats. These data were collected by a single group -of
Investigators. Published data on background rates of lung carcinomas 1n
female Osborne-Mendel rats were used 1n addition to assay vehicle controls
to provide a non-zero Incidence for the zero dose used In the two-stage
model. The relative potencies obtained by comparison of maximum likelihood
estimates are given In Table VIII-19. Relative potencies for PAH-conta1n1ng
mixtures were used to test addUlvlty assumptions.
Rugen et al. (1989) assessed relative carcinogenic potencies of PAH as a
basis for proposing acceptable exposure levels (AEL) 1n drinking water.
They used the published slope factor of 11.53 per(mg/kg)/day for benzo-
[ajpyrene (U.S. EPA, 1980d) and a 10~s risk level to determine a 0.028
pg/1 concentration as the AEL. Relative potencies or relative tumor
-doses (RTD) were determined as follows:
RTD
where:
d, = dosage of chemical 1
n, = tumor frequency after m months of exposure to d,
d~ - dosage of chemical 2
n2 = tumor frequency after m months of exposure to d2
04450 VIII-75 11/15/91
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TABLE VIII-19
Relative Potencies for PAH and PAH-Contalnlng M1xturesa»b
Material Relative Potency
Benzo[a]pyrene 1.0
Benzo[b]fluoranthene 0.1228
Benzo[J]fluoranthene 0.0523
Benzo[k]fluoranthene 0.0532
Benzo[e]pyrene 0.0070
Indeno[l,2,3-c,d]pyrene 0.2780
Benzo[g,h,1]perylene 0.0212
Anthanthrene 0.3160
Flue gas from coal-fired furnace 0.0542
Diesel engine exhaust 0.0028
Gasoline engine exhaust 0.0217
Sldestream cigarette smoke 0.0030
aRelat1ve potencies based on maximum likelihood estimates from lung
Implantation data.
bTable adapted from Clement Associates (1990b)
04450 VIII-76 11/15/91
-------
Direct comparisons of dose and tumor frequency were made for several skin-
painting assays. No model was applied to the data but rather single doses
were chosen as the basis for comparison. Table VIII-20 lists the RTD values
and data on which they were based.
One end use of a series of relative potency would be 1n the evaluation
of PAH-contalnlng mixtures (a TEF approach). This 1s an alternative to
assuming that all carcinogenic PAHs are equlpotent with benzo[a]pyrene. All
the means of comparing PAHs described 1n this chapter Indicate that benzo-
[ajpyrene and d1benz[a,h]anthracene are of comparable potency as carcinogens
and the other PAHs described In this document less active. In the absence
of synerglstlc Interactions risk estimates of PAH mixtures based on
one-to-one benzo[a]pyrene equivalency will be overestimates.
A critical review and analysis of the above has not yet been done by
U.S. EPA. This will be necessary before a TEF or other comparative approach
•
to the derivation of quantitative estimates for PAH can be recommended. At
this time It 1s thus recommended that a quantitative cancer risk estimate be
proposed only for benzo[a]pyrene.
Interactions With Other Chemicals. As reviewed In the Synerglsm
and/or Antagonism Section 1n Chapter V, several of the PAHs have been deter-
mined to be affected by the presence of other PAHs or other substances.
Much of the research 1n this area has focused on the promotion or Inhibitory
effect of noncardnogens such as pyrene on a known animal carcinogen, often
benzo[a]pyrene. The route of administration 1s typically nonoral (dermal,
subcutaneous Injection or Inhalation). There are data for both enhancement
04450 VI11-77 09/23/91
-------
o
.*»
f»
CTI
TABLE VIII-20
Comparison of Carcinogenic Potency of PAH Using Relative Tumor Dose*
»— 1
1
CD
PAH
Benzo[a]pyrene
D1benz[a.h]anthracene
Benzo[a]pyrene
Benzo[ J]f luoranthene
Benzo[b]f luoranthene
Benzo[a]pyrene
Indeno[ 1 , 2, 3-c , d Jpyrene
Benzo[a]pyrene
Benz[a]anthracene
Benzo[a]pyrene
Benz[a]anthracene
Observation Time
(months)
7
7
8
8
8
10
10
18
18
15
15
Concentration
(X)
0
0
0
0
0
0
0
0
1
0
0
.01
.01
.01
.1
.1
.005
.5
.02
.0 •
.001
.02
Tumor Incidence
(X)
75
45
. 85
65
20
50
30
50
14
33
2.5
Relative
Tumor Dose
1
1.67
1
13.1
42.5
1
167
1
179
1
264
Reference
Wynder
Wynder
Wynder
Wynder
Wynder
Wynder
Wynder
and
and
and
and
and
and
and
Blngham and
Blngham and
Wynder
and
Hoffman
Hoffman
Hoffman
Hoffman
Hoffman
Hoffman
Hoffman
Falk.
Falk.
Hoffman
Blngham and Falk.
. 1959a
. 1959a
. 1959a
. 1959b
. 1959b
. 1959a
. 1961
1969
1969
. 1959a
1969
*Table adapted from Rugen et al.. 1989
o
(A
co
-------
and,. Inhibition of biologic activity when PAHs are tested as simple mixtures
and as components of complex mixtures. The outcomes are dependent upon such
variables as strain and species, whether the PAHs are given concurrently
(tests for cocardnogenesls) or applied sequentially (tests for Initiation
and promotion) and the route employed.
It has been postulated that PAHs act by similar mechanisms as Initiators
of cardnogenlclty, which allows an assumption of simple similar action and
dose addltlvUy. This assumption, however, does not take Into account the
data, albeit limited, on both PAH promotional and Inhibitory effects.
