United States Office of Water EPA 440/5-80-069
Environmental Protection Regulations and Standards October 1980
Agency Criteria and Standards Division
Washington DC 20460 £,.
SEPA Ambient
Water Quality
Criteria for
Polynuclear Aromatic
Hydrocarbons
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AMBIENT WATER QUALITY CRITERIA FOR
POLYNUCLEAR AROMATIC HYDROCARBONS
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
U.S. Environmental Protection Agency
Region 5, Library (PL-12J)
77 West Jackson Boulevard, 12th Floor
Ctacago, IL 60604-3590
A
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
''^/
'^ A
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 19/9J.
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
A
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
David J. Hansen, ERL-Gulf Breeze
U.S. Environmental Protection Acencv
Maximal ian Toxicology and Human Health Effects:
Joseph Santodonato (author)
Syracuse Research Corporation
Debdas Mukerjee (doc. tngr.) ECAO-Cin
U.S. Environmental Protection Agency
Bonnie Smith (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Steven D. Lutkenhoff, ECAO-Cin
U.S. Environmental Protection Agency
James Selkirk
Oakridge National Laboratory
Yin Tak Woo
Julian Andelman
University of Pittsburgh
Edmund LaVoie
American Health Foundation
S.D. Lee, ECAO-Cin
U.S. Environmental Protection Agency
Leo Newland
Texas Christian University
Jerry F. Stara, ECAO-Cin
U.S. Environmental Protection Agency
Roy E. Albert, CAG*
U.S. Environmental Protection Agency
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, P. Gray, R. Rubinstein.
*CAG Participating Members: Elizabeth L. Anderson, Larry Anderson, Ralph Arnicar,
Steven Bayard, David L. Sayliss, Chao W. Chen, John R. Fowle III, Bernard Haberman,
Charalingayya Hiremath, Chang S. Lao, Robert McGaughy, Jeffrey Rosenblatt,
Dharm V. Singh, and Todd W. Thorslund.
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TABLE OF CONTENTS
Page
Criteria Summary
Chemical Abbreviations Used Within This Document A-l
Introduction A-2
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Criteria B-2
References B-5
Mammalian Toxicology and Human Health Effects C-l
Exposure C-l
Ingestion from Water C-l
Ingestion from Food C-10
Inhalation C-28
Dermal C-37
Pharmacokinetics C-37
Absorption C-37
Distribution C-38
Metabolism C-39
Excretion C-49
Effects C-50
Acute, Subacute, and Chronic Toxicity C-50
Synergism and/or Antagonism C-57
Teratogenicity C-63
Mutagenicity C-64
Carcinogenicity C-72
Criterion Formulation C-108
Existing Guidelines and Standards C-108
Current Levels of Exposure C-109
Special Groups at Risk C-lll
Basis and Derivation of Criterion C-117
References C-124
Appendix C-128
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CRITERIA DOCUMENT
POLYNUCLEAR AROMATIC HYDROCARBONS
CRITERIA
Aquatic Life
The limited freshwater data base available for polynuclear aromatic hy-
drocarbons, mostly from short-term bioconcentration studies with two com-
pounds, does not permit a statement concerning acute or chronic toxicity.
The available data for polynuclear aromatic hydrocarbons indicate that
acute toxicity to saltwater aauatic life occurs at concentrations as low as
300 ug/1 and would occur at lower concentrations among species "hat are more
sensitive than those tested. No data are available concerning the chronic
toxicity ot poynuclear aromatic hydrocarbons to sensitive saltwater aauatic
life.
Human Health
For the maximum protection of human health from the potential carcino-
genic effects due to exposure of polynuclear aromatic hydrocarbons through
ingestion of contaminated water and contaminated aauatic organisms, the am-
bient water concentration should be zero based on the non-threshold assump-
tion for this chemical. However, zero level may not be attainable at the
present time. 'Therefore, the levels which may result in incremental in-
-'5 -6
crease of cancer risk over the lifetime are estimated at 10 ", 10 , and
10 . \The corresponding recommended criteria are 28.0 ng/1, 2.8 ng/1, and
•i*-1"*
0.28 ng/1, respectively. If the above estimates are made for consumption of
aouatic organisms only, excluding consumption of water, the levels are 311.0
ng/1, 31.1 ng/1, and 3.11 ng/1, respectively.
VI
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CHEMICAL ABREVIATIONS USED WITHIN THIS DOCUMENT
Abbreviation Chemical
A: anthracene
ANT: anthranthrene
BaA: benz[a]anthracene
BaP: benz[a]pyrene
BbFL: benzo[b]fluoranthene
BeP: benzo[elpyrene
BjFL: benzofj]fluoranthene
BkFL: benzo[k]fluoranthene
BPR: benzo[g,h,i]perylene
CH: chrysene
CR: coronene
DBA: dibenz[a,h]anthracene
DMBA: 7,12-dimethylbenz[a]anthracene
F: fluorene
FL: fluoranthene
IP: indeno[l,2,3-cdlpyrene
MCA: 3-methylcholanthrene
NA: naphthanlene
P: pyrene
PA: phenanthrene
PAH: polynuclear aromatic hydrocarbons
PR: perylene
A-l
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INTRODUCTION
Polynuclear aromatic hydrocarbons (PAH) are a diverse class of compounds
consisting of substituted and unsubstituted polycyclic and heterocyclic aro-
matic rings. PAH are formed as a result of incomplete combustion of organic
compounds with insufficient oxygen. This leads to the formation of C-H free
radicals which can polymerize to form various PAH. Among these PAH are com-
pounds such as benzo[a]pyrene, and benz[alanthracene.
PAH are present in the environment from both natural and anthropogenic
sources. As a group, they are widely distributed in the environment, having
been detected in animal and plant tissue, sediments, soils, air., and surface
water (Radding et al. 1976); Shackelford and Keith (1976) report that PAH
have been detected in surface waters, finished drinking water, industrial
effluents, ambient river water, well water, and ground water.
PAH will adsorb strongly onto suspended particulates and biota and that
their transport will be determined largely by the hydrogeologic condition of
the aouatic system. PAH dissolved in the water column will probably undergo
direct photolysis at a rapid rate. The ultimate fate of those which accumu-
late in the sediment is believed to be biodegradation and biotransformation
by benthic organisms (U.S. EPA, 1979).
A-2
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The general
follows:
Molecular weight
Melting point
Vapor pressure (20°C)
Solubility in water
(25°C)
Log octanol/water
partition coefficients
' al properties of acenaphthylene and fluorene are as
Acenaphthylene
152.2ia
92°Ca
10-3 to 10-2torrb
3.93
4.07d
Fluorene
116.153
116-117°ca
10-3 to 10-2torrb
1.98 mg/ic
1.69 mg/ld
4.18d
a) Weast, 1977.
b) Estimated, based on data for structurally similar compounds.
c) Mackay and Shiu, 1977.
d) Calculated as per Leo, et al. 1971.
e) May and Wasik, 1978.
The general physical properties of anthracene and phenanthrene are as
follows:
Molecular weight
Melting point
Vapor Pressure (20°C)
Solubi1ity in water
(25°C)
Log octanol/water
partition coefficients
Anthracene
178.23f
216°Cf
1.95xlO-4torrf
0.045 mg/lh
0.073 mg/19
4.45f
Phenanthrene
178.23f
101°C
6.8xlO-4torrf
1.00 mg/lh
1.29 mg/19
4.46^
f) Radding, et al. 1976.
g) Mackay and Shiu, 1977.
h) May and Wasik, 1978.
A-3
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The physical properties of polycyclic aromatic hydrocarbons are as follows:
Molecular
weight
Melting
point
Vapor Pressure
(20°C)
Solubility in
water (25°C)
Benzo[a]
Anthracene
228.281
155-157°Ci
5xlO-9torri
0.014 mg/lJ
.009 mg/10
Benzo[bl
Fluoranthene
252.32"!
167-168°C"I
lO-ll-l^on-
NA
Benzofk]
Fluornathene
252.320
217°CO
9.59xlO-Htorrk
NA
Chrysene
228.28k
256°Ck
10-H-10-6torr"i
0.002 mg/lJ
0.002 mg/ld
Pyrene
2020
150°CP
6.85xlO-7torrk
0.14 mg/lJ
0.132 mg/lQ
Log Octanol/
Partition
Coefficient
5.61k
6.57"
6.84"
5.61k
5.32"
i) Smith et al. 1978.
j) Mackay and Shiu, 1977.
k) Radding et al. 1976.
1) IARC, 1973.
m) Estimated based on data for sturcturally similar compounds.
n) Calculated as per Leo et al. 1971.
o) Weast, 1977.
p) Cleland and Kingsbury, 1977.
a) May and Wasik, 1978.
NA = No data found.
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cn
The general physical properties of the polycyclic aromatic hydrocarbons having 5 or more aro-
matic rings which are discussed in this chapter are shown below.
Molecular weight
Melting point
Vapor pressure
(torr)
Solubility in
water (25*C)
Loo octanol /water
Benzo[g,h,i)
perylene
276>*
222*Ct
-lo-io". y
0.00026 mg/iw
7.23Z
Benzo[a]
pyrene
252*
179*CS
5xlO-9s,v
0.0038 rag/I"
6.04aa
Dibenzo[a,h)
anthracene
278. 36^
270" Cr
-10-10u,y
0.0005 rag/I*
5.97Z
Indeno[l,2,3-cd]
pyrene
276. 34^
162.5-164'Cr
-I0-10u,y
NA
7.66Z
partition
coefficient
r) Weast, 1977.
s) Smith et al. 1978.
t) Cleland and Kingsbury, 1977.
u) 20'C
v) 25'C
w) Mackay and Shiu, 1977.
x) Davis, et al. 1942.
y) Estimated, based on data for structurally similar compounds.
z) Calculated according to Leo, et al. 1971.
aa) Radding, et al. 1976.
NA = No data found.
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REFERENCES
Cleland, J.6. and G.L. Kingsbury. 1977. Multimedia environmental goals for
environmental assessment, Vol. II. MEG charts and background information.
U.S. Environ. Prot. Agency, (Office of Research and Develop.), Washington,
D.C. EPA-600/7-77-136b. p. 451.
Davis, W.W., et al. 1942. Solubility of carcinogenic and related hydrocar-
bons in water. Jour. Am. Chem. Soc. 64: 108.
International Agency for Research on Cancer. 1973. IARC monographs on the
evaluation of carcinogenic risk of the chemical to man. Certain polycyclic
aromatic hydrocarbons and heterocyclic compounds Vol. 3. Benzofblfluoran-
thene. p. 69. IARC, Lyon. p. 271.
Leo, A., et al. 1971. Partition coefficients and their uses. Chem. Rev.
71: 525.
Mackay, D. and W.Y. Shiu. 1977. Aoueous solubility of polynuclear aromatic
hydrocarbons. Chem. Eng. Data. 22: 399.
Mackay, 0. and A.W. Wolkoff. 1973. Rate of evaporation of low-solubility
contaminants from water bodies to atomosphere. Environ. Sci. Technol.
8: 611.
May, W.E. and S.P. Wasik. 1978. Determination of the solubility behavior
of some polyaromatic hydrocarbons in water. Anal. Chem. 50(7): 997.
A-6
-------
May, W.E. and S.P. Wasik. 1978. Determination of the aoueous solubility of
polynuclear aromatic hydrocarbons by a coupled chromatographic techniaue.
Anal. Chem. 50(11): 997
Radding, S.B., et al. 1975. The environmental fate of selected polynuclear
aromatic hydrocarbons. U.S. Environ. Prot. Agency, (Office of Toxic Sub.),
Washington, O.C. EPA-560/5-75-009. p. 122.
Shackelford, W. and L.H. Keith. 1976. Frequency of organic compounds
identified in water. U.S. Environ. Prot. Agency, (Office of Research and
Development), Athens, Georgia. EPA-600/4-76-062. p. 618.
Smith, J.H., et al. 1978. Environmental pathways of selected chemicals in
freshwater systems; Part II: Laboratory Studies. U.S. Environ. Prot.
Agency, Athens, Georgia. EPA-600/7-78-074. p. 432.
U.S. EPA. 1979. Water-related environmental fate of 129 priority pollu-
tants. EPA-440/4-79-0296.
Weast, R.C. (ed.) 1977. Handbook of Chemistry and Physics, 58th edition.
CRC Press Inc., Cleveland, Ohio. p. 4, p. 2398.
A-7
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Aquatic Life Toxicology*
INTRODUCTION
No standard freshwater toxicity tests have been reported for any poly-
nuclear aromatic hydrocarbon not already discussed in criterion documents on
specific compounds (e.g., fluoranthene and acenaphthene). There are some
data for bioconcentration during tests with model ecosystems or for short
periods of time.
As was true for freshwater organisms, no standard toxicity tests with
saltwater organisms have been conducted with any polynuclear aromatic hydro-
carbon. There are a variety of data for bioconcentration during short expo-
sures.
EFFECTS
Miscellaneous
Lu, et al. (1977) conducted studies with benzo[a]pyrene in a terres-
trial-aquatic model ecosystem and observed bioconcentration factors after 3
days ranging from 930 for the mosquitofish to 134,248 for Daphnia pulex
(Table 1). Bioconcentration factors for Daphnia magna and Hexagenia sp. for
a shorter time were 200 to 3,500 (Table 1).
The bioconcentration factors for polynuclear aromatic hydrocarbons by
saltwater species are lower than those observed with freshwater organisms
but may be due to the short exposure periods (Table 1). A polychaete worm
was exposed to various crude oil fractions and 96-hour IC50 values were
between 300 and 1,000 ug/1 (Neff, et al. 1976a).
*The reader is referred to the Guidelines for Deriving Water Quality Cri-
teria for the Protection of Aquatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation The following tables
contain the appropriate data that were found in the literature, and at the
bottom of each table are calculations for deriving various' measures of tox-
icity as described in the Guidelines.
B-l
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CRITERIA
The limited freshwater data base available for polynuclear aromatic hy-
drocarbons, mostly from short-term bioconcentration studies with two com-
pounds, does not permit a statement concerning acute or chronic toxicity.
The available data for polynuclear aromatic hydrocarbons indicate that
acute toxicity to saltwater aquatic life occurs at concentrations as low as
300 vg/1 and would occur at lower concentrations among species that are more
sensitive than those tested. No data are available concerning the chronic
toxicity of polynuclear aromatic hydrocarbons to sensitive saltwater aquatic
life.
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Table 1. Other data for polynuclear aromatic hydrocarbons
Specl es
Alga,
Oedoqonlum cardlacum
Snai 1,
Physa sp.
Cladoceran,
Daphnla put ex
Mosquito,
Culex plpiens
qulnquefasclatus
Mosqultof Ish,
Gambusia afflnis
Protozoa,
Parameclum caudatum
Cladoceran,
Daphnla maqna
Cladoceran,
Daphnla pulex
Mayf ly,
Hexaqenla sp.
Bluegi 1 1,
Lepomls macrochlrus
Chemical
Benzolalpyrene
Benzolalpyrene
Benzolalpyrene
Benzolalpyrene
Benzolalpyrene
Anthracene
Anthracene
Anthracene
Anthracene
Durat 1 on
FRESHWATER
3 days
3 days
3 days
3 days
3 days
60 mln
1 hr
24 hrs
28 hrs
Benzo-(a)-anthracene 6 mos
Result
Effect (ug/l) Reference
SPECIES
Model ecosystem, - Lu, et al. 1977
bi oconcentrat 1 on
Model ecosystem, - Lu, et al. 1977
bl oconcentrat Ion
factor = 82,231
Model ecosystem, - Lu, et al. 1977
bl oconcentrat ion
factor = 134,248
Model ecosystem, - Lu, et al. 1977
b loconcentrat Ion
factor = 1 1,536
Model ecosystem, - Lu, et al. 1977
b loconcentrat Ion
90% lethal photo- 0.1 Epstein, 1963
dynamic response
B loconcentrat Ion - Herbes, 1976
factor = 200
Bioconcentration - Herbes & Risl, 1978
factor = 760
Bioconcentration - Herbes, 1976
factor = 3,500
Ql% mortality 1,000 Brown, et al. 1975
Eastern oyster,
Crassostrea vlrglnlca
Benzolalpyrene
SALTWATER SPECIES
14 days
B ioconcentrat ion
factor = 242
Couch, et al.
In press
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Table t. (Continued)
Spec Ies
Clam,
Rang I a cuneata
Clam,
Rang!a cuneata
Clam,
Rang I a cuneata
Mudsucker,
G\ I I ichthys mirabi Us
Tidepool sculpin,
Oliogocottus maculosus
Sand dab,
Citharichthys stigmacus
Polychaete worm,
Neanthes arenaceodentata
Polychaete worm,
Neanthes arenaceodentata
Polychaete worm,
Neanthes arenaceodentata
Chemical
Duration
BenzolaIpyrene 24 hrs
Benzol a Ipyrene 24 hrs
Chrysene 24 hrs
BenzolaIpyrene 96 hrs
(edible tissue)
BenzolaIpyrene 1 hr
(edi ble t issue)
BenzolaIpyrene 1 hr
(edible t issue)
Crude oiI extract 95 hrs
(f luorene)
Crude oil fraction 96 hrs
(phenanthrene)
Crude oiI fraction 96 hrs
(I-methyl-
phenanthrene)
Effect
B ioconcentrat ion
factor = 8.66
Bioconcentration
factor = 236
B ioconcentrat ion
factor =8.2
Bioconcentration
factor = 0.048
Bioconcentrat ion
factor = 0.13
B ioconcentrat ion
factor = 0.02
LC50
LC50
LC50
Result
(yg/l) Reference
Neff, et al. 1976a
Neff, et al. 1976b
Neff, et al. 1976a
Lee, et al. 1972
Lee, et al. 1972
Lee, et al. 1972
1,000 Neff, et al. 1976a
600 Neff, et al. I976a
300 Neff, et al. 1976a
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REFERENCES
Brown, E.R., et al. 1975. Tumors in fish caught in polluted waters: Pos-
sible explanations. Comparative Leukemia Res. 1973, Leukemogenesis. Univ.
Tokyo Press/Karger, Basel, p. 47.
Couch, J.A., et al. The American Oyster as an Indicator of Carcinogens in
the Aquatic Environment. Jn: Pathobiology of Environmental Pollutants -
Animal Models and Wildlife as Monitors. Storrs, Conn. National Academy of
Sciences. (In press)
Epstein, S.S., et al. 1963. The photodynamic effect of the carcinogen,
3,4-benzypryene, on Paramecium caudatum. Cancer Res. 23: 35.
Herbes, S.E. 1976. Transport and Bioaccumulation of Polycyclic Aromatic
Hydrocarbons (PAH) in Aquatic Systems. _In: Coal Technology Program Quart-
erly Progress Report for the Period Ending December 31, 1975. Oak Ridge
National Lab., Oak Ridge, Tennessee. ORNL-5120. p. 65.
Herbes, S.E. and G.F. Risi. 1978. Metabolic alteration and excretion of
anthracene by Daphnia pulex. Bull. Environ. Contam. Toxicol. 19: 147.
Lee, R.G., et al. 1972. Uptake, metabolism and discharge of polycyclic
aromatic hydrocarbons by marine fish. Mar. Biol. 17: 201.
3-5
-------
Lu, P., et al. 1977. The environmental fate of three carcinogens; benzo-
(a)-pyrene, benzidine, and vinyl chloride evaluated in laboratory model
ecosystems. Arch. Environ. Contain. Toxicol. 6: 129.
Neff, J.M., et al. 1976a. Effects of Petroleum on Survival., Respiration
and Growth of Marine Animals, _In: Sources, Effects and Sinks of Hydrocar-
bons in the Aquatic Environment. Proc. of a Symposium, American Uni-
versity, Washington, D.C. Amer. Jnst. of Biol. Sci.
Neff, J.M., et al. 1976b. Accumulation and release of petroleum-derived
aromatic hydrocarbons by four species of marine animals. Mar. Biol.
38: 279.
B-6
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Mammalian Toxicology and Human Health Effects
EXPOSURE
Ingestion from Water
The uptake of polynuclear aromatic hydrocarbons (PAH) in
humans from water occurs through the consumption of drinking water.
In the United States, the sources of drinking water are ground
waters and surface waters, such as lakes and rivers. Although a
small amount of PAH originates from natural or endogenous sources,
the predominant sources of PAH in surface waters are man-made. The
discharges of raw and industrial sewage, atmospheric fallout and
precipitation, road runoff, and leaching from polluted soils, all
of which contain substantial PAH concentrations (Andelman and
Suess, 1970), contribute to the PAH contamination in surface
waters. Other than leaching from soils, the only source of PAH in
ground water is of endogenous origin. Borneff (1977) estimated
that low-level contaminated river and lake waters contain five
times higher PAH concentrations than ground water, whereas in
medium-level polluted river and lake waters this value may be 10 to
20 times higher. The concentration of PAH in ground water obtained
by various authors is given in Table 1.
The PAH level in surface waters was determined by a number of
German, English, and Russian workers. In all of these methods, the
PAH were solvent extracted from the water, subjected to clean-up
procedures and analyzed either by TLC-spectrofluorometry or by
u.v.-spectrophotometry. These values are presented in Table 2.
Keegan (1971) analyzed the PAH content in three relatively
unpolluted U.S. river waters by removing the PAH from water by sol-
C-l
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TABLE 1
PAH Concentration in Ground Water
o
Source
G. Finthen,
Germany,
Mainz ,
Germany
Unspeci f ied
locations in
Germany
Average of 12
German ground
waters*
Champaign,
111.*
Elkhart,
Ind.*
Fairborn,
0.*
Concentration, yg/1
Bap Carcinogenic Total
PAH PAH
0.002
0.005
0.0004 0.003 0.04
0.06
N.D.a 0.003 0.007
0.004 0.004 0.02
0.0003 0.0008 0.003
Reference
Bornef f,
Bornef f,
Bornef f
1964
Bornef f
1969
Basu and
Basu and
Basu and
1964
1964
and Kunte,
and Kunte,
Saxena, 1977
Saxena, 1977
Saxena, 1977
*These are results of 6 specified PAH
aN.D.: not detected
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TABLE 2
Concentration of PAH in Surface Waters
Source
Rhine River
at Mainz
River Main at
Seligenstadt
River Danube
at Ulm
River Gersprenz
at Munster
River Aach at
Stockach
River Schussen
0
i
W River Plyussa:
at Shale-oil
effluent discharge
site 3,500 m
downstream
at Navy
water intake
BaP
0.08
0.0024
0.0006
0.0096
0.017
0.01
12
1
0.1
Concentration, yg/1
Carcinogenic
PAH
0.49
0.155
0.067
0.047
0.95
0.20
Total
PAH
1.12
0.48
0.24
0.14
2.5
1.0
Reference
Borneff and
1 f\ f A
1 U *•» a
J_ J \J ™
Borneff and
1964
Borneff and
Borneff and
1964
J_ 3< \f ~
Borneff and
Borneff and
1 rt £ C
1965
Dikun and
Makhinenko
Dikun and
Makhinenko
Dikun and
fc4 -*. 1* 1* £ a** *•* w^ l^ ^%
Kunte,
Kunte,
Kunte,
Kunte,
Kunte,
Kunte,
, 1963
, 1963
1 O£1
A river:
15 ra below coke
by-product
discharge site
500 m downstream
Thames River
at Kew Bridge
at Albert Bridge
at Tower Bridge
8-12
2-3
0.13
0.16
0.35
0.18
0.27
0.56
0.50
0.69
1.33
Fedorenko, 1964
Fedorenko, 1964
Harrison, et al.
1975
Harrison, et al.
1975
Harrison, et al.
1975
-------
vent extraction. The extract was subjected to clean-up and the PAH
were analyzed by TLC-spectrofluorometry. Only samples from the
Oyster River showed detectable amounts of four PAH. No PAH could
be detected in the other two water samples from the Cocheco and
Winnepesaukee Rivers.
The PAH levels in surface waters used as raw water sources for
drinking water, and the effects of treatments of these waters on
PAH levels, are shown in Table 3.
According to Borneff (1977), in surface waters, one-third of
the total PAH is bound to larger suspended particles, a third is
bound to finely dispersed particles, and the last third is present
in dissolved form. The particle-bound portion of PAH can be re-
moved by sedimentation, flocculation, and filtration processes.
The remaining one-third dissolved PAH usually requires oxidation
for partial removal/transformation. The use of C12, C102, 03, and
u]v] light for this purpose has been studied. According to Borneff
(1977), 50 to 60 percent of BaP can be removed by chlorination of
water. However, the total PAH is reduced to a smaller degree by
chlorination. C102 on the other hand, reduces BaP concentration by
90 percent. But at BaP concentrations lower than 10 ppt, C102 no
longer functions as an oxidant for the transformation of BaP. The
transformation of PAH is faster with 03, but the use of O., requires
intensified prepurification to prevent oxidation of other chemi-
cals. Filtration with activated carbon has been suggested by
Borneff (1977) as the best method for PAH removal/transformation
during water treatment. The reduction of BaP concentration with
activated carbon was 99 percent efficient in actual field tests
C-4
-------
TABLE 3
Concentrations of PAH in Raw and Treated Surface Water
used as Drinking Water Sources
Concentration, |ig/l
Source
Hivor Rhine
River Rhine
Lake Constance
Lake Constance
n
1 Engl ish River
linylish River
Monongahela River
at Pittsburgh
same as above
Ohio River at
lluntington, W. Va
same as above
Ohio River at
Wheeling, W. Va.
same as above
Delawater Rivei at
L'hi lade 1 phi a
same as above
Lake Winnebago at
Apple ton, Wis.
same as above
These are average
These values are
Treatment
Untreated
Bank and acti-
vated carbon
filtered
Untreated
Rapid sand
filtration
chlor ination
Untreated
Filtration and
chlor ination
Untreated
Treated
Untreated
Treated*3
Untreated
Treated*1
Untreated
Treated*1
Untreated
Treated11
of five determinations
estimates on the basis
BaP
0.082
0.0005
0.0013
0.0017
0.06b
0.009
0
0
0
0
0
0
0
0
0
0
.04
.0004
.006
.0005
.21
.002
.04
.0003
.0006
.0004
Carci nogenic
PAH
0.485
0.015
0.030
0.017
0.37°
0.051°
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
14
002
020
002
57
Oil
16
002
002
002
with the exclusion of a sixth high val
of average PAH adsorption in reservoir.
Total
PAH
1.11
0.13
0.065
0.053
0.73b
0.24
0.
0.
0.
0.
1.
0.
0.
0.
0.
0.
ue.
hr.
60
003
058
007
59
14
35
015
007
,006
r-a r rr i n
Reference
Borneff and Kunte
Borneff and Kunte
Borneff and Kunte
Borneff and Kunte
Harrison, et al.
Harrison, et al.
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
oci e n i c f
and
and
and
and
and
and
and
and
and
and
rom
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena ,
Saxena,
non-care
, 1964
, 1964
, 1964
, 1964
1976
1976
1978
1978
1978
1978
1977
1977
1978
1978
1977
1977
inogen ic
These values may be a little higher due to the inability or separation UL on ,-..<= "atV "
(1The treatment included flocculation, activated carbon addition, filtration, pll control,
-------
(Borneff, 1977). with the exception of Appleton, wis. drinking
water, this finding of Borneff (1977) has been validated by the
work of Basu and Saxena (1977, 1978), who demonstrated an 88 to 100
percent reduction of PAH in U.S. drinking waters by the use of
activated carbon. In the case of Appleton, wis. water, the initial
PAH level in raw water was very low. Therefore, it can be concluded
that below a certain minimum concentration, activated carbon may
not be very effective for PAH removal/transformation.
As some derivatives of BaP and other PAH are formed during the
disinfection of water with oxidizing agents and u.v. radiation, it
is of interest to examine briefly the carcinogenicity of such
derivatives. With the exception of alkylated derivatives, most BaP
derivatives at best have only weak carcinogenic activity (Butenandt
and Dannenberg, 1956). However, 10-chloro-compounds do cause
tumors (Andelman and Suess, 1970). The quinones, some of which are
also formed during chlorination (Andelman and Suess, 1970) do not
produce tumors (Butenandt and Dannenberg, 1956) , and may,, in fact,
inhibit the activity of other carcinogens (Buu-Hoi, 1959). The
possibility of transformation of PAH into other carcinogenic com-
pounds during water treatment processes is an area which remains
largely unexplored.
The PAH content in U.S. drinking waters was analyzed by Basu
and Saxena (1977, 1978). Six representative PAH recommended by the
World Health Organization (WHO, 1970), as the measure of PAH con-
tamination in drinking water, were monitored in this study (BbFL
was replaced by BjFL) and the average concentration of PAH was
found to be 13.5 ng/1. The U.S. EPA also conducted the National
C-6
-------
Organic Monitoring Survey (NOMS) (U.S. EPA, 1977) to determine the
frequency of occurrence and the levels of PAH in U.S. drinking
water supplies. Of the 110 water samples analyzed, none showed any
PAH other than fluoranthene. Seventeen out of 110 samples analyzed
showed positive fluoranthene values with an average of 20 ng/1 con-
centration. It should be mentioned that the detection limit of PAH
in this study was as high as 50 ng/1. The PAH levels in various
drinking waters are shown in Table 4.
Finished waters from various treatment sites are transported
to the consumers through a variety of pipelines. Borneff (1977)
reported a 10-fold increase in PAH concentration from beginning to
end of a water supply pipe that resulted from the paint used on the
water pipes. Leaching of PAH from the coating materials used on
the pipes could possibly cause an increase in their concentration
in the water reaching consumers. In other instances, PAH could be
adsorbed from the water onto the surface of the pipes causing a
decrease in their concentration. In the United States, two kinds
of pipes are commonly used as distribution lines for transporting
treated waters. These are cast/ductile iron, asbestos/cement
pipes, and a combination of these. The effect of contact with
these pipes on the quality of drinking water in terms of PAH con-
centration was studied by Basu and Saxena (1977). Because of the
intermixing of the pipes, it is difficult to draw definite conclu-
sions from their results. However, it seems likely that in in-
stances where an enhancement of PAH concentration was observed, the
tar/asphalt coating of the pipes was responsible for the increase.
Cement-coated pipes, on the other hand, produced lower PAH concen-
trations, possibly due to adsorption of PAH from the water.
C-7
-------
TABLE 4
PAH Levels in a Few Drinking Waters
o
i
00
Concentration/ ng/1
Source
Mixed tap water at
Mainz, Germany
Water at:
Syracuse, N.Y.
Buffalo, N.Y.
New York, N.Y.
Lake George, N.Y.
Endicott, N.Y.
Hammondsport , N.Y.
Pittsburgh, Pa.
Philadelphia, Pa.
Huntington, W. Va .
Wheeling, W. Va.
New Orleans, La.
Appleton, Wis.
Champaign, 111,
Fairborn, Ohio
Elkhart, Ind.
0
0
0
0
0
0
0
0
0
2
1
0
N
0
N
BaP
.3
.2
.5
.3
.2
.3
.4
.3
.5
.1
.6
.4
.D.b
. 1
.D.b
Carcinogenic
PAH
0
0
3
1
1
1
1
2
2
11
1
2
1
0
0
.3
.2
.9
.5
.1
.5
.9
.0
.0
.3
.6
.4
.2
.8
.3
Total
PAH
7
1
0
6
4
8
3
2
14
7
138
2
6
2
2
0
.0
.1
.9
.4
.2
.3
.5
.8
.9
.1
.5
.2
.1
.8
.9
.3
Reference
Bornef f ,
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
Basu
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
1964
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena ,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
Saxena,
1978
1978
1978
1978
1978
1978
1978
1978
1978
1977
1978
1977
1977
1977
1977
Only the six WHO (1970) - recommended PAH were analyzed, with the exception that BbFL
replaced BjFl. PAH were concentrated by passing 60 liters of drinking water through
polyurethane foams. The eluate from the foams was subjected to cleanup and analyzed
for PAH by TLC-spectrofluorometry.
N.D.: not detected.
-------
There are very few epidemiological studies concerning the cor-
relation between cancer and drinking water. It was, nevertheless,
noted that four London boroughs, supplied largely by well water,
had lower cancer mortalities than most of the other boroughs, which
were supplied with surface water (Stocks, 1947). Another study
concluded that the highest cancer death rates occurred in communi-
ties supplied by river water, followed by communities supplied by
well water, and health water (Diehl and Tromp, 1953; Tromp, 1955).
However, none of these studies attempted to correlate cancer mor-
bidity with concentrations of PAH. Finally, it should be noted
that one epidemiological study of the incidence of gastric cancer
concluded that social factors and the kinds of soils present re-
duced the correlations otherwise obtained with the type of domestic
water supply (Wynne-Griffith and Davies, 1954; Davies and Wynne-
Griffith, 1954).
Although the levels of PAH detected in U.S. drinking waters
are well below the WHO (1970) recommended limit of 200 parts per
trillion (ppt), the health hazards associated with repeated expo-
sure (more effective than an equivalent single dose (Payne and
Hueper, 1960) of carcinogens through drinking water should not be
underestimated. Shabad and Il'nitskii (1970) stated that the
amount of carcinogenic PAH consumed by man from water is typically
only 0.1 percent of the amount he consumes from foods. If the total
PAH uptake from food is taken as 4.15 mg/year (Borneff, 1977), the
human uptake of PAH from drinking water should not exceed 4
ug/year. Assuming the PAH concentration value of 13.5 ng/1 in U.S.
drinking water (Basu and Saxena, 1977,1978), and a daily consump-
C-9
-------
tion of 2.5 liters of drinking water, the yearly intake of PAH from
U.S. drinking water would be 12.3 ug or 0.3 percent of the total
food intake. Nevertheless, the accumulation of PAH in edible
aquatic org'-iisms through polluted surface waters can greatly in-
crease the:-- amount in foods, including fish, some mollusks, and
edibl-:- ale fie (Andelman and Snodgrass, 1974) . The use of contami-
nated wate; ;or irrigation can also spread PAH into other vegetable
foodstuffs .Thabad and Il'nitskii, 1970). Therefore, it is impor-
tant to monitor the PAH levels in surface waters not used as drink-
ing water sources as well as drinking waters, in order to estimate
accurately the human intake of PAH.
Ingest ion from Food
PAH formed through both natural and man made sources can enter
the food chain of man. There is considerable disagreement, how-
ever, concerning the contribution of each of these sources to the
total PAH contamination in foods. From their work with marine
algae and fishes obtained from polluted and unpolluted sources,
Harrison, et al. (1975) concluded that endogenous synthesis may be
the important factor for PAH contamination in these species.
Others, however, believe that the endogenous formation of PAH
occurs to such a limited extent that it is completely masked by the
accumulation of PAH from the environment (Payer, et al. 1975). The
latter conclusion was verified by Shabad and Smirnov (1972). it
has been demonstrated by these authors that plants near an airport
contained 10 to 20 times more BaP than areas remote from the run-
way. The results of Dunn and Stich (1976) indicated a correlation
between the PAH level in mussels with industrial, urban, and recre-
C-10
-------
ational activity. The highest occurrence of BaP in marine organ-
isms in the areas adjacent to the sea lanes tends to support the
view that exogenous sources are the predominant factor for PAH con-
tamination in foods.
The primary routes of entry for PAH in foods are surface ad-
sorption and biological accumulation from the environment (Binet
and Malet, 1964). The adsorption of PAH from the soil by various
plant roots and translocation to the shoots is well documented (Lo
and Sandi, 1978). Similarly, the absorption of PAH by other marine
organisms has been demonstrated by Lee, et al. (1972). Oysters and
clams collected from moderately polluted waters also concentrate
PAH via absorption (Cahnmann and Kuratsune, 1957; Guerrero, et al.
1976). The waxy surface of some plant leaves and fruits can con-
centrate PAH through surface adsorption (Hetteche, 1971; Kolar, et
al. 1975). Kolar, et al. (1975) have shown that the concentration
of BaP in vegetation is proportional to the exposure time during
the growing season (bioaccumulation through adsorption) and the
structure of the surface of the plant (surface adsorption). The
above-ground parts of the vegetables contain more BaP than under-
ground parts. Only about 10 percent of the externally deposited
BaP in lettuce, kale, spinach, leeks, and tomatoes can be removed
by cold water washing (Kolar, et al. 1975).
Food additives and food packaging materials such as paraffin
waxes containing PAH, contribute to the enhancement of PAH levels
in processed foods. For example, Swallow (1976) found that paraf-
fin wax wrapping for food contained PaA, CH, BeP, and BaP at levels
of 29 ppb, 2 ppb, 0-48 ppb, and 2 opb, respectively. Certainly,
C-ll
-------
some of these PAH in the packing material can diffuse into the
food. Hexane, a commercial solvent used to extract edible vege-
table oils, is also a source of PAH contamination. PAH present in
food-grade carbon blacks used for food processing can be transport-
ed to the food products. Curing smoke and other pyrolysis products
used during cooking add to the level of PAH in food. However, in
raw foods which require cooking, the largest source of PAH contami-
nation originates from the cooking process itself.
In order to summarize the available data on PAH levels, vari-
ous foods have been categorized following the pattern of USDA-FDA
for total diet samples (Martin and Duggan, 1968). These are shown
in table form later in the text. It should be recognized that the
data presented in the tables are neither exhaustive nor absolute.
Not all the PAH detected by the various authors are listed in these
tables. Only the most frequently detected PAH are listed. The
concentration values given in these tables are subject to consider-
able variation. The PAH concentrations in uncooked foods largely
depend on the source of food. For example: vegetables, fruits,
and fishes obtained from a polluted environment can be expected to
contain higher concentrations of PAH. Therefore, the PAH content
is subject to regional variation. In the case of raw foods which
require cooking, the method of cooking is largely responsible for
the PAH content in the food and is subject to regional or even per-
sonal variation. Therefore, the frequency of occurrence of PAH in
a particular food is dependent on a number of factors. The results
presented in Tables 5 and 6 represent only the values where the
sample showed detectable levels of PAH.
C-12
-------
TABLE 5
PAH Concentrations (ppb) in a few Vegetable Oils and Margarine
o
1
1— <
oo
A PA
Corna
Coconut 36 51
Margar inec
Sunflower0
Soybean3
Olive9
Peanut9
FL P BaA
3.1 0.
18.0 15.0 2.
1.
29.
13d
1.3 1.6 0.
3.2 2.6 1.
3.3 2.9 1.
8
0
4-
5
9
0
1
BeP
0
2
0
1
4
1
0
.7
.0
.5-
.2
.0
.6
.4
BaP BPR CH
0
0
6
8
1
0
0
.7 0.6
12
.2-
.8
.0 4.0
.4 1.0
.5 0.9
.6 0.9
Howard, et al. 1966c
'Biernoth and Rost, 1967
'Swallow, 1976
This value represents concentration of BaA and CH
-------
TABLE 6
PAH Concentrations (ppb) in Smoked and Nonsmoked Fish
Fish
Smoked eela
Smoked lumpfish
Smoked trout3
Smoked herring
Smoked herring
(dried)
Smoked salmon
Smoked sturgeon
Smoked whitefish
/-*
Smoked whiting
o Smoked redfish
i-1 Smoked cod
Electric smoked
mackerel
Gas smoked.
mackerel
F A PA FL
9.0 4.0 37.0 4.0
5.0 t 10.0 2.0
67.0 26.0 52.0 12.0
3.0
1.8
3.2
2.4
4.6
1.5 4.1 4.0
2.6 1.9 9.0 5.2
8.2 2.3 11.0 2.6
P
6.0
1.0
5.0
2.2
1.8
2.0
4.4
4.0
0.5
3.0
0.6
3.6
4.0
BaA BeP BaP PR
tb 1.0
t t 0
t t
1.7 1.2 1.0
0.5 0.4
0.8
4.3
6.6 0.7
0.3 0.3
4.0 0.4
1.2 0.5 0.2 t
0.6 0.2 0.3 t
BPR
1.0
2.4
2.2
0.2
0.3
Non-smoked
haddock5 1.6 0.8
Non-smoked
her r ing
(salted) 0.8 1.0
Non-smoked
salmon 1.8 1.4
^Thorsteinsson, 1969; Dungal, 1961
Howard, et al. 1966a
^Malanoski, et al. 1968
Masuda and Kuratsune, 1971
t = trace
-------
It has been claimed by Zitko (1975) that PAH are not bioaccu-
mulated along the food chain. However, Bj^rseth (1978) demonstrat-
ed that both common and horse mussels bioaccumulated PAH, although
not to the same degree. Dunn and Stitch (1976) have shown that mus-
sels cannot metabolize BaP upon their removal from water. In
water, mussels released 79 percent of naphthalene in three days,
with a half-life of 1.3 days. The BaP released from both clams and
mussels in water takes place with a half-life of two to five weeks
(Dunn and Stitch, 1976).
The human intake of PAH through the digestive system has been
estimated by Borneff (1977). According to this estimate the human
intake of PAH per year is about 3 to 4 mg from fruits, vegetables,
and bread, 0.1 mg from vegetable fats and oils, and about 0.05 mg
from smoked meat or fish and drinking water.
Vegetable Fats, Oils, and Shortening: Several PAH have been
found in edible oils by European workers (Howard and Fazio, 1969).
The PAH levels in a few vegetable oils and margarine are presented
in Table 5. PAH other than those shown in Table 5 have been report-
ed in these oils (Swallow, 1976). Since the concentration of PAH
in vegetable oils depends on the nature of refinement of the crude
oil (Grimmer and Hildebrandt, 1967), one can expect variations in
their concentrations. Heating of the oils also leads to a slight
increase in PAH concentrations. For example, Lijinsky and Shubik
(1965b) did not detect any PAH in uncooked Wesson and Crisco oil.
However, oil used previously for deep-frying of food showed 1.4 ppb
BaP, 12 ppb FL, and 6 ppb pyrenes (Lijinsky and Ross, 1967; Malano-
ski, et al. 1968).
C-15
-------
Swallow (1976) determined the level of PAH in butter and found
the concentration of BaA + CH, BaP, IP + DBA, and BPR to be 1 ppb.
In a total diet study with a composite sample containing the fats,
oils, and shortening, Howard, et al. (1968) found less than 0.5 ppb
of seven PAH. However, Borneff (1977) estimated that the human
intake of PAH from vegetable fats and oils amounted to 0,,1 mg/year.
Fish and Other Marine Foods: Raw fish from unoolluted waters
usually do not contain detectable amounts of PAH, but smoked or
cooked fish contain varying levels of PAH. In addition to the ori-
gin of the fish, (polluted or unpolluted water) , the amount of PAH
in smoked fish depends on various parameters, such as type of
smoke, temperature of combustion, and degree of smoking (Draudt,
1963) .
The skin of fish apparently serves as a barrier to the migra-
tion of PAH into the body tissues. This was postulated by Malano-
ski, et al. (1968) from their observations that the BaP level in
the skin was much higher than in the interior of cooked fish.
The PAH levels in various smoked and unsmoked fish are shown
in Table 6. In addition to the fish presented in this table, var-
ious other marine organisms had been tested for PAH content. For
example, cooked squid and prawns had BaP concentrations of 1.04 ppb
and 0.08 ppb, respectively (Shiraishi, et al. 1975). Various other
edible marine organisms were investigated and found to contain PAH.
Swallow (1976) analyzed smoked oysters and determined the levels of
BaA + Ch, BbFl + BkFL + BjFL, IP + DBA and BPR to be 19 ppb, 8 ppb,
9 ppb, 7 ppb, and 3 ppb, respectively. Cooked scallops were found
to contain 9.9 ppb BaP (Shiraishi, et al. 1975). Shiraishi, et al.
C-16
-------
(1973) detected 0 to 31.3 ppb BaP in various Japanese seaweeds.
However, no BaP was detected in crab (Shiraishi, et al. 1975). The
absence of BaP in crab is corroborated by the work of Lee, et al.
(1976) , who found no evidence of PAH storage by any of the crab
tissues.
A bioconcentration factor (BCF) relates the concentration of a
chemical in aquatic animals to the concentration in the water in
which they live. The steady-state BCFs for a lipid-soluble com-
pound in the tissues of various aquatic animals seem to be propor-
tional to the percent lipid in the tissue. Thus the per capita
ingestion of a lipid-soluble chemical can be estimated from the per
capita consumption of fish and shellfish, the weighted average per-
cent lipids of consumed fish and and shellfish, and a steady-state
BCF for the chemical.
Data from a recent survey on fish and shellfish consumption in
the United States were analyzed by SRI International (U.S. EPA,
1980). These data were used to estimate that the per capita con-
sumption of freshwater and estuarine fish and shellfish in the
United States is 6.5 g/day (Stephan, 1980). In addition, these
data were used with data on the fat content of the edible portion of
the same species to estimate that the weighted average percent lip-
ids for consumed freshwater and estuarine fish and shellfish is 3.0
percent.
No measured steady-state BCF is available for any of the fol-
lowing compounds, but the equation "Log BCF = (0.85 Log P) - 0.70"
can be used (Veith, et al. 1979) to estimate the steady-state BCF
for aquatic organisms that contain about 7.6 percent lipids (Veith,
C-17
-------
1980) from the octanol/water partition coefficient (P). The log P
values were obtained from Hansch and Leo (1979) or were calculated
by the method described therein. The adjustment factor of
3.0/7.6 = 0.395 is used to adjust the estimated BCF from the 7.6
percent lipids on which the equation is based to the 3.0 percent
lipids that is the weighted average for consumed fish and shellfish
in order to obtain the weighted average bioconcentration factor for
the edible portion of all freshwater and estuarine aquatic orga-
nisms consumed by Americans (Table 7). Caution must be excercised
in application of common practice in obtaining the BCP as described
here, because the ecological impact of PAH is not well understood
at this time. Numerous studies show that despite their high lipid
solubility, PAH show little tendency for bioaccumulation in the
fatty tissues of animals or man (Lee, et al. 1972; Ahokas, et al.
1975; Graf, et al. 1975). This observation is not unexpected, in
light of convincing evidence to show that PAH are rapidly and ex-
tensively metabolized. Since only low levels of PAH are detected
in plants and lower organisms, (Radding, et al. 1976), transfer of
PAH through the food chain does not seem likely. The direct impact
of PAH on plants, animals, or the ecological balance of nature is
difficult to evaluate, since few data are available which suggest
that adverse effects may occur. Thus it is appropriate in the case
of PAH to use the octanol-water partition coefficient for estima-
tion of the BCF. Instead a more realistic value of 30, based on the
work of Lu, et al. (1977) in fish, is recommended for criteria der-
ivation.
C-18
-------
TABLE 7
Calculated Bioconcentration Factors of PAH
Based upon the Octanol/water Partition Coefficient
Chemical
Acenaphthalene
Anthracene
Benzo(a) pyrene
Phenanthrene
3-Methylcholanthrene
1-Methylphenanthrene
Dibenzofuran
Fluoranthene
Fluorene
Benz (a) anthracene
1 , 12-Benzoperyl'ene
Benzo(K) fluoranthene
Benzo(B) fluoranthene
Chrysene
Dinbenza (A,H) anthracene
2 ,3-Phenylene pyrene
Pyrene
Dibenz (A,H) acridine
Log P
3.74
4.45
6.06
4.46
6.97
5.00
4.12
4.90
4.18
5.61
6.51
6.06
6.06
5.61
6.77
6.51
4.88
5.73
Estimated Steady
State BCF
301
1,210
28,200
1,230
168,000
3,550
634
2,920
713
11,700
68,200
28,200
28,200
11,700
113,000
68,200
2,800
14,800
Weighted
Average BCF
119
478
11,100
486
66,400
1,400
250
1,150
282
4,620
26,900
11,100
11,100
4,620
4,460
26,900
1,110
5,850
C-19
-------
Meat and Meat Products: Raw meat does not normally contain
PAH, but smoked or cooked meat may contain varying amounts of PAH
(Lo and Sandi, 1978). Table 8 shows the concentration of PAH de-
tected in a few meats and meat products. The higher concentration
of PAH in charcoal broiled ribs (containing more fats) than in
charcoal broiled steaks tends to support the idea that the most
likely source of PAH is the melted fat. These fats drip on the heat
source and are pyrolyzed. The PAH compounds in the smoke are then
deposited on the meat as the smoke rises (Lijinsky and Shubik,
1965a). Many factors, such as degree of smoking, and the tempera-
ture of combustion affect the composition and concentration of PAH
in cooked meat (Howard, et al. 1966a) In addition to the pyrolysis
of fats, incomplete combustion of charcoal can also contribute to
the PAH content in broiled meat. Thus, the source of heat used for
cooking is responsible for the PAH concentration in cooked meats.
These effects are indicated in Table 9.
In North America, except for smoked ham, most smoked meats
contained much less carcinogenic PAH than European samples (Howard,
et al. 1966a,b). The high incidence of stomach carcinoma in Ice-
land has been explained by the high concentration of BaP in smoked
trout and mutton which are consumed in large quantities in the area
(Bailey and Dungal, 1958). On the other hand, very low concentra-
tions of PAH in Norwegian bologna sausages (see Table 8) are prob-
ably indicative of the tradiation of light smoking of food in Nor-
way (Frethein, 1976).
About 60 to 75 percent of the BaP in smoked food has been found
to be in the superficial layer of meat (Thorsteinsson, 1969). This
C-20
-------
PAH Concentrations (ppb) in a Few Smoked Meat and Meat Products
Meat A PA
Charcoal broiled 21.0
steaks3
Barbecued ribs3 7.1 58.0
Smoked beef ,
(chipped)
Smoked ham
Smoked pork
(roll)b
Smoked frank-
furters
Barbecued beef6
._ Smoked hot
o c
i sausages
to ,
M Smoked mutton 13.0 104.0
Smoked mutton
sausagesd 2.0 17.0
Smoked bologna6
Smoked salami 0.7 Dg
Smoked Morta-
dellaf 2.6 D
Heavily smoked
baconf 20.0 D
FL
43.0
49.0
0.6
14.0
3.1
6.4
2.0
18.0
6.0
5.6
22.0
35.0
P
35.0
42.0
0.5
11.2
2.5
3.8
3.2
1.5
8.0
2.0
5.2
15.0
27.0
BaA BeP
1.4 5.5
3.6 7.5
0.4
2.8 1.2
1.5 2.0
13.2 1.7
0.5
2.0 5.0
0.5 t
0.04- 5.0
0.55
0.6 0.2
2.8 1.8
29.0 D
BaP PR BPR CH
5.8 0.9 6.7 0.6
10.5 1.5 4.7 ±.2
3.2 1.4
3.5 4.3 9.6
0.4 1.0
th
t
0.04- 0.04- 0.04- 0.15-r
0.08 0.07 0.20 1.201
2.0
0.8 3.2 D 1.2
0.7 0.1 0.4 3.4
3.6 0.9 3.0 D
, Lijinsky and Shubik, 1965a
Howard, et al. 1966a,b; Panalaks, 1976
dMalanoski, et al. 1968
Thorsteinsson, 1969
Frethein, 1976; Panalaks, 1976
Lo and Sandi, 1978
^D = detected
.t = trace
1compound unseparated
-------
n
i
K>
to
TABLE 9
Effect of Different Cooking Variables on the Concentration of PAH (ppb) in Cooked Meat
Meat Effect
Charcoal broiled
hamburger3 Fat Content
Fat,c hotd
Lean,6 hot
No-drip pan
Charcoal broiled
hamburger3 Heating
f , ^ temperature
Lean, hot
Lean, cool
Broiled T-bone
steak3 Heat
Charcoal, hot source
Flame, hot
Smoked hamb Degree of
Light Smoke
Heavy
FL
13.3
0.3
0.2
0.3
1.3
19.8
19.0
4.0-
14.0
48.0-
156.0
P
7
I
0
1
0
19
20
2
11
35
161
.7
.6
.1
.6
.6
.1
.0
.0-
.0
.0-
.0
BaA BeP BaP BPR CH CR
2.7 2.6 14.9 1.7 1.0
.9
t
0.9 0.3
31.0 17.6 50.4 12.4 25.4 8.0
3.9 5.7 4.4 6.2 2.0 9.0
0.5- 0-2.0 3.0- 0-1.4 0-3.0
3.0 4.0
6.0- 4.0- 3.8- 2.5- 12.0-
33.0 26.0 55.0 25.0 66.0
1Lijinsky and Ross, 1967
3Filipovic and Toth, 1971; Toth and Blass, 1972
d
:Fat: 21% fat
Hot: 7 cm. from heat source
'Lean: 7% fat
Cool: 25 cm from heat source
-------
low penetration has also been noted by Rhee and Bratzier (1970) ,
who observed that in smoked bologna sausages, the BaP is located
within 1.5 mm from the surface. Cellulose casings can be used as a
more effective barrier to BaP permeation during smoking of frank-
furters than animal casing (Simon, et al. 1969) .
In addition to meat and meat products, liquid smoke flavorings
used during the cooking of meat have been found to contain a vari-
ety of PAH. Lijinsky and Shubik (1965b) have detected BaP, FL, P,
BPR, BaA, and CH in liquid smoke at concentrations of 1 opb, 16 ppb,
7 ppb, 1 ppb, 12 ppb, and 6 ppb, respectively. In liquid hickory
smoke flavoring, Youngblood and Blumer (1975) found the total con-
centration of PAH as 9,400 ppm. The high level of PAH present in
the resinous condensate in liquid smoke flavoring indicates the
importance of its efficient removal from the aqueous flavoring
prior to its use in foodstuffs (white, et al. 1971).
Vegetables, Fruits, Grains and Cereal Products, Sugar and Ad-
juncts, and Beverages: Various Eurooean and Japanese workers have
reported the presence of BaP and other PAH in these products; their
results are summarized in Table 10. Studies in this field in North
America are lacking. Test results indicate that surface adsorption
and root uptake are the principal modes of PAH accumulation in
vegetables (Binet and Mallet, 1964). The frizzy leaf of kale, for
example, has a large surface area and holds dust particularly well.
PAH are adsorbed by the wax layer and protected against solar reac-
tions (Hetteche, 1971). In kale, Hetteche (1971) found the concen-
tration of PAH to be the following: PA, 70-586 ppb; A, 2.4-97.5
ppb; P, 36.2-510 ppb; FL, 53.6-1,196 ppb; BaA, 11.2-230 ppb; CH,
C-23
-------
o
I
ro
TABLE 10
BaP Content in Fruits and Other Foods
Fruits
Apple
Apple
Banana
Banana peel
Grape
Grape
Japanese pear
Pear
Persimmon
Pineapple
Plums
Plums
Dried prunes
Manderin orange
Orange peel
Strawberry
Pumpkin
Concentration
(ppb)
0.
8.
0.
0.
0.
0.
0.
1.
0.
0.
0.
29.
02
3
02
03
2
02
05
9
02
02
04
7
Comments
Polluted
environment
Polluted
environment
Polluted
environment
References
Shira
Kolar
Shira
Shira
Kolar
Shira
Shira
Kolar
Shira
ishi ,
, et
ishi ,
ishi ,
, et
ishi ,
ishi,
, et
ishi ,
Shiraishi ,
Shiraishi ,
Polluted
environment
0.2 to 1.5
0.
0.
N.
N.D. to
03
15
D.a
trace
Kolar
I ARC,
, et
1973
Shiraishi ,
Shira
Shira
Shira
ishi ,
ishi ,
ishi ,
et
al.
et
et
al.
et
et
al.
et
et
et
al.
et
et
et
et
al.
1975
al.
al.
1975
al.
al.
1975
al.
al.
al.
1975
al.
al.
al.
al.
1975
1975
1975
1975
1975
1975
1975
1975
1975
1975
1975
1974
-------
Table 10 (cont.)
Grains & Cereal Products
Product
Concentration
(ppb)
Comments
References
Wheat grain
Wheat sprouts
Cereals
Barley
Oats
Polished rice
Rye seedling
0.1
60.0
0.2 to 4.1
0.3
0.2
N.D.a
10.0 to 20.0
Polluted
environment
Polluted
environment
Polluted
environment
8 other PAH
identified
Kolar, et al. 1975
Siddiqui and Wagner, 1972
IARC, 1973
Kolar, et al. 1975
Kolar, et al. 1975
Shiraishi, et al. 1973
Graf and Nowak, 1966
(^ Lentil seedlings
*"" Sesame seeds
Product
Charred biscuits
Caramel
Chocolate
10.0 to 20.0
N.D.
Sugar
Concentration
(ppb)
11.0-72.0
N.D.a
0.2-1.7
8 other PAH
identified
and Adjuncts
Comments
4 other PAH
quantified
Graf and Nowak, 1966
Shiraishi, et al. 1973
References
Kuratsune, 1956
Shiraishi, et al.
Fabian, 1965
1973
-------
Table 10 (cont.)
o
i
K>
CTl
Vegetables
Vegetable
Parsley leaf and
stem
Red clover
Mushroom
Lettuce
Lettuce
Spinach
Spinach
Spinach
Radish leaves
Radish roots
Radish roots
Tomatoes
Tomatoes
Cabbage
Cabbage
Chinese cabbage
Potatoes
Potatoes
Sweet potatoes
Sweet pepper
Cauliflower
Bean paste
Kidney bean
Carrot
Cucumber
Eggplant
Onion bulb
Onion greens
Concentration
(ppb)
24.3
7.5
7.0
8.6
N.D.
6.2
1.3
7.4
5.3
1.2
N.D.a
0.1
0.2
12.3 to 20.9
N.D.
0.05
N.D. to 0.01
0.2
N.D.
N.D.
5.1
N.D.
N.D.
N.D. to 0.02
N.D.
N.D.
N.D. to 0.01
0.01
Comments
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
References
Kolar, et al.
Kolar, et al.
Kolar, et al.
Kolar, et al.
Shiraishi, et
Kolar, et al.
Shiraishi, et
IARC, 1973
Kolar, et al.
Kolar, et al.
Shiraishi, et
Kolar, et al.
IARC, 1973
Kolar, et al.
Shiraishi, et
Shiraishi, et
Shiraishi, et
Kolar, et al.
Shiraishi, et
Shiraishi, et
Kolar, et al.
Shiraishi, et
Shiraishi, et
Shiraishi, et
Shiraishi, et
Shiraishi, et
Shiraishi, et
Shiraishi, et
1975
1975
1975
1975
al. 1974
1975
al. 1973
1975
1975
al. 1974
1975
1975
al. 1974
al. 1974
al. 1974
1975
al. 1974
al. 1974
1975
al. 1973
al. 1973
al. 1973
al. 1973
al. 1973
al. 1974
al. 1974
-------
Table 10 (cont.)
Beverages
o
i
K)
Beverage
Tea leaves
Black tea aroma
Roasted coffee
(moderate dark)
Roasted coffee
(darkest)
Coffee sootsc
Concentration
(ppb)
Comments
References
Dark rum
Whiskey
1.0
0.04
3 quinolines
Swallow, 1976
IARC, 1973; Nishimura
3.9 to 21.3
N.D.
N.D. to 4.0
200.0-440.0
detected
7 quinolines
detected
and Masuda, 1971
IARC, 1973
Vitzthum, et al. 1975
Kuratsune and Hueper,
1960
Kuratsune and Hueper,
1958, 1960
Kuratsune and Hueper,
1958
N.D. = Not detected
This is the volatile components of black tea.
•»
'These are the soots generated during direct and indirect roasting of coffee beans.
-------
28.6-395 ppb; BeP, 3.8-67.2 ppb; BaP, 0.9-48.6 ppb; PR, N.D.-7 ppb;
BPR, 1.2-46.4 ppb; and CR 0.1-7.2 ppb.
The concentration of BaP in vegetables is directly proportion-
al to exposure time during the growing season and structure of the
surface of the plant. The above-ground parts contain more BaP than
underground parts. Washings with cold water do not remove more
than 10 percent of the BaP (Kolar, et al. 1975). Fruits grown in
polluted environments show a high degree of PAH contaminsition main-
ly through adsorption on the waxy surface.
In smoked Gouda cheese, Panalaks (1976) found 0.5 ppb BaP and
Howard, et al. (1966a) found 2.8 ppb FL and 2.6 ppb P. The unsmoked
cheese contained lower levels of PAH. Grimmer (1974) analyzed
baker's yeasts and determined the level of PAH. The values are
shown in Table 11.
Inhalation
A variety of PAH have been detected in ambient air in the
United States and elsewhere in the world. Because of its carcino-
genic properties, BaP has been most extensively monitored and has
frequently been used as an indicator of ambient PAH. The presumed
correlation between the concentration of BaP and other PAH, how-
ever, does not always exist. For example, a study by Kertesz-
Saringer and Morlin (1975) found little or no relationship between
BaP and other PAH in Budapest air. Gordon (1976) and Gordon and
Bryan (1973) came to a similar conclusion from their work with
ambient Los Angeles air.
The concentration and the nature of PAH in ambient air are
dependent on a number of factors. In general, the PAH concentra-
C-28
-------
TABLE 11
PAH Concentrations (ppb) in a Variety of Baker's Yeast3'
PAH
PA
A
P
FL
BaA
CH
BeP
BaP
PR
French
17.8-34.60
2.6-13.6
11.6-19.6
18.5-21.2
9.8-23.3
8.1-13.4
8.0-10.6
8.0-12.2
0.9-1.2
German
67.0
4.8-10.2
11.5-35.0
17.2-66.8
2.5-15.8
4.2-14.0
3.1-14.3
1.8-13.2
N.D]-0.5
Scottish
1,620
567
327
93-
203
50
40.4
6.2
16.7
Russian
7.2
4.7
16.9
32.1
10 '.8
11.1
8.7
0.5
6.0
Source: Grimmer, 1974
This is baker's yeast as opposed to dietary or brewer's yeast.
C-29
-------
tion is lowest during the summer months and highest during the
winter, (Sawicki, et al. 1962) probably due to commercial and resi-
dential heating during winter (U.S. EPA, 1974). However, there are
some exceptions. Cleveland, Ohio, for instance, does not follow
the high winter-low summer pattern (U.S. EPA, 1974). It has been
suggested that this may be due to significant industrial emissions
that are uniform throughout the year (U.S. EPA, 1974).
The nature and relative amounts of individual PAH in ambient
air are also dependent on the source of these compounds. Thus, the
content of PAH sampled in an industrial area is a composite of the
emissions from various industrial and transportation sources within
the area. For example, Gordon (1976), from his study of the rela-
tive PAH concentration pattern for different areas in Los Angeles,
found a correlation between coronene concentration and automobile
emissions. Similarly, Greinke and Lewis (1975) had demonstrated
that emissions from coke ovens contain lower amounts of certain
methyl-substituted PAH than emissions from petroleum pitch vola-
tiles. Bartle, et al. (1974) also used a PAH profiling technique
for the identification of air pollution sources, such as coal burn-
ing, vehicular emissions, and oil and gas burning.
Meteorological factors have a dominant effect on PAH concen-
trations. For example, Lunde and Bj^rseth (1977) demonstrated that
under favorable wind conditions PAH from downtown London could be
transported to Norway. The tendency of atmospheric inversion to
increase the PAH levels in urban areas has also been shown (Hoff-
mann and Wynder, 1977).
C-30
-------
The annual average ambient BaP concentrations for different
U.S. urban and rural locations during the period 1966-70 have been
compiled by U.S. EPA report (Santodonato, et al. 1978). The aver-
age BaP concentrations in U.S. urban and rural areas obtained from
this U.S. EPA study are shown in Table 12.
An interesting trend has developed from the National Air Sur-
veillance Network (NASN) monitored BaP values listed in Table 12.
As can be seen, the average BaP concentrations in urban areas de-
creased from 3.2 ng/m in 1966 to 2 . 1 ng/m in 1970, approximately
a 30 percent decrease. The decrease is more dramatic (i.e., 80
percent) between the period 1966 to 1976. Even the concentrations
in rural areas indicate a downward trend. This decline in BaP con-
centration is believed to be due primarily to decreases in coal
consumption for commercial and residential heating, improved dis-
posal of solid wastes, and restrictions on open burning (Faoro and
Manning, 1978). A further observation that can be made from Table
12 is the 5- to 10-fold difference in BaP concentration between
urban and rural locations.
The NASN study did not include the determination of concentra-
tions of other PAH. The summer and winter averages of ambient PAH
concentrations for seven urban locations were determined by
Sawicki, et al. (1962) . The averages of summer and winter data
from this work are presented in Table 13.
The average of total PAH concentrations for all cities listed
in Table 13 is 46.4 ng/m . However, these values were obtained
from ambient air sampled in 1958-59 and probably have decreased
during subsequent years. If an 80 percent decrease of total PAH
C-31
-------
TABLE 12
Average BaP Concentrations (ng/m ) in U.S. Urban
and Rural Areas During 1966-76a
Period
Urban
Rural
1966
3.2
0.4
1970
2.1
0.2
1976
0.5
h
0.1°
Source: Santodonato, et al. 1978
^This value is the average of two rural locations.
C-32
-------
TABLE 13
Summer-Winter Average of Ambient PAH Concentrations (ng/m )
*>
in the Air of Selected Cities
o
1
CO
co
City
Atlanta
Birmingham
Detroit
Los Angeles
Nashville
New Orleans
San Francisco
BPR
7.0
13.2
21.3
10.2
10.2
6.0
5.1
BaP
4.5
15.7
18.5
2.9
13.2
3.1
1.3
BeP
3.1
8.0
14.2
4.4
7.6
4.8
1.7
BkFL
3.7
8.8
12.5
3.1
8.0
2.9
1.0
P
3.4
9.6
19.4
3.2
15.3
1.3
1.0
CR
3.4
3.0
4.1
7.1
3.0
14.8
3.3
PR
0.8
3.8
3.9
0.8
2.3
0.6
0.2
A
0.4
1.3
1.2
0.1
1.0
0.1
0.1
Total
26.3
63.4
95.1
31.8
60.6
33.6
13.7
Source: Sawicki, et al. 1962
-------
concentration is assumed (as in the case of BaP), the present ambi-
ent PAH concentration in the U.S. urban areas can be extrapolated
as 9.3 ng/m3. Although the concentration of BaP and some other PAH
might have decreased in past decades, the concentration of corenene
and some other PAH may not have maintained the same trend. This
could be due to the higher number of automobiles in current use.
Therefore, this 80 percent decrease figure may or may not be valid
for all PAH.
The concentrations of PAH in recent years in individual U.S.
cities have been determined by a number of authors. The lowest and
highest values of these determinations published during the period
1971-77 are shown in Table 14.
The exact amount of human PAH intake from all modes is diffi-
cult to determine because of the different modes of inhalation due
to smoking, occupational exposure, or exposure to ambient air.
Considering only exposure to ambient air, one needs an average PAH
concentration in air in order to determine the PAH intake through
inhalation. In the absence of national average data for PAH equiv-
alent to NASN data on national average BaP levels, the yearly aver-
age data for Los Angeles are used for the derivation of PAH intake
due to inhalation. These values are given in Table 15.
It can be seen from Table 15 that the yearly intake of total
PAH, carcinogenic PAH, and BaP through inhalation is 39.8 yg, 9.9
yg, and 1.9 yg, respectively. It should be recognized that these
data are based on the average ambient air concentration of one city
and probably will not reflect the true U.S. average. It is note-
worthy, however, that the total ambient PAH concentration of 10.9
C-34
-------
TABLE 14
PAH Concentration Range in U.S. Cities Determined
by Various Authors in Recent Years
o
i
u>
LF1
Compound
NA
A
BaA
PA
FL
BbFL
BjFL
BkFL
P
BaP
BeP
IP
CH
PR
BPR
CR
Concentration,
Range, ng/m
0.052
0.068
0.18
0.011
0.10
0.1
0.01
0.03
0.18
0.13
0.9
0.03
0.6
0.01
0.2
0.2
- 0.350
- 0.278a
- 4.6
- 0.340
- 4.1
- 1.6
- 0.8
- 1.3
- 5.2
- 3.2
- 4.6
- 1.34
- 4.8
- 1.2
- 9.2
- 6.4
Reference
Krstulovic, et al. 1977
Lunde and Bjjzirseth, 1977
Fox and Staley, 1976; Cordon, 1976
Krstulovic, et al. 1977
Fox and Staley, 1976; Hoffman
and Wynder, 1977
Gordon and Bryan, 1973
Gordon and Bryan, 1973
Gordon and Bryan, 1973
Fox and Staley, 1976; Gordon and
Bryan, 1973
Colucci and Begeman, 1971; Fox
and Staley, 1976
Gordon, 1976; Fox and Staley,
Gordon, 1976; Gordon and Bryan
Gordon, 1976; Fox and Staley,
Gordon and Bryan, 1973
Gordon and Bryan, 1973
Gordon and Bryan, 1973
1976
, 1973
1976
lThis Norwegian value is included because no recent U.S. data are available.
-------
TABLE 15
Average Ambient PAH Concentration in U.S. and
Daily Intake of PAH Through Inhalation3
o
1
PAH
Ambient cone. , ng/m
Inhalation intake/day, ng°
aThese values are based on
BaP
0.5
5.0
the study of Gordon,
Carcinogenic
PAHb
2.7
27.0
1976.
Total PAH
10.9
109.0
-------
ng/m derived u '-his work is very close to the earlier extrapo-
lated value of 9.3 ng/m .
Dermal
No direct information is available on the importance of dermal
absorption in total human exposure to PAH. PAH can be absorbed
across the skin by animals. For those humans exposed to only ambi-
ent levels of PAH, dermal absorption is not likely to be a signifi-
cant route of entry.
PHARMACOKINETICS
There are no data available concerning the pharmacokinetics of
PAH in humans. Nevertheless, it is possible to make limited as-
sumptions based on the results of animal studies conducted with
several PAH, particularly BaP. The metabolism of PAH in human and
animal tissues has been especially well-studied, and has contrib-
uted significantly to an understanding of the mechanisms of PAH-
induced cancer.
Absorption
The demonstrated toxicity of PAH by oral and dermal adminis-
tration (Smyth, et al. 1962) indicates that they are capable of
passage across epithelial membranes. The high lipid solubility of
compounds in this class supports this observation. Animal studies
with structurally-related PAH such as benzo(a)pyrene (BaP), chry-
sene, 7,12-dimethylbenz(a)anthracene (DMBA), benz(a)anthracene,
and 3-methylcholanthrene (MCA) confirmed that intestinal transoort
readily occurs, primarily by passive diffusion (Rees, et al. 1971).
In addition, there is ample evidence to indicate that benzo(a)py-
rene, and presumably other PAH, are easily absorbed through the
lungs (Kotin, et al. 1969; Vainio, et al. 1976).
C-37
-------
Distr ibution
The tissue distribution and accumulation of PAH have not been
studied in humans. It is known, however, that several PAH (e.g.,
benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, 3-methylcholan-
threne, phenanthrene) become localized in a wide variety of body
tissues following their absorption in experimental rodents (Kotin,
et al. 1969; Bock and Dao, 1961; Dao, et al. 1959; Flesher, 1967) .
Relative to other tissues, PAH localize primarily in body fat and
fatty tissues (e.g., breast) (Schlede, et al. 1970a,b; Bock and
Dao, 1961).
Disappearance of BaP from the blood and liver of rats follow-
ing a single intravenous injection was very rapid (Schlede, et al.
1970a). The concentration of BaP in the blood one minute after a 10
pg injection was 193 +_ 29 ng/ml; after five minutes concentration
of BaP in the blood was 31+1 ng/ml. Similarly, in the liver, the
half-time for BaP disappearance was about ten minutes. In both
blood and liver, however, the initial rapid elimination phase was
followed by a slower disappearance phase, lasting six hours or
more. In the same experiment, disappearance of BaP from the brain
was slower than from blood or liver, and the concentration of BaP
in fat increased during the six-hour observation period. Schlede,
et al. (1970a) concluded that a rapid equilibrium occurs for BaP
between blood and liver, and that rapid disappearance from the
blood is due to both metabolism and distribution into tissues.
This contention is supported by data (Schlede, et al. 1970b) show-
ing that pretreatment with BaP (which induces microsomal enzyme
activity) accelerates both the rate of BaP disappearance from all
C-38
-------
tissues and the excretion of BaP metabolites into the bile. The
ability of BaP to stimulate its own metabolism may have important
implications for human situations, where lifelong exposure to PAH
is known to occur.
With certain PAH, passage into the fetus following intragas-
tric or intravenous administration to pregnant rats has been vari-
able (Shendrikova and Aleksandrov, 1974).
Metabolism
In the past, the relative lack of chemical reactivity for
tumorigenic PAH has been puzzling in light of their dramatic bio-
logical effects. Early attempts to explain the carcinogenicity of
various PAH utilized physico-chemical calculations (Pullman and
Pullman, 1955). These early hypotheses were based on the assump-
tion that those regions of the molecule favoring substitution or
addition reactions would preferentially react with critical cellu-
lar target sites to initiate a carcinogenic transformation. This
concept, however, did not prove successful for PAH.
More recently it was learned that PAH are metabolized via
enzyme-mediated oxidative mechanisms to form reactive electro-
philes (Lehr, et al. 1978). For many of the PAH, certain "bioacti-
vated" metabolites are formed having the capability for covalent
interaction with cellular constituents (i.e., RNA, DNA, proteins)
and ultimately leading to tumor formation (see Effects section).
The obligatory involvement of metabolic activation for the
expression of PAH-induced carcinogenesis has prompted the investi-
gation of PAH metabolism in numerous animal models and human tis-
sues. From these studies has emerged an understanding of the gen-
>39
-------
eral mechanisms involved in PAH biotransformation. It is now known
that PAH are metabolized by the cytochrome P-450-dependent micro-
somal mixed-function oxidase (MFO) system, often designated aryl
hydrocarbon hydroxylase (Conney, 1967; Marquardt, 1976; Sims, 1976;
Gelboin, et al. 1972). The activity of this enzyme system is read-
ily inducible by exposure to chemicals and is found in most mamma-
lian tissues, although predominantly in the liver (Bast, et al.
1976; Chuang, et al. 1977; Andrews, et al. 1976; Cohn, et al. 1977;
Wiebel, et al. 1975; Grundin, et al. 1973; Zampaglione and Manner-
ing, 1973). The MFO system is involved in the metabolism of endo-
genous substrates (e.g., steroids) and the detoxification of many
xenobiotics. Paradoxically, however, the MFO system also catalyzes
the formation of reactive epoxide metabolites from certain PAH,
possibly leading to carcinogenesis in experimental mammals (Sims
and Grover, 1974; Selkirk, et al. 1971, 1975a; Sims, 1976; Thakker,
et al. 1977; Levin, et al. 1977a; Lehr, et al. 1978; see Effects
section). A second microsomal enzyme, epoxide hydrase, converts
epoxide metabolites of PAH to vicinal glycols, a process which may
also play a critical role in carcinogenic bioactivation. Figur.e 1
presents a schematic representation of the various enzymes involved
in activation and detoxification pathways for BaP. At present this
also appears to be representative of the general mechanism for PAH
metabolism.
A discussion of the metabolism of PAH in mammalian species,
including man, is best approached by examining in detail the chemi-
cal fate of the most representative and well-studied compound in
the PAH class, namely BaP. The metabolism of BaP has been exten-
0-40
-------
sr,
(ENOOPLASMIC
RETICULUM)
GLUTATHIONE
«
TRANSFSRASE
iCYTOSOL)
CYTOCHROME P--JSO
MIXED - FUNCTION OXIOASc (MFOI
SiP OXIDES
•*• 8uP PHENOLS
EPOXIOE
HYORASc
IENOOPUASMIC
RETICULUM)
B.iP QUIMONSS
sulface?
;:lucuronides
8..P OIHYOROOIOLSiPROPOSED PROXIMATE CARCI.\OG = '
MFO
UDP-GLUCURQWOSVI. TRAKSFcRASE
(EiVOOPI.AS.%:iC
B.tP niOL EPOXIOES
(PROPOSED ULTIMATE
H20-SOLUBLE CONJUGATES
(UcTOXIFICATION PROCUCTS)
FIGURE 1
Enzymatic Pathways Involved in the Activation
and Detoxification of BaP
C-41
-------
sively studied in rodents, and the results of these investigations
provide useful data which can be directly compared to and contrast-
ed with the results of more limited studies employing human cells
and tissues. Therefore, separate discussions are based upon the
available experimental evidence regarding PAH metabolism in gener-
al, and BaP metabolism in particular, in both animals and man.
Metabolism of PAH in Animals: The metabolites of PAH produced
by microsomal enzymes in mammals can arbitrarily be divided into
two groups on the basis of solubility. In one group are those
metabolites which can be extracted from an aqueous incubation mix-
ture by an organic solvent. This group consists of ring-hydroxy-
lated products such as phenols and dihydrodiols (Selkirk, et al.
1974; Sims, 1970), and hydroxymethyl derivatives of those PAH hav-
ing aliphatic side chains, such as 7,12-dimethylbenz(a)anthracene
(Boyland and Sims, 1967) and 3-methylcholanthrene (Stoming, et al.
1977; Thakker, et al. 1978). In addition to the hydroxylated
metabolites are quinones, produced both enzymatically by microsomes
and non-enzymatically by air oxidation of ohenols. Labile metabol-
ic intermediates such as epoxides can also be found in this frac-
tion (Selkirk, et al. 1971, 1975a,b; Sims and Grover, 1974; Yang,
et al. 1978) .
In the second group of PAH metabolites are the water soluble
products remaining after extraction with an organic solvent. Many
of these derivatives are formed by reaction (conjugation) of hy-
droxylated PAH metabolites with glutathione, glucuronic acid, and
sulfate. Enzyme systems involved in the formation of water-soluble
metabolites include glutathione S-transferase, UDP-glucuronosyl
C-42
-------
transferase, and sulfotransferases (Bend, et al. 1976; Jerina and
Daly, 1974; Sims and Grover, 1974). Conjugation reactions are be-
lieved to represent detoxification mechanisms only, although this
group of derivatives has not been rigorously studied.
The metabolite profile of BaP which has recently been expanded
and clarified by the use of high pressure liquid chromatography is
depicted in Figure 2. This composite diagram shows three groups of
positional isomers, three dihydrodiols, three quinones, and several
phenols. The major BaP metabolites found in microsomal incubations
are 3-hydroxy-BaP, 1-hydroxy-BaP, 7-hydroxy-BaP, and 9-hydroxy-
BaP. The BaP-4,5-epoxide has been isolated and identified as a
precursor of the BaP-4,5-dihydrodiol. Other studies indicate that
epoxides are the precursors of the 7,8-dihydrodiol and 9,10-dihy-
drodiol as well. Considerable evidence has recently become avail-
able which implicates the diol epoxide, 7/^,8?T-dihydro^7,8-dihy-
droxybenzo(a)pyrene-9,lO'X-oxide, as an ultimate carcinogen de-
rived from BaP (Jerina, et al. 1976; Kapitulnik, et al. 1977b,
1978a,b; Levin, et al. 1976a,b; Yang, et al. 1978).
Since the resonance properties of PAH make ring openings dif-
ficult, enzymatic attack in the microsomes functions to open double
bonds and add an oxygen-containing moiety, such as a hydroxyl
group, to give it more solubility in aqueous media (e.g., urine)
and thus facilitate removal from the body. In the formation of
metabolic intermediates by oxidation mechanisms, relatively stable
PAH are converted to unstable products (i.e., epoxides). Thus,
nucleophilic attack of this reactive intermediate, through the for-
mation of a transient carbonium ion, would be greatly enhanced.
C-43
-------
o
i
1 9,10-epox 9, 10-diol
[s.lO-diol
[7,8,9,10
)— [2,3-epwl''
~OM L J
3-OH
6-PHENOXY
RADICAL
BENZO(a)PYRENE
/I
6-OH-Me
PJP
010]
-7,8 epox|
-lelrol]
7,8-epox
9,10-epox
6-OH
HO
^
"Q&
OH
7 8-epox
7 8-diol
7-OH
I
CONJUGATES
BOUND MACROMOLECULES
DNA
RNA
PROTEIN
FIGURE 2
Metabolites of Benzo(a)pyrene
-------
Arylations of this type are common to many classes of carcinogenic
chemicals. Therefore, the microsomal cytochrome P-450-containing
MFO system and epoxide hydrase play a critical role in both the
metabolic activation and detoxification of many PAH.
Various forms of liver microsomal cytochrome P-450 can be iso-
lated from animals treated with different enzyme inducers (Wiebel,
et al. 1973; Nebert and Felton, 1976; Conney, et al. 1977a,b; Lu,
et al. 1978). Moreover, the metabolite profiles of BaP can be
qualitatively altered depending on the type of cytochrome P-450
present in the incubation mixture (Lu, et al. 1976; Wiebel, et al.
1975). This observation has important implications in considering
the carcinogenic action of certain PAH toward tissues from animals
of different species, sex, age, nutritional status, and exposure to
enzyme-inducing chemicals. Limited evidence is also available
indicating that multiple forms of epoxide hydrase exist among ani-
mal species, which may also influence the pattern of PAH metabolism
with respect to carcinogenic bioactivation (Lu, et al. 1978) .
Comparative Metabolism of PAH in Animals and Man: An impor-
tant consideration in evaluating the health hazards of PAH is
whether metabolism in various animal tissues and species is indica-
tive of the pattern of PAH metabolism in the target organs of
humans. Moreover, it is essential to determine whether differences
occur in the metabolism of PAH by: (a) different tissues in the
same animal; and (b) different animals of the same species.
Numerous studies have shown that the qualitative and quantita-
tive differences exist in the metabolism of BaP by different tis-
sues and animal species (Sims, 1976; Leber, et al. 1976; Wang, et
C-45
-------
al. 1976; Pelkonen, 1976; Kimura, et al. 1977; Selkirk, et al.
1976) . For the most part, however, interspecies extrapolations of
qualitative patterns of PAH metabolism appears to be a valid prac-
tice. On the other hand, marked differences in patterns of tissue-
specific metabolism may prevent the reliable extrapolation of data
from hepatic to extrahepatic (i.e., target organ) tissues. These
differences may also exist in human tissues (Conney, et al. 1976) .
Freudenthal, et al. (1978) recently examined the metabolism of
BaP by lung microsomes isolated from the rat, Rhesus monkey, and
man. Metabolite profiles obtained by high pressure liquid chroma-
tography are shown in Figure 3. Their results confirmed previous
observations regarding the existence of considerable individual
variation in BaP metabolism among samples from the same species.
In addition, it was apparent that qualitative and quantitative
interspecies variation also existed (Table 16). Nevertheless, the
qualitative differences between man and the other animal species
were by no means dramatic, and probably do not compromise the
validity of extrapolations concerning PAH metabolism.
The metabolite pattern obtained for BaP in human lymphocytes
is similar to that obtained with human liver microsomes (Selkirk,
et al. 1975b), and human lymphocytes (Booth, et al. 1974). How-
ever, in cultured human bronchus (24 hrs) and pulmonary alveolar
macrophages an absence of phenols (i.e., 3-hydroxy-BaP) and paucity
of quinones were observed (Autrup, et al. 1978) . Instead, a rela-
tive abundance of the trans-7,8-diol metabolite of BaP was demon-
strated. This result is noteworthy in light of the possiblity that
the 7,8-diol is capable of further oxidative metabolism to an ulti-
C-46
-------
_• 26
.. 24
U9
UJ
H-
20
g
>-
iu
O
16
12
D
BP
3.6-
DIONE
1.6-
DIONE
6,12-DIONE
3 - OH - BP
0 20 40 60 80 100 120 140 160 180 200 220
FRACTION
20 40 60
80 100 120 140 160 180 200 220
FRACTION
30
28
24
20
16
12
I ' I
4.5-
DIOL
3,6-
QUINONE
1.6- I 6.12-
OUINONE R .QUINONE
I ' I
3 - OH - BP
BP
20 40 60 80 100 120 140 160 180 200 220 240
FRACTION
FIGURE 3
Comparative Metabolism of Benzo(a)pyrene by Lung Microsomes
from Rat, Rhesus Monkey, and Human
Source: Freudenthal, et al. 1978
C-47
-------
TABLE 16
Metabolite Percentages of BP Metabolites from Rat, Rhesus, and Human Lung Microsomal Assays*
O
I
*>.
00
Metabolite percentages
(pmoles metabolite/pinoles total metabolites x 100)
Metabolite
Pre-9, 10
9, lO-Diol
A
U
-------
mate carcinogenic form of BaP. It is not known whether a longer
incubation period would have changed the pattern of metabolite for-
mation.
Excretion
There is no direct information available concerning the excre-
tion of PAH in man. Limited inferences can be drawn from animal
studies with PAH, however.
As long ago as 1936, researchers recognized that various PAH
were excreted primarily through the hepatobiliary system and the
feces (Peacock, 1936; Chalmers and Kirby, 1940). However, the rate
of disappearance of various PAH from the body, and the principal
routes of excretion are influenced both by structure of the parent
compound and the route of administration (Heidelberger and Weiss,
1951; Aitio, 1974a,b). Moreover, the rate of disappearance of a
PAH [i.e., benzo(a)pyrene] from body tissues can be markedly stimu-
lated by prior treatment with inducers of microsomal enzymes fe.g.,
benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, 3-methylcholan-
threne, chrysene] (Schlede, et al. 1970a,b). Likewise, it has been
shown that inhibitors of microsomal enzyme activity, such as para-
thion and paraoxon, can decrease the rate of BaP metabolism in cer-
tain animal tissues (Weber, et al. 1974). From the available evi-
dence concerning excretion of PAH in animals, it is apparent that
extensive bioaccumulation is not likely to occur.
C-49
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EFFECTS
Acute, Subacute, and Chronic Toxicity
The potential for PAH to induce malignant transformation domi-
nates the consideration given to health hazards resulting from ex-
posure. This is because overt signs of toxicity are usually not
produced by many PAH until the dose is sufficient to oroduce a high
tumor incidence. Although the emphasis on carcinogenic!ty is cer-
tainly justified when dealing with public health issues concerning
PAH, one must recognize that nonneoplastic lesions may also result
from environmental and occupational contact. Such effects can be
seen with low doses of carcinogenic PAH and with those compounds
which possess no tumorigenic activity. Numerous PAH have demon-
strated carcinogenic activity when administered to laboratory ani-
mals by various routes of administration. However, since many PAH
have not been tested for biologic activity it is not possible to
list all carcinogenic PAH. A summary of those PAH which are thus-
far known to be carcinogenic in animals is provided in surveys pub-
lished by the U.S. Public Health Service (Hartwell and Shubik,
1951; Shubik and Hartwell, 1957, 1969; Tracor Jitco, Inc., 1974,
1976) . Since only a small percentage of PAH compounds are known to
be carcinogenic, measurements of total PAH (i.e., the sum of all
multiple fused-ring hydrocarbons having no heteroatoms) cannot be
equated with carcinogenic risk. When the term "total PAH" is used
it is necessary in each case to specify the compounds being con-
sidered .
As long ago as 1937, investigators knew that carcinogenic PAH,
produced systemic toxicity as manifested by an inhibition of body
C-50
-------
growth in rats and mice (Haddow, et al. 1937). Tissue damage re-
sulting from the administration of various PAH to experimental ani-
mals is often widespread and severe, although selective organ de-
struction may occur (e.g., adrenal necrosis, lymphoid tissue dam-
age). Few investigators, however, have attempted to ascertain the
molecular mechanism of PAH-induced cytotoxicity. Nevertheless,
current opinion favors the concent that normally proliferating tis-
sues (intestinal epithelium, bone marrow, lymphoid organs, testis)
are preferred targets for PAH, and this susceptibility may be due
to a specific attack on DNA of cells in the S phase of the mitotic
cycle (Philips, et al. 1973). Additional factors which may have an
important bearing on the adverse effects resulting from PAH expo-
sure are primary and secondary alterations in enzyme activity and
immunologic competence. Moreover, these toxicant-induced changes
may play an important role in the eventual induction of neoplasia.
Target organs for the toxic action of PAH are diverse, due
partly to extensive distribution in the body and also to the selec-
tive attack by these chemicals on proliferating cells. Damage to
the hematopoietic and lymphoid systems in experimental animals is a
particularly common observation. Yasuhira (1964) described severe
degeneration of the thymus and marked reduction in weight of the
spleen and mesenteric lymph nodes of CF-^ Swiss and C57BL mice given
a single intraperitoneal injection of MCA (0.3 to 1.0 mg) between
12 hours and 9 days after birth. Degeneration of young cells in the
bone marrow and retardation of thyroid gland development were also
noted. Newborn mice were highly susceptible to the toxic effects
of MCA, with many animals dying from acute or chronic wasting dis-
-------
ease following treatment. Among surviving CF-, mice, numerous thy-
momas eventually developed; none were evident, however, in C57BL
mice despite serious thymic damage.
DMBA is well-known for its effects on the bone marrow and lym-
phoid tissues. With single feedings (112 or 133 mg/kg body weight)
to female Sprague-Dawley rats, age 50 days, DMBA induced nancyto-
penia by causing a severe depression of hematopoietic and lymphoid
precursors (Cawein and Sydnor, 1968). Maturation arrest occurred
at the proerythroblast levels; no injury to the stem cells or the
formed elements in the peripheral blood was evident. The fact that
only the more rapidly proliferating hematopoietic elements were
vulnerable to attack by DMBA led the authors to suggest that inhi-
bition of DNA replication may be involved in the toxicologic re-
sponse.
Philips and coworkers (1973) provided strong support for the
argument that DMBA-induced cytotoxicity is mediated via an inter-
action with DNA. Female Sprague-Dawley rats receiving 300 mg/kg of
body weight of DMBA orally and male rats receiving an intravenous
injection of 50 mg/kg of body weight of DMBA displayed iniury to
the intestinal epithelium, extreme atrophy of the hematopoietic
elements, shrinkage of lymphoid organs, agranulocytosis, lympho-
penia, and progressive anemia. Mortality among rats receiving DMBA
by gastric intubation (females) was about 65 percent. In rats
given 50 mg/kg of body weight of DMBA intravenously, incorporation
14
of C-labeled thymidine into DNA of small and large intestine,
spleen, bone marrow, cervical lymph nodes, thymus, and testis was
significantly inhibited. This inhibition was as high as 90 percent
C-52
-------
in several organs at six hours which indicated a strong inhibition
of DNA synthesis. Consequently, the authors postulated that DNA in
S phase cells is particularly susceptible to DMBA attack. This
phenomenon probably applies for other carcinogenic PAH as well.
Another lesion, characteristic of that produced by X-rays, is
the severe testicular damage induced by DMBA in rats (Ford and
Huggins, 1963). Single intravenous injections of DMBA (0.5 to 2.0
mg) given to adolescent (25 days of age) rats caused transient de-
generative changes in the testis which were most evident 38 to 40
days after treatment. Essentially the same effects were produced
in adult rats, age 60 days, given DMBA orally (20 mg) and intrave-
nously (5 mg). Lesions of the testes were highly specific and in-
volved destruction of spermatogonia and resting spermatocytes, both
of which are the only testicular cells actively synthesizing DNA.
Neither the remaining germinal cells nor the interstitial cells
were damaged by DMBA. Surprisingly, no testicular damage was pro-
duced by single feedings of BaP (100 mg), MCA (105 mg), or 2-aceto-
aminophenanthrene (40 mg).
It is well known that the application of carcinogenic polycy-
clic hydrocarbons to mouse skin leads to the destruction of seba-
ceous glands, hyperplasia, hyperkeratosis, and even ulceration
(Bock, 1964). Sebaceous glands are the skin structures most sensi-
tive to polycyclic hydrocarbons, and assay methods for detection of
carcinogens have been based on this effect. Although a good corre-
lation can be obtained between carcinogenic activity and sebaceous
gland suppression for many PAH [e.g., MCA, DMBA[ BaP, DBA, benz(a)-
anthracene], such an effect is neither necessary nor sufficient for
C-53
-------
carcinogenesis. However, workers exposed to PAH-containing materi-
als such as coal tar, mineral oil, and petroleum waxes are known to
show chronic dermatitis, hyperkeratoses, etc. (Hueper, 1963; NAS,
1972) , though the possible significance of these skin disorders to
human cancer is not known.
In female animals, ovotoxicity has been reported to result
from the administration of PAH. DMBA was shown to cause the de-
struction of small oocytes and to reduce the numbers of growing and
large oocytes after oral administration to mice (Kraup, 1970) .
More recently a report was published that destruction of primordial
oocytes in mice by injection of MCA was correlated with the genetic
capability for PAH-induced increases in ovarian aryl hydrocarbon
hydroxylase activity (Mattison and Thorgeirsson, 1977). Thus, the
ovarian metabolism of PAH and ovotoxicity are apparently linked.
A toxic reaction which is apparently unique to DMBA is the
selective destruction of the adrenal cortex and induction of adre-
nal apoplexy in rats (Boyland, et al. 1965). Adrenal apoplexy,
increased adrenal gland weight, and increased adrenal hemoglobin
content were induced in female Sprague-Dawley rats by a single
intragastric dose of 30 mg DMBA. The same amount of adrenal damage
could be produced by a 5 mg dose of the principal DMBA oxidative
metabolite, 7-hydroxymethol-12-methylbenz(a)anthracene. Other
DMBA metabolites produced no adrenal damage, thus indicating that a
specific reactive intermediate may be responsible for this phe-
nomenon.
Repeated injections of benz(a)anthracene derivatives to mice
and rats have produced gross changes in the lymphoid tissues.
C-54
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Hoch-Ligeti (1941) administered DBA, benz(a)anthracene, and an-
thracene to mice in weekly subcutaneous injections for 40 weeks.
Analysis of lymph glands removed at weekly intervals showed an in-
crease of reticulum (stem) cells and an accumulation of iron in all
treated groups of animals. Lymphoid cells were reduced and lymph
sinuses dilated in all groups, although these effects were more
common in mice receiving DBA. The weights of the spleens in mice
treated with DBA were significantly reduced in comparison to con-
trols and those animals receiving benz(a)anthracene or anthracene.
A more detailed study on the effects of repeated administra-
tion of DBA on lymph nodes of male rats was reported in 1944 (Las-
nitzki and Woodhouse, 1944). Subcutaneous injections given five
times weekly for several weeks caused normal lymph nodes to undergo
hemolymphatic changes. These changes are characterized by the pre-
sence of extravascular red blood cells in the lymph spaces and the
presence of large pigmented cells. These changes were not observed
by Hoch-Ligeti (1941) in mice, but could be Produced in rats by BaP
and MCA in addition to DBA. The noncarcinogen, anthracene, on the
other hand, did not produce as dramatic a change in the lymph nodes
of rats.
In light of the concern over PAH-induced neoplasms of the
respiratory tract,' an understanding of early pathological altera-
tions and preneoplastic "lesions in this tissue has particular sig-
nificance .
In a study conducted by Reznik-Schuller and Mohr (1974), BaP-
induced damage to the bronchial epithelium of Syrian golden ham-
sters was examined in detail using tissue sections. Animals were
C-55
-------
treated intratracheally with 0.63 mg BaP (total dose) dispersed in
a solution of saline, dodecylsulfate, Tris-HCl, and EDTA once week-
ly for life. Animals were serially sacrificed at weekly intervals
following the first month of treatment, and sections of the bronchi
were examined microscopically. In the first animals sacrificed,
minimal focal cell proliferation in the area of the basement mem-
brane was evident in the bronchial epithelium. By 7 weeks, cyto-
plasmic vacuolization of both goblet and ciliated cells had oc-
curred. Epithelial and basal cell proliferation continued for sev-
eral weeks and led to the formation of three-to four-layered hyper-
plastic regions by the llth week. Epithelial cells began to pene-
trate through the basement membrane by the 12th week, and within
two or more weeks the bronchial epithelium began to grow continu-
ously into the surrounding lung tissues. Microscopic bronchogenic
adenomata had developed by the 20th week. These tumors consisted
primarily of ciliated cells and goblet cells, with only a few basal
cells present. The apparently small amount of basal cell prolifer-
ation may have been the reason why squamous metaplasia was not ob-
served by the time the experiment had ended after 21 weeks. Squa-
mous metaplasia and keratinization were found in the trachea, but
not in the bronchi, after 21 weeks of treatment. Although these
investigators found no increase in the number of alveolar macro-
phages, others have reported numerous alveolar macrophage responses
in BaP-treated hamsters as well as focal areas of accumulated
macrophages containing a yellow pigment having unknown biological
significance (Henry, et al. 1973; Saffiotti, et al. 1968).
C-56
-------
Epithelial proliferation and cell hyperplasia in the absence
of necrosis and/or marked inflammation is a common observation in
the tracheobronchial mucosa of animals directly exposed to carcino-
genic PAH. This phenomenon was shown with repeated exposures of
DMBA, BaP, and dibenzo(a,i)pyrene in hamsters (Reznik-Schuller and
Mohr, 1974; Saffiotti, et al. 1968; Stenback and Sellakumar,
1974a,b).
Numerous investigators have demonstrated that carcinogenic
PAH can produce an immunosuppressive effect. This effect was first
observed by Malmgren, et al. (1952) using high doses of MCA and
DB(a,h)A in mice. Subsequent studies established that single car-
cinogenic doses of MCA, DMBA, and BaP caused a prolonged depression
of the immune response to sheep red blood cells (Stjernsward, 1966,
1969). Noncarcinogenic hydrocarbons such as benzo(e)pyrene and
anthracene reportedly had no immunosuppressive activity. In a re-
cent review on immunosuppression and chemical carcinogenesis, sub-
stantial evidence was presented to indicate that the degree of
immunosuppression was correlated with carcinogenic potency for PAH
(Baldwin, 1973). Both cell-mediated and humoral immune reactions
are affected by PAH.
Synergism and/or Antagonism
It is well-known that the development of PAH-induced tumors in
epithelial and non-epithelial tissues can be altered by: (1) com-
ponents in the diet, (2) inducers and inhibitors of microsomal
enzymes, (3) other co-administered noncarcinogenic or weakly car-
cinogenic chemicals, and (4) the vehicle used to deliver a carcino-
genic PAH to experimental animals. These factors tend to compli-
C-57
-------
cate the extrapolation of animal dose-response data to human situa-
tions. On the other hand, these observations in animals reinforce
the belief that similar interactions occur with regard to the
action of PAH in humans.
Early studies conducted by Falk and coworkers (1964) indicated
that the carcinogenic effect of BaP on subcutaneous injection in
mice could be markedly inhibited by the simultaneous administration
of various noncarcinogenic PAH. Similarly, they showed that neu-
tral extracts of particulate air pollutant fractions also produced
inhibitory effects on BaP-induced tumorigenesis. However, when
Pfeiffer (1973, 1977) conducted similar studies with BaP and DBA in
the presence of 10 noncarcinogenic PAH, no inhibitory effect was
evident. Moreover, an increased tumor yield resulted from injec-
tion of mixtures containing increasing amounts of the components.
This effect, however, was less dramatic than if BaP were adminis-
tered alone, and it paralleled the dose-response curve for DBA act-
ing singly.
Many studies on cocarcinogenesis have been concerned with the
identification of tumor accelerating substances present in ciga-
rette smoke. These compounds are generally tested for cocarcino-
genic activity by repeated application to mouse skin together with
low doses of BaP. A positive response would be obtained in cases
where the tumor yield of the combination exceeds that produced by
either agent alone at the same doses. Van Duuren and coworkers
(1973, 1976) established that a pronounced cocarcinogenic effect
could be obtained with catechol and the noncarcinogens, oyrene,
BeP, and benzo(g,h,i)perylene. Doses of 12, 15, 21, and 2,000 yg
C-58
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of these compounds, respectively, were applied three times a week
for 52 weeks to female ICR/Ha Swiss mice. Each animal also re-
ceived 5 yg of BaP in 0.1 ml acetone with each dose of test sub-
stance. Although phenol has been regarded as a tumor-promotor in
the two-stage carcinogenesis sytem (Van Duuren, 1976), this com-
pound has a slight inhibitory effect on BaP carcinogenesis when
administered in combination. These results, therefore, indicated
that tumor-promotors and cocarcinogens may not have the same mode
of action, and that the two terms should not be used interchange-
ably. Other PAH (e.g., fluoranthene, pyrene, pyrogallol) also pos-
sess cocarcinogenic activity but have no tumor-promoting activity
(Van Duuren, 1976). Additional studies by Schmeltz, et al. (1978)
established that most of the naphthalenes found in cigarette smoke
(250 yg, three times a week) have an inhibitory effect on skin
tumorigenesis as induced by BaP (3 ug, three times a week). On the
other hand, several of the alkylnaphthalenes tested (dimethyl-,
trimethyl-, tetramethyl-) enhanced the carcinogenic activity of BaP
on mouse skin.
Numerous investigators have shown that antioxidants are effec-
tive inhibitors of PAH-induced tumor development. This action has
been demonstrated with selenium (Shamberger, 1970; Shamberger and
Rudolph, 1966; Riley, 1969), dl- o<-tocopherol (vitamin E) (Sham-
berger, 1970; Shamberger and Rudolph, 1966), and ascorbic acid
(Shamberger, 1972) in mice treated with DMBA and croton oil. The
carcinogenic action of MCA has been reduced by tocooherol-rich
diets in rats and mice (Jaffe, 1946; Haber and Wissler, 1962). The
antioxidant food additives butylated hydroxytoluene (BHT), ethoxy-
C-59
-------
quin, and butylated hydroxyanisole (BHA) have inhibited lung,
breast, and gastric tumor formation induced in rats and mice by
various carcinogens in the diet (Wattenberg, 1972, 1973; Watten-
berg, et al. 1976). The sulfur-containing antioxidants (disulfur-
am, dimethyldithiocarbamate, and benzyl thiocyanate) inhibited
DMBA-induced mammary cancer in rats when they were added to the
diet; in the mouse, disulfuram prevented the formation of forestom-
ach tumors induced by BaP in the diet, but had no effect on BaP-
induced pulmonary adenoma (Wattenberg, 1974). The agricultural
herbicide, maleic hydrazide, and its precursor, maleic anhydride,
can inhibit the initiating activity of DMBA in the mouse skin two-
stage carcinogenesis system (Akin, 1976).
Rahimtula and coworkers (1977) examined the abilities of sev-
eral antioxidants to affect BaP hydroxylation by rat liver micro-
somal mixed-function oxidases. Their results indicated that anti-
oxidants can markedly inhibit BaP hydroxylation by an apparently
direct action on microsomal oxidation mechanisms. Furthermore, all
of the antioxidants tested reduced the bacterial mutagenicity of
BaP in the presence of rat liver microsomes and cofactors. The
authors suggested that antioxidants may exert their protective
effect In vivo by inhibiting the formation of carcinogenic inter-
mediates from PAH. This conclusion, however, seems to conflict
with data indicating that inducers of increased BaP hydroxylase
activity can also inhibit tumor formation (Wattenberg and Leong,
1970). However, flavones are also inhibitors of BaP metabolism _in
vitro, thereby indicating that their specific effects depend uoon
how and where they are used. These investigators found that sev-
C-60
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eral synthetic and naturally occurring flavones when incorporated
in the diet (3 to 5 mg/g) or applied to the skin caused a profound
increase in BaP hydroxylase activity in the small intestine and
skin, respectively. In addition, pulmonary adenoma formation re-
sulting from oral administration of BaP was totally prevented, and
skin tumors initiated by BaP application to mice were significantly
reduced ( ) 50 percent) by treatment with the synthetic flavone,
P -naphthoflavone. Pulmonary tumor formation was also reduced 50
percent by incorporation of the naturally occurring flavone, quer-
cetin pentamethyl ether, into the diet. Sullivan and coworkers
(1978) recently demonstrated that BHA, BHT, phenothiazine, pheno-
thiazine methosulfate, and ethoxyquin all can reduce the quantita-
tive yield of BaP metabolites in incubations with rat liver micro-
somes. The possibility that only specific components of the drug
metabolizing enzyme system may be induced by antioxidants has not
been fully explored.
In addition to flavones, other naturally occurring compounds
have exhibited protective effects against PAH-induced tumor forma-
tion. Retinoids have clearly been shown to play a role in reducing
carcinogen-induced tumors (Nettesheim, et al. 1975; Cone and Net-
tesheim, 1973; Chu and Malmgren, 1965; Smith, et al. 1975). Net-
tesheim and Williams (1976) recently examined whether inadequate
vitamin A consumption may predispose individuals to carcinogenesis,
or whether increased vitamin A intake exerts a protective effect
against neoplasia. They found that a diet deficient in vitamin A
increased the formation of MCA-induced metaplastic lung nodules in
female Fisher 344 rats, even though adequate amounts of the vitamin
C-61
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were stored in the liver. On the other hand, moderate amounts of
the vitamin A added to the diet markedly reduced the development of
MCA-induced lesions of the lung. High doses of the vitamin qiven
intragastrically provided no additional protection, however.
Further studies on naturally occurring antineoclastic com-
pounds were recently reported by Wattenberg (1977). Benzyl iso-
thiocyanate and phenethyl isothiocyanate, both found in cruciferous
plants such as cabbage, brussel sprouts, cauliflower, etc., inhib-
ited DMBA-induced mammary cancer in Sprague-Dawley rats. When
added to the diet together with DMBA, these compounds inhibited the
development of forestomach tumors and pulmonary adenomas in female
ICR/Ha mice. Similar anticarcinogenic actions were obtained when
BaP was incorporated into the diet. These results lead to inter-
esting speculation regarding the role and importance of diet in
human susceptibility to environmental carcinogens. In cases where
dietary constituents can alter the metabolism of xenobiotics such
as PAH, then the anticarcinogenic effect may result from an altera-
tion of steady state levels of activated versus detoxified metabo-
lites.
Studies have shown that not only can specific substances in
the diet affect the response to Carcinogens, but decreased protein
content in the diet may also decrease the activation of carcinogens
(Czygan, et al. 1974) . The feeding of protein-deficient diets to
male mice decreased liver weights and reduced cytochrome P-450 con-
tent in the total liver. Diets deficient in both protein and cho-
line produced even further reductions in liver weight and cyto-
chrome P-450 content. Liver microsomes isolated from these animals
C-62
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displayed a decreased ability to activate dimethylnitrosamine to a
mutagen in the Ames Salmonella test system, which paralleled the
reduction in cytochrome P-450 content produced by the diet. Con-
versely, the inactivation of the direct-acting (ultimate) carcino-
gen, N-methyl-N'-nitro-N-nitrosoguanidine, was reduced in liver
microsomes from mice receiving a protein-deficient diet.
In humans fed charcoal-broiled beef, the metabolism of the
drug phenacetin was enhanced; in pregnant rats a similar diet stim-
ulated the activity of AHH in the placenta and liver (Conney, et
al. 1977a,b). Further studies showed that high-protein diets en-
hanced the metabolism of antipyrene and theophylline in man, while
a high-carbohydrate diet depressed the rate of metabolism of these
drugs. Additional agents in man's environment which inhibit AHH
activity include certain organophosphate pesticides, piperonyl
butoxide, carbon tetrachloride, ozone, carbon monoxide, nickel car-
bonyl, and nickel, tin, cobalt, and other metals (Conney, et al.
1977a,b).
Teratogenicity
No information is available concerning the possible teratogen-
ic effects of PAH in man. Furthermore, only limited data are
available regarding the teratogenic effects of PAH in experimental
animals.
BaP had little effect on fertility or the developing embryo in
several mammalian and nonmammalian species (Rigdon and Rennels,
1964; Rigdon and Neal, 1965). On the other hand, DMBA and its
hydroxymethyl derivatives apparently are teratogenic in the rat
(Currie, et al. 1970; Bird, et al. 1970). However, DMBA is not gen-
erally regarded as an environmental contaminant.
C-63
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Mutagenicity
No reliable way presently exists to measure whether PAH may
induce heritable mutations in humans. However, the concept that
carcinogenesis is an expression of an alteration in the genetic
material of a cell (i.e., somatic mutation) implies that a formal
relationship exists between mutagenesis and carcinogenesis (Nery,
1976; Miller, 1978). The results obtained with several in. vitro
mutagenesis test systems, particularly the Ames Salmonella typhi-
murium assay, support the belief that most carcinogenic chemicals
are mutagenic as well. For PAH, the Ames assay has been very effec-
tive in detecting those parent structures and their biotransforma-
tion products which possess carcinogenic activity (McCann, et al.
1975; Teranishi, et al. 1975; McCann and Ames, 1976; Sugimura, et
al. 1976; Wislocki, et al. 1976b; Wood, et al. 1976a;' Tokiwa, et
al. 1977; Brookes, 1977). The Ames assay, however, may not be 100
percent effective in detecting all PAH carcinogens, nor does the
assay provide a reliable quantitative measure of carcinogenic
potency or tumor-initiating activity.
The availability of Salmonella typhimurium strains for the
detection of chemically induced mutations and the use of microsomal
preparation to provide metabolic activation has made possible an
investigation of the mechanisms of PAH-induced mutagenesis. In
particular, an exhaustive survey of the mutagenicity of all the
possible oxidative metabolites of BaP has helped to confirm the
belief that diol epoxide intermediates are the ultimate mutagens/
carcinogens derived from PAH (Jerina, et al. 1976; Wood, et al.
1976a,b; Wislocki, et al. 1976a,b; Thakker, et al. 1976; Levin, et
al. 1977a,b). These results are summarized in Table 17.
C-64
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TABLE 17
Comparison of Inherent Mutagenic Activity of Thirty BaP Derivatives in
Salmonella typhimurium TA98 and in Chinese Hamster V79 Cells9'
o
i
a\
ui
Compound
Relative % Activity
Strain TA98
BaP 1,6-, 3,6-, 6,12-, 4,5-, 11,12-quinone
BaP 4,5-, 7,8-, 9,10-, 11,12-dihydrodiol
BaP
V79
Diol epoxide-1
Diol epoxide-2
H4 9,10-epoxide
H4 7,8-epoxide
BaP 4,5-oxide
BaP 7,8-oxide
BaP 9,10-oxide
BaP 11,12-oxide
6-HOBaP
12-HOBaP
1-HOBaP
3-HOBaP
2-, 4-, 5-, 7-, 8-, 9-, 10-, 11-HOBaP
100
35
95
10
20
1
1
0.
5
1.
0.
0.
-------
Further examination of the mutagenic activity of PAH and their
derivatives has been conducted in mammalian cell culture systems.
These systems operate with concentrations of test compounds which
are lower than those used in the Ames assay. This work has been
conducted primarily with Chinese hamster cell lines, either V79
cells derived from male lung tissue or CHO cells derived from the
ovary. These cells, however, do not possess a microsomal enzyme
system and thus co-cultivation with lethally irradiated rodent
embryo cells which retain metabolic activity is required for test-
ing of PAH.
Using this system, Huberman and Sachs (1974, 1976) demonstrat-
ed that a number of carcinogenic PAH produced forward mutations
involving three genetic markers: (1) ouabain resistance; (2) tem-
perature sensitivity; and (3) 8-azaguanine resistance. Noncarci-
nogenic PAH such as BeP, phenanthrene, and pyrene were not mutagen-
ic. In addition, studies by Huberman indicated that a correlation
could be shown between the degree of carcinogenicity and the fre-
quency of induced somatic mutations (Huberman, et al. 1977). The
demonstration that covalent binding of carcinogenic PAH with DMA of
V79 cells was the same as occurs in vivo further strengthened the
argument that genetic interaction (i.e., somatic mutation or gene
depression) may be involved in tumor formation (Newbold, et al.
1977) .
The use of Chinese hamster V79 cells to test the rnutagenicity
of BaP metabolites has contributed significantly to an understand-
ing of the molecular action of PAH (Huberman, et al. 1976a,b, 1977;
Malaveille, et al. 1975; Newbold and Brooks, 1976; Jerina, et al.
C-66
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1976). Comparison of the mutagenic activities of the optically
pure ( + ) and (-)-enantiomers of BaP 7,8-dihydrodiol revealed that,
in the presence of a metabolic activating system, the (-)trans,
7,8-dihydrodiol was the most active mutagen (Huberman, et al.
1977) . These results are consistent with the fact that the
(-) trans 7,8-dihydrodiol is the only BaP enantiomer by rat liver
microsomes (Yang, et al. 1977), and that it is highly carcinogenic
to newborn mice (Kapitulnik, et al. 1978a,b). Because the (-)trans
7,8-dihydrodiol had no mutagenic activity in the absence of enzymes
required for PAH metabolism, it was apparent that the BaP 7,8-diol-
9,10-epoxide, which is derived from this intermediate, is an ulti-
mate mutagen/carcinogen. Studies by Wood, et al. (1977a) on the
mutagenicity to V79 cells by the four optically pure enantiomers of
the BaP 7,8-diol-9-10-epoxides supported this belief. None of the
triols and tetrols which are derived from BaP diol epoxides were
mutagenic to V79 cells, and thus represent probable detoxification
products (Huberman, et al. 1977).
The current belief that neoplastic transformation may arise
from a chemically induced somatic mutation was made even more con-
vincing by the recent studies of Huberman and coworkers (1976b).
They demonstrated for the first time that BaP and BaP 7,8-dihydro-
diol can induce both neoplastic transformation and mutagenesis
(ouabain resistance) in the same culture of normal diploid hamster
embryo cells. The concentrations for transformation and mutagene-
sis were the same, and showed a dose-response effect in both trans-
formation and ouabain resistance for BaP 7,8-dihydrodiol.
C-67
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In further adaptation of the cell-mediated mutagenesis system,
V79 cells are metabolically activated by rat liver homogenates con-
taining microsomes and cofactors (Krahn and Heidelberger, 1977).
The mutagenic activity of BaP, MCA, DMBA, and benz(a)anthracene in
this system showed a limited correlation with their respective car-
cinogenic potencies. It should be noted, however, that the selec-
tion of a particular activating system (i.e., microsomes v. feeder
cells) may have a significant influence on the test results.
The analysis of chromosomal aberrations and sister chromatid
exchanges (SCEs) is often recommended as a screening technique for
potential mutagens and carcinogens. Several investigators have
examined the effects of PAH on the chromosomes of mammalian cells.
Early studies indicated that variations in chromosome number and
structure may accompany tumors induced by BaP, MCA, and DMBA in the
rat, mouse, and hamster (Kato, et al. 1975). However, in cultured
human leukocytes exposed to DMBA, chromosome damage was not the
same as that produced in hamster cells. Although it is argued that
chromosome changes in PAH-induced tumors are all specific (Levan
and Levan, 1975; Ahlstrom, 1974), others (Popescu, et al. 1976;
Nery, 1976) claim that detectable chromosome changes are not speci-
fic for the carcinogenic agent nor are they a prerequisite for neo-
plastic growth. Moreover, an increased rate of SCEs can be pro-
duced by BaP in cultured human lymphocytes (Rudiger, et al. 1976?
Schonwald, et al. 1977) but this increase is not correlated with
different rates of BaP metabolism (Rudiger, et al. 1976), a sur-
prising result in light of the known importance of metabolic acti-
vation for BaP mutagenicity. BaP-induced SCEs rates did not differ
C-68
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between lymphocytes taken from normal humans and those from pa-
tients with lung cancer (Schonwald, et al. 1977). In recent stud-
ies with cultured Chinese hamster cells exposed to DMBA, BaP, and
MCA, none of the chemicals produced chromosome breaks and only DMBA
could successfully induce SCEs (Abe and Sasaki, 1977). Although it
cannot be denied that PAH cause chromosome damage, it is not clear
whether this effect may represent an epigenetic phenomenon which is
merely secondary to mutagenesis and neoplastic transformation.
Furthermore, in cases where a chemically induced mutation is
"silent" (i.e., neutral amino acid substitution), there is no rea-
son to believe that detectable chromosome damage should occur.
In recent comparisons of three cytogenetic tests, (1) induc-
tion of chromosome aberrations, (2) induction of micronuclei, and
(3) jji vivo induction of sister chromatid exchanges, the last test
proved to be the most sensitive with carcinogenic polycyclic hydro-
carbons (Bayer, 1978) . Since positive results were also obtained
with phenanthrene, the usefulness of sister chromatid exchange as a
screening technique for carcinogen detection is limited. BaP was
positive in the sister chromatid exchange test, weakly active in
the chromosome aberration test, and negative in the micro-nucleus
test. On the other hand, DMBA was clearly positive in all three
tests. The conclusion was that cytological tests do not provide
reliable correlations with all carcinogens tested and thus cannot
be used alone in mutagenicity/carcinogenicity evaluations.
Damage to the genome resulting from chemical insult can theo-
retically also be detected by examining DNA repair (Stich and
Laishes, 1973). The suggestion that DNA repair is applicable as a
C-69
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screening procedure for evaluating potential chemical mutagens is
based on the assumption that the level of DNA repair synthesis in a
cell reflects the extent of DNA damage produced by a chemical.
Indeed, unscheduled incorporation of H-thymidine into nuclear DNA
of normal human cells exposed to epoxides of benz(a)anthracene and
MCA has been observed (Stich and Laishes, 1973). However, since a
metabolic activation system was not present in this .'system, the
parent hydrocarbons showed no activity. More recent studies con-
firmed that K-region epoxides of BaP, DMBA, and DBahA caused DNA
damage in human skin fibroblasts which was repaired with the same
system used for repairing lesions induced by ultraviolet radiation
(Maher, et al. 1977). As would be expected, the parent hydrocar-
bons exerted no effect. More important, results were obtained
which indicated that the DNA repair process itself does not induce
mutations, but rather that mutagenesis occurs before the DNA lesion
can be excised.
DNA repair synthesis in human fibroblasts (Regan, et al. 1978;
Stich, et al. 1975,1976; San and Stich, 1975), rat Liver cells
(Williams, 1976), and Chinese hamster V79 cells (Swenberg, et al.
1976) has been successfully used for the detection of chemical car-
cinogens, including numerous PAH. However, the percentage of car-
cinogens giving positive results for DNA repair is considerably
less than in the cell transformation or microbial mutagenesis
assays. Nevertheless, tests with human skin fibroblasts showed
that DNA repair synthesis results from exposure to BaP 7,8-diol-
9,10-epoxides, whereas BaP 4,5-, 9,10-, and 11,12-oxides did not
produce DNA damage which was repairable by the ultraviolet excision
C-70
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repair system (Regan, et al. 1978). These results support the con-
cept that diol epoxide metabolites of PAH are ultimate mutagens.
Tumors induced _in vivo by PAH are commonly associated with
chromosome abnormalities in the neoplastic cells. In particular,
sarcomas induced by DMBA, MCA, and BaP in the rat display karyotype
variations which were reportedly nonrandom and distinctly different
from sarcomas induced by Rous sarcoma virus (Levan and Levan, 1975;
Mitelman, et al. 1972) . The chromosome patterns of DMBA-induced
sarcomas were found to be identical with those observed in rat leu-
kemias (Mitelman and Levan, 1972) and in primary carcinomas of the
auricular skin (Ahlstrom, 1974) induced by DMBA.
Considerable evidence is also available to indicate that chro-
mosome alterations in PAH-induced tumors in vivo are not consistent
either in frequency or in pattern. DMBA-induced tumors (fibrosar-
coma, squamous carcinoma, lymphosarcoma) of the uterine cervix in
ICR mice revealed various karyotypic profiles (Joneja and Coulson,
1973; Joneja, et al. 1971). These tumors displayed diploid, aneu-
ploid, tetraploid, and octaploid chromosome constitutions. Tumors
induced in mice with MCA and dibenzo(a,i)pyrene also showed a wide
variation in chromosome constitution (Biedler, et al. 1961; Hell-
strom, 1959). Mice treated with 30 ug DMBA, a dose sufficient to
produce a 100 percent incidence of thymic lymphomas, did not reveal
an excess of chromosome abnormalities in bone marrow or thymus
(Ottonen and Ball, 1973) . Even at higher doses (60 ug DMBA) , the
incidence of abnormal chromosomes did not significantly differ from
controls. Subcutaneous tumors in Syrian hamsters induced by single
injections of BaP (0.1 ug) or DMBA (0.1 mg), and cultured cell
C-71
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populations derived from these tumors, failed to reveal common
karyotypic changes (DiPaolo, et al. 1971a,b) . Tumor cells had
aneuploid, diploid, and hypotetraploid chromosome constitutions;
further karyotype rearrangements occurred with subsequent growth in
vitro.
In humans, the presence of the "Philadelphia" chromosome in
myeloid leukemia appears to be the only example of a human chromo-
some abnormality which is tumor-specific (Nowell and Hungerford,
1960). In PAH-induced experimental tumors, lymphatic leukemia in
mice produced by DMBA also displays consistent chromosome abnormal-
ities (Joneja and Coulson, 1973) . Beyond this common feature, con-
vincing data have not been presented to indicate that somatic cells
exposed to PAH may suffer characteristic or reproducible damage to
the genome. Instead, random karyotypic mutants of transformed
.cells are thought to be selected in response to growth pressures in
the host environment (e.g., tissue necrosis, infection, anoxia,
lack of nutrition) (Joneja and Coulson, 1973).
Carcinogenicity
Animal data: Numerous polycyclic aromatic compounds are dis-
tinctive in their ability to produce tumors in skin and most epi-
thelial tissues of practically all species tested. Malignancies
are often induced by acute exposures to microgram quantities of
PAH. Latency periods can be short (four to eight weeks) and the
tumors produced may resemble human carcinomas. Carcinogenesis
studies involving PAH have historically involved primarily effects
on the skin or lungs. In addition, subcutaneous or intramuscular
injections are frequently employed to produce sarcomas at the in-
C-72
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jection site. Ingestion has not been a preferred route of adminis-
tration for the bioassay of PAH (Santodonato, et al. 1980).
Concern over potential human cancer risk posed by PAH present
in the atmosphere stems from studies demonstrating that crude ex-
tracts of airborne particulate matter can be carcinogenic to ani-
mals (Hoffmann and Wynder, 1976; Wynder and Hoffman, 1965; Hueper,
et al. 1962; Kotin, et al. 1954). Fractions soluble in benzene or
benzene-methanol produced tumors in mice by skin painting or sub-
cutaneous injection. Both the aromatic and oxygenated neutral sub-
fractions were active as complete carcinogens, and indicated the
presence of numerous carcinogenic materials, including non-PAH.
Since the carcinogenicity of the total organic particulates and
aromatic neutral subfractions could be explained only partly by the
presence of BaP, its usefulness as a measure of carcinogenic risk
from air pollution may be limited.
From investigations in which nolycyclic carcinogens were
painted on the skin of mice has emerged the two-stage theory of
skin carcinogenesis (Berneblum, 1941; Van Duuren, 1969, 1976). The
first stage, initiation, results from the ability of a carcinogen
to effect a permanent change within a cell or cell population fol-
lowing a single application. The measure of carcinogenic potency
is often regarded as the capacity for tumor initiation. However,
some weak or inactive complete carcinogens can be active as tumor
initiators (e.g., dibenz(a,c)anthracene, 1-methylchrysene, benz-
(a)anthracene). The second stage, promotion, is a prolonged pro-
cess which does not necessarily require the presence of a carcino-
gen, but nevertheless a chemical stimulus must be supplied (e.g.,
C-73
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by croton oil). A complete carcinogen is one which, if applied in
sufficient quantity, can supply both initiating and promoting stim-
uli (e.g., DMBA, BaP). The formation of skin tumors by polycyclic
hydrocarbons may also be influenced by inhibitors and accelerators
(cocarcinogens), thus complicating the interpretation of experi-
mental data.
The tumorigenic effects of PAH when applied to the skin of
animals have been known for decades. Iball (1939) collected the
results of a series of experiments to arrive at a method for com-
paring the carcinogenic potencies of various polycyclic aromatic
chemicals. His results, presented in Table 18, express tumorigenic
potency in mouse skin as the ratio of percent tumor incidence to
the average latency period. This expression, commonly referred to
as the Iball index, is still used as a means of comparing the rela-
tive activity of carcinogens. An important data compilation on
agents tested for carcinogenicity has more recently been oublished
by the U.S. Public Health Service (Publication No. 149) which lists
the results of tests on hundreds of chemicals in numerous animals
including rodent, avian, and amphibian species.
Experimental models for respiratory carcinogenesis have major
limitations in that the delivery of carcinogens to the tracheobron-
chial tree in measured amounts and their adequate retention at the
target tissue are poorly controlled. Therefore, the conduct of
dose-response studies on lung tumor induction has been seriously
hampered. Moreover, the possible relevance of the two-stage theory
of carcinogenesis to lung cancer has not been clearly established.
Many of the bioassay data on PAH-induced lung cancer have been de-
>74
-------
n
i
-~j
t_n
TABLE 18
Carcinogenic Compounds in Descending Order of Potency*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11 .
12.
13.
14.
15.
16.
17.
18.
19.
20.
21 .
22.
23.
24.
Compound
7 , 12-nimethylbenz (a) anthracene
3-Methylcholanthrene (a)
3-Methylcholanthrene (b)
3-Methylcholarithrene
(a and b added together)
Benzo(a)pyrene (from pitch)
Benzo(a) pyrene (synthetic)
Benzo (a) pyrene
(5 and 6 added together)
Cholanthrene
5 , 6-Cyclopenteno-benz (a) anthracene
2-Methyl-benzo(c) phenanthrene
1 0-Methyl-benz (a) anthracene
5,6-Dimethyl-benz (a) anthracene
6-Isopropyl-benz(a)anttuacene
nibenzo(c,q)carbazo)c
Di ben zo (a, h) pyrene
5-Methyl-benz (a) anthracene
5-Kthyl-benz (a) anthracene
n i ben z ( a, h) anthracene
Benzo(c) phenanthrene
Oibenzo(a,q) carbazole
5-n-Propyl-benz (a) anthracene
Dibenz (c,h) acrid ine
3-Methyl-dibenz (a,h) anthracene
Dibenz (a ,h) acrid ine
Totals
Number
of Mice
Alive when
First Tumor
20
18
8
26
10
9
19
49
14
16
18
19
1 5
19
17
8
9
65
18
9
20
28
25
25
Number
of
Tumors
13
18
5
23
10
7
17
28
13
12
12
16
11
9
10
7
7
41
12
4
6
11
7
6
305
Percentage
of
Tumors (A)
65
100
62.5
88.5
100
78
89 5
57
93
75
66.5
84
73.5
47.5
59
87 5
77.5
63
67
44 . 5
30
39.3
28
24
Papilloma
6
1
0
1
2
2
4
5
1
5
2
0
1
4
0
o
2
8
5
1
->
2
i
2
60
Epi thelioma
7
17
5
22
8
5
23
12
7
10
16
10
5
30
5
33
7
3
9
4
245
Average
Latent
Period(B)
43
99
1C!
109
127
i no
112
1 Q A
155
147
220
204
1 A 1
205
285
239
387
•)c -i
192
357
350
Index
(A/B x 100)
151
101
80
7Q
51
48
45
38
36
29
28
27
26
17
16
11
9
7
*Somce: Ibal], 1939
-------
rived from animal model systems employing various modes of admin-
istration (inhalation, intratracheal instillation, intravenous in-
jection), and the use of carrier particles (e.g., ferric oxide) for
the delivery of the carcinogen to the bronchial epithelium. Thus,
the results obtained from these studies cannot always be directly
compared. The most commonly employed method for the study of PAH-
induced lung cancer involves intratracheal instillation of test
material in the Syrian golden hamster.
Following the identification of the first carcinogenic hydro-
carbon from soot (BaP) an intensive effort was mounted to isolate
the various active components of carcinogenic tars (IARC, 1973).
From the earliest studies conducted, the realization emerged that
carcinogenic PAH are structurally derived from the simple angular
phenanthrene nucleus (Arcos and Argus, 1974). However,, unsubsti-
tuted PAH with less than four condensed rings that have been tested
have not shown tumorigenic activity. Furthermore, of the six pos-
sible arrangements with four benzene rings, only two of these com-
pounds are active: benzo(c)phenanthrene and benz(a)anthracene.
The unsubstituted penta- and hexacyclic aromatic hydrocarbons are
clearly the most potent of the series. These include BaP, DBahA,
dibenzo(a,h)pyrene, dibenzo(a,i)pyrene, dibenzo(a,l)pyrene, di-
benzo(a,e)pyrene, benzo(b)fluoranthene, and benzo(j)fluoranthene.
Somewhat less potent as carcinogens are the dibenzanthracenes and
dibenzophenanthrenes. Only a few heptacyclic hydrocarbons show
carcinogenic activity. These include phenanthro(2',3':3,4')py-
rene, peropyrene, and dibenzo(h,rst)pentaphene. Beyond seven un-
substituted aromatic rings, there are very few known carcinogenic
C-76
-------
hydrocarbons. However, many physico-chemical and enzymatic para-
meters must be dealt with in respect to carcinogenic PAH. Factors
such as solubility and intracellular localization to achieve meta-
bolic activation are likely to be important determinants of the
true carcinogenicity of a particular PAH.
Among the unsubstituted polycyclic hydrocarbons containing a
nonaromatic ring, a number of active carcinogens are known. The
most prominent examples of this type of compound are cholanthrene,
11,12-ace-benz(a)anthracene, 8,9-cyclopentanobenz(a)anthracene,
6,7-ace-benz(a)anthracene, acenaphthanthracene, 1,2,5,6-tetrahy-
drobenzo(j)cyclopent(f,g)aceanthrylene, and "angular" steran-
threne. All of these compounds retain an intact conjugated phenan-
threne segment.
The addition of alkyl substituents in certain positions in the
ring system of a fully aromatic hydrocarbon will often confer car-
cinogenic activity or dramatically enhance existing carcinogenic
potency. In this regard, Arcos and Argus (1974) noted that mono-
methyl substitution of benz(a)anthracene can lead to strong carci-
nogenicity in mice, with potency depending on the position of sub-
stitution in the decreasing order, 7 > 6 > 8^12 > 9. A further en-
hancement of carcinogenic activity is produced by appropriate di-
methyl substitution of benz(a)anthracene. Active compounds are
produced by 6,8-dimethyl-, 8,9-dimethyl-, 8,12-dimethyl-, 7,8-
dimethyl-, and 7,12-dimethyl-substitution. The latter compound is
among the most potent PAH carcinogens known, although it has not
been shown as a product of fossil fuel pyrolysis. Methyl substitu-
tion in the angular ring of benz(a)anthracene, however, tends to
C-77
-------
deactivate the molecule, although 4,5-dimethylbenz(a)anthracene
may be an exception. Carcinogenic trimethyl- and tetrctmethylbenz-
(a)anthracenes are known, and their relative potencies are compara-
ble to the parent 7,12-DMBA. In general, free radical synthesis of
polycyclic hydrocarbons by pyrolysis does not favor alkyl side
chain formation.
Alkyl substitution of partially aromatic condensed ring sys-
tems may also add considerable carcinogenic activity. The best
example of this type of activation is 3-methylcholanthrene, a high-
ly potent carcinogen.
With alkyl substituents longer than methyl, carcinogenic!ty
tends to decrease, possibly due to a decrease in transport through
cell membranes. However, different positions in the benz(a)anthra-
cene molecule will vary with respect to the effect of n-alkyl sub-
stitution on carcinogenicity. Benz(a)anthracene is especially sen-
sitive to decreased carcinogenicity caused by the addition of bulky
substituents at the 7-position, and is indicative of a once widely-
held view for most polycyclics that high reactivity of the meso-
phenanthrenic region (now called the "K-region") was a critical
determinant for carcinogenicity. Current studies show that the
K-region is not involved in critical binding to DNA.
Partial hydrogenation of the polycyclic aromatic skeleton can
generally be expected to decrease carcinogenic potency. This was
shown with various hydrogenated derivatives of BaP, benz(a)anthra-
cene, and MCA. On the other hand, the carcinogenicity of DBahA,
dibenzo(a,i)pyrene, and dibenzo(a,h)pyrene is not significantly
altered by meso-hydrogenation. This may be due to the fact that
C-78
-------
extensive resonance capability is preserved. Moreover, 5,6-dihy-
dro-DBahA actually displayed a fourfold increase in carcinogenicity
in comparison to the parent hydrocarbon (Arcos and Argus, 1974),
possibly due to the hydrophilicity and ease of intracellular trans-
port of its dihydrodiol derivative.
For many years, investigators have sought a common molecular
feature among PAH carcinogens which would serve to explain their
biological activity. The "electronic theory of carcinogenesis" has
relied upon an analysis of the influence of electron density at
specific molecular regions to explain unique reactivity with cellu-
lar constituents. A basic assumption arising from the work of the
Pullmans and others (Pullman and Pullman, 1955) was that a meso-
phenanthrenic region ("K-region") of high 7T -electron density and
with a propensity for addition reactions was a critical structural
feature for polycyclic carcinogens. In expanding this hypothesis,
further biological significance was attributed to the concomitant
presence of a rather unreactive meso-anthracenic region ("L-re-
gion") for high carcinogenicity. In addition, a region of compara-
tively low reactivity which characteristically undergoes metabolic
perhydroxylation (corresponding to the 3,4-positions of benz(a)an-
thracene) has been designated the M-region. According to the the-
ory, only binding of the K-region to critical cellular sites would
cause tumor formation; protein binding at the L-region causes no
tumorigenic effect, while inactivation is produced by metabolic
C-79
-------
perhydroxylation in the M-region. The three regions of reactivity
are readily distinguished in the benz (a) anthracene skeleton:
t. M-region of metabolic
jr*" perhydroxylation
•
_/ \ :•'
L-region ' _ ,*v-^~=-"K- region
The electronic K-L theory of carcinogenic reactivity has encoun-
tered numerous inconsistencies, primarily because these relation-
ships were derived from physico-chemical properties of the parent
hydrocarbon and gave no consideration to the biological, effects of
activated metabolites.
Advances in recent years have focused attention on the poten-
tial reactivity of diol epoxide metabolites of PAH, and their ease
of conversion to triol carbonium ions. Under the assumption that
diol epoxides, which are more readily converted to carbonium ions,
will be better alkylating agents to produce carcinogenesis and
mutagenesis, the "bay region" theory has been proposed (Lehr, et
al. 1978; Wood, et al. 1977b) . Examples of a "bay region" in a
polycyclic hydrocarbon are the regions between the 10 and 11 posi-
tions of BaP and the 1 and 12 positions of benz (a) anthracene:
Bay region Bay region
Benzo(a)pyrene Benz(a)anthracene
The theory predicts that diol epoxides in which the oxirane oxygen
forms part of a "bay region" (e.g., BaP 7,8-diol-9,10-epoxide)
will be more reactive and hence more carcinogenic than diol epox-
ides in which the oxirane oxygen is not situated in a "bay region."
C-80
-------
Experimentally, the "bay region" diol epoxides of benz(a)anthra-
cene, BaP, and chrysene were more mutagenic ii\ vitro and/or tumori-
genic than other diol epoxide metabolites, their precursor dihydro-
diols, the parent hydrocarbons, or other oxidative metabolites.
Moreover, quantum mechanical calculations were in accord with the
concept that reactivity at the "bay region" is highest for all the
diol epoxides derived from polycyclic hydrocarbons.
The bay region concept has received enough confirmation to
lead to suggestions that an analysis of theoretical reactivity in
this manner may be useful in screening PAH as potential carcinogens
(Smith, et al. 1978). Among several indices of theoretical reac-
tivity examined, the presence of a bay region for a series of PAH
displayed a high degree of correlation with positive carcinogenic
activity (Table 19).
The carcinogenic activity of BaP has been studied extensively
in various animal model systems. In recent years, research on BaP
has been expanded to include an examination of the tumorigenic
activity of various BaP metabolites. These efforts were directed
at the objective of identifying a BaP derivative which acts as the
principal ultimate carcinogen resulting from metabolic activation
(Levin, et al. 1976a,b, 1977a,b; Slaga, et al. 1976, 1977; Kapitul-
nik, et al. 1976a,b; Wislocki, et al. 1977; Conney, et al.
1977a,b).
Studies on the activity of BaP and its derivatives as complete
carcinogens on mouse skin (Table 20) and as tumor initiators (Table
21) revealed that marked differences in tumorigenic potency exist.
The apparent lack of activity for the BaP 7,a-diol-9,10-epoxides,
C-81
-------
o
I
oo
TABLE 19
Reactivity Indices for Polycyclic Hydrocarbons*
K- L—
Compound region? region?
Naphthalene
Anthracene - +
Tetracene - +
Pentacene - +
Hexacene - +
BA + 4-
Benzo(a) tetracene + +
Phenanthrene +
Benzo(c) phenanthrene +
Chrysene +
Benzo(b)chrysene + +
Picene +
Triphenylene
Benzo(g)chrysene +
Dibenz (a, c)anthracene - +
Dibenz (a, j)anthracene + +
Dibenz (a, h) anthracene + +
Naphtho(2,3-b)pyrene + +
Benzo(a) pyrene +
Benzo(e)pyrene +
Dibenzo(a,l)pyrene +
Dibenzo(a»i)pyrene +
Dibenzo(a,e)pyrene +
Dibenzo(a/h)pyrene +
Tr ibenzo ( a, e, i) pyrene
Carcinogenicity Index
Bay Arcos and Jerina, et al.
region Argus (1974) (1972)
0
0_
- 0
- 0
~
+ 5 +
+
+ 0
+ 4 +
O i
+ 3 +
f\
+ 0
+ 0
i 1 T i i
+ 17 ++
. *5 _l_
+ 3 +
A J_
+ 4 +
*")/>• i i
+ 26 ++
a 27 ++
, "I *J I L i I
-|- / J TTTT
f\ _l_
i 1 -f-
4- 33 ++
M Jl 111 _l_
+ 74 ++++
-)- 50 ++4-
n /% 1 L 1 _l_
4- 70 ++++
T ^ I*
4- 16 ++
*Source: Smith, et al. 1978
aThis compound does not strictly possess a bay region but does contain a "pseudo" bay region,
bJerina, et al. (1972) have assigned this as ++++.
-------
TADLE 20
Skin Tumors in Mice Treated with Benzo(a)pyrene and Derivatives
O
I
oo
OJ
Treatment3
OaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
1-IIOBaP
2-IIOBaP
3-IIOBaP
4-HOBaP
5-HOBaP°
6-IIOBaP
7-HOBaP°
8-IIOBaP
c
9-HOBaP
_ c
10-HOBaP
11-llOBaP
12-llOBaP
BaP 4,5-oxide
BaP 4,5-oxiile
BaP 7,8-oxide
13aP 7,8-oxide
BaP 7,8-oxide
BaP 7,8-oxide
BaP 9,10-oxide
BaP 11,12-oxiae
BaP 11,12-oxlde
Total No.
Animals
25
30
26
30
27
30
30
30
30
30
30
30
25
29
29
26
26
28
30
27
26
28
28
23
30-39
30-39
30-39
30
30
30-39
30-39
28
17
Dose,
li mo lea
0.4
0.4
0.4
0.15
0.1
0.1
0.1
0.1
0.05
0.025
0.02
0.02
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
O.I
0.4
0.3
0.15
0.1
0.4
0.4
0.1
Mice with
Tumors, %
100
100
92
100
96
38
50
91
59
7
4
0
0
100
0
0
0
0
0
0
0
0
14
0
4
6
94
53
18
9
0
0
0
Total No.
Skin Tumors
32
34
34
40
28
13
15
24
20
2
1
0
0
37
0
0
0
0
0
0
0
0
4
0
1
2
37
16
5
3
0
0
0
Reference
Wislocki, et al. 1977
Wislocki, et al. 1977
Albert, et al. 1978
t.evln, et al. I976a,h
Wislocki, et al. 1977
Levin, et al. 1977a,b
Levin, et al. 1977a,b
Levin, et al. 1977a,b
Levin, et al. 1977a,b
Levin, et al. J977a,b
Levin, et al. I977a,b
Levin, et al. 1977a,b
Wislocki, et al. 1977
Wislocki, et al. 1977
Wislocki, et al. .1977
Albert, et al. 197Q
Albert, et al. 1978
Albert, et al. 1978
Albert, et al. 1970
Albert, et al. 1970
Albert, et al . 1978
Albert, et al. 1.970
Wislocki, ct al. 1.977
Wislocki, et al. 1.977
Levin, et al. 1976a
Levin, et al. 1976a
Levin, et al. 1976a
Levin, et al. ]97Ca
Levin, ct al. 1976a
Levin, et al . 1976a
Levin, et al. 1976a
Wislocki, et al. 1977
Wislocki , et al . 1077
-------
TABLE 20 (cont.)
O
I
00
Treatment3
BaP 7,8-dihydro-
diol
BaP 7,8-dihydro-
diol
BaP 7,8-dihydro-
diol
BaP 7,8-dihydro-
diol
BaP 7,8-dihydro-
diol
(±,-7/,8«-ni-
eopxy-7,8,9,10-
tetrahydrobenzo
(a) pyrene
(diol epoxide 1)
diol epoxide 1
diol epoxide 1
~hydroxy-9 o<, ,10<=<-
epoxy-7,8,9,10-
tetrahydrobenzo
(a) pyrene
(diol epoxide 2)
diol epoxide 2
diol epoxide 2
rotal No.
Animals
30
30
30
30
30
30
30
30
30
30
30
nose,
limoles
0
0
0
0
0
0
0
0
0
0
0
.3
.15
.1
.05
.025
.4
.1
.02
.4
.1
.02
Mice with
Tumors, %
100
100
92
76
7
0
0
0
13
7
0
Total No'b Reference
Skin Tumors
42
40
28
24
2
0
0
0
3
2
0
Levin,
Levin,
Levin,
Levin,
Levin,
Levin,
Levin,
Levin,
Levin,
Levin,
Levin,
et
et
et
et
et
et
et
et
et
et
et
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
1976b
I976b
I976a
1976a
1976a
1976a
1976a
1976a
1976,1
1976a
1976a
aFemale C57BL/6J mice wore treated with BaP or BaP derivatives (0.02-0.4 nmole) once every 2 weeks tor 60 weeks
by topical application to the shaved skin of the back.
bSkin tumors consisted mostly of squamous cell carcinomas; other skin tumors were fibro-sarcomas, papiUomas,
and keratocanthomas.
°Mice were treated once every 2 weeks for 56 weeks.
-------
TABLE 21
Summary of the Skin Tumor Initiation Activities of. Benzo(a)pyrene and its Metabolites3
O
I
oo
Ln
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
Initiator
4 , 5-epoxide
7 ,8-epoxide
9, 10-epoxide
11 , 12-epoxide
7 /o,8o<-diol-9o<,lOo<-epoxide
7 /.ScK-diol-g^lO^-epoxide
7,8-dihydrodiol .
(-)-BaP 7,8-dihydrodiol"
(+)-BaP 7,8-dihydrodiol
No.
Mice
30
30
30
30
29
29
30
29
28
29
30
30
Dose,
hmoles
200
200
200
200
200
200
200
200
200
200
100
100
Weeks of
Promotion
23
30
21
23
23
30
30
30
30
30
21
21
Mice with
Tumors, %
94
92
77
20
81
15
38
69
7
86
77
23
Papillomas/
Mouse
4
5
2
0
1
0
0
1
0
5
3
0
.8
.3
.6
.2
.9
.15
.45
.5
.07
.0
.8
.43
neference
Slaqa,
Slaga,
Lev in.
Slaga,
Slaga,
Slaga,
Slaqa,
Slaga,
Slaga,
Slaga,
Levin,
Levin ,
et
et
et
et
et
et
et
et
et
et
et
et
al .
a).
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
1976
1977
1977b
1976
1976
1977
1977
1977
1977
1977
1977b
1977b
b
Female CD-I mice were treated with a single dose of initiator dissolved in acetone, acetone: NH.OII (1,000;]),
or dimethyl sulfoxideracetone (1;3) and followed 1 week later by twice-weekly applications of 10 pg of TPA.
Promotion was by twice-weekly applications of 16 hmoles of TPA beginning 11 days after treatment with initiator.
-------
despite their exceptional mutagenicity, may be due to poor skin
penetration of adult mouse skin because of high chemical reactiv-
ity. Indeed, as a carcinogen in newborn mice the (-) enantiomer of
BaP, 7,8-dihydrodiol, and the 7,8-diol-9,10-epoxide derived there-
from are far more active than the parent hydrocarbon (Kapitulnik,
et al. 1977a,b,c,d, 1978a,b). These studies on the newborn mouse
clearly indicate the role of a BaP 7,8-diol-9,10-epoxide as an
ultimate carcinogenic metabolite of BaP.
Further dose-response information on the sarcomagenic activ-
ity of BaP by subcutaneous injection to rats and mice is summarized
in Table 22.
Temporal relationships for the development of BaP-induced skin
cancers in mice have been examined by Albert, et al. (1978) . Their
results showed that increasing weekly doses of BaP caused a short-
ening of the latency period for carcinoma formation. Furthermore,
it was determined that the development of papillomas as a precursor
lesion to carcinoma formation occurred only at higher BaP doses
(e.g., 32 and 64 ug/week) . At the lower dose levels (8 and 16
yg/week), carcinomas appeared de novo without precursor papilloma
formation.
The carcinogenicity of BaP by oral intake has not been studied
as throughly as for other routes of administration. Nevertheless,
tumors of various sites result when BaP is administered orally to
rodents (Table 23).
With oral, intratracheal, and intravenous routes of adminis-
tration, BaP is less effective than other PAH (e.g., DMBA, MCA,
dibenz(a,h)anthracene) in producing carcinomas. On the other hand,
C-86
-------
TABLE 22
Induction of Sarcoma by Benzo(a)pytene
o
1
CD
-J
Species
Kat
(Sprague-Dawley)
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
No. and (Sox)
13 (female)
14 (male)
16 (female)
9 (?)
10 (?)
12 (?)
15 (?)
Total Dose
H moles
6.0a
7.1b
7.1b
15.9°
5.0C
0.5C
0.002°
Animals with
Sarcoma, %
100
93
50
66.6
70
66.6
0
Average Latency,
Days
101 + 2.7
129
160
112
122
155
N.A.d
Reference
Flesher, et al. 1976
Buu-ltoi, 1964
nuu-Hoi, 1964
Gottschalk, 1942
Gottschalk, 1942
Gottschalk, 1942
Gottschalk, 1942
Administered as 0.2 pinole dissolved in 0.1 ml sesame oil by subcutaneous injection on alternate days for 30 doses
beginning at 30 days of age.
Administered as three injections of 2.4 (jmoles each, given at 1 month intervals.
"Administered as a single injection under the skin of the abdomen, dissolved in 0.5 ml of neutral olive oil.
Not applicable.
-------
TABLE 23
Carcinogenicity of nenzo(a)pycene by Oral Administration to Various Mammals*
Compound
Species
Dose
Route of
Administration
Eefects
BaP
O
I
CO
00
Mouse
Mouse
(age 17-116 days)
Mouse
Mouse
(age 18-30 days)
Rat
(Sprague-Dawley;
age 105 days)
Hamster
0.2 mg in PEG9
50-250 ppm
250 ppm
250 ppm
2.5 mg per day
2-5 mg bi-weekly
Hamster
500 ppm
Intragastric
Dietary
(110-197 days)
Dietary
Dietary
(140 days)
Oral
Intragastr ic
Dietary
(4 days per week
for up to 14 mo.)
14 tumors of the forestomach in
5 animals out of 11
^70% incidence of stomach tumors
at 50-250 ppm for 197 days;
no tumors with diets containing
up to 30 ppm for 110 days
100% stomach tumor incidence
when diet was fed for 30 days;
5-7 days of feeding, 30-40%; 2
to 4 days of feeding, 10 percent;
1 day of feeding, 0 percent
Leukemias, lung adenomas, and
stomach tumors produced
Papillomas developed in the
esophagus and forestomach in
3 out of 40 animals
5 stomach papillomas in 67 ani-
mals treated for 1-5 months;
7 papillomas and 2 carcinomas
in 18 animals treated for 6-9
months; 