Presentation of any PAH risk assessment based on add1t1v1ty must, therefore,
be qualified; theoretically both under- and over-estimation of risk could
result from an assumption of addltlvUy.
Existing Guidelines. Recommendations and Standards
A drinking water standard for PAHs as a class has been developed (U.S.
EPA, 1980d). The World Health Organization European Standards for Drinking
/
Water recommends a concentration of PAHs not to exceed 0.2 yg/i (WHO,
1970). This recomendatlon 1s based on the composite analysis of six PAHs 1n
drinking water. . Fluoranthene, benzo[a]pyrene, benzo[g,h,1]perylene, benzo-
[b]flubranthene, benzo[k]fluoranthene and 1ndeno[l,2,3-cd]pyrene were used.
These six were not chosen on the basis of potential health effects, but
because they were considered useful Indicators of the presence of PAH
pollutants (Borneff and Knute, 1969). A quantitative risk estimate of 11.53
(mg/kg/day)'1 based on the data of Neal and Rlgdon (1967) was derived In
U.S. EPA (1980d). From this, the criterion for the ambient water quality
for PAHs associated with human lifetime cancer risk of 10~5 was estimated
to be 28 ng/l (U.S. EPA, 1982).
04450 VIII-79 09/23/91
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In the occupational environment, a Federal standard has been promulgated
for coke oven emissions, based primarily on the presumed effects of the
carcinogenic PAHs contained 1n the mixture as measured by the benzene
soluble fraction of total partlculate matter (U.S. EPA, 1984b). Similarly,
the AC6IH (1988) recommends a workplace exposure limit for coal, tar pitch
volatlles, based on the benzene-soluble fraction containing carcinogenic
PAHs. NIOSH has also recommended a workplace criterion for coal tar
products (coal tar, creosote and coal tar pitch), based on measurements of
the cyclohexane extractable fraction. These criteria are summarized below:
Substance Exposure Limit Agency
Coke oven emissions 0.15 mg/m3 (soluble NIOSH (1973b)
fraction), 8-hour OSHA (1985)
time-weighted average
Coal tar products 0.1 mg/m8, 10-hour NIOSH (1973a,
time-weighted average 1977)
Coal tar pitch 0.2 mg/m8, (benzene ACGIH (1988)
Volatlles soluble fraction), 8-hour
time-weighted average
Special Groups at Risk
There are several human subpopulatlons that are considered to be at
Increased risks from exposure to PAHs. The largest subpopulatlon comprises
those who are occupationally exposed to one or more of the PAHs. . Coke oven
emissions and coal tar pitch volatlles Include PAHs such as naphthalene,
acenaphthene and fluoranthene. Industries that expose workers to pesti-
cides, coal tar, creosote and fossil fuel oils (I.e., steelworkers, roofers
and auto mechanics) appear to be at higher risk. Persons living In Indus-
trial .areas, or where exposure to auto exhaust Is greater and smokers are
also considered to be at an Increased risk from PAH exposure.
04450 VIII-80 11/15/91
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It 1s known that PAHs are dependent upon metabolism to reactive
electrophlles for their activity and that Individuals of a species, humans
Included, vary 1n their capacity to undertake this metabolism. It 1s also
known that the 1ndudb1lHy of AHH 1n human tissues 1s genetically
determined. The degree to which 1nduc1b1l1ty or nonlndudblHty of AHH
figures as a risk factor for human cardnogenesls, however, Is not clear.
•
Another genetic predisposing factor may be the Individual's capacity to
repair certain types of DNA damage that can be caused by PAH metabolites 1n
a fashion that 1s error-free and does not lead to fixation of mutagenlc
events.
Summary
There were no data reported suitable for calculation of 1-day, 10-day or
longer-term health advisories. DWELs for several PAHs were determined on
the basis of 90-day studies. They are the following: anthracene, 10.5
mg/l; fluoranthene, 1.4 mg/i; fluorene, 1.4 mg/l; pyrene, 1.0 mg/l.
/
Welght-of-evldence determinations for likelihood of human cardnogenlc-
1ty were these: Group D, not classifiable as to human cardnogenldty —
acenaphthylene, anthracene, benzo[g,h,1]perylene, fluoranthene, fluorene,
naphthalene, phenanthrene and pyrene; group B-, probable human carcinogen
— benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]-
pyrene, chrysene, d1benz[a,h]anthracene and 1ndeno[l,2,3-cd]pyrene.
04450 VIII-81 11/15/91
-------
Data from three studies wherein benzo[a]pyrene was given orally were
modeled by several procedures. Both a two-stage and the linearized multi-
stage procedures were used to calculate upper bound risk estimates from the
data of Neal and Rlgdon (1967). Data from both male and female rats as well
as various subsets of the published data from the dietary portion of the
study by Brune et al. (1981) were used. An extrapolation was also done from
the observed response 1n Chouroullnkov et al. (1967). The upper bounds
judged to be most acceptable range from 4.5 per(mg/kg)/day to 9.0
per(mg/kg)/day. A geometric mean of the acceptable upper bound estimates 1s
5.8 per(mg/kg)/day or 1.65xlO"4 per wg/l. Concentrations of benzo-
[a]pyrene corresponding to lifetime risks of 10"5, 10~6 and 10~7 were
determined to be 6.0xlO~2, 6.0xlO~8 and 6.0xlO~« vg/l,
respectively. These risk estimates were found to be acceptable by the
Carcinogen Assessment Verification Endeavor (CRAVE) Work Group 12/04/91.
04450 VIII-82 12/10/91
-------
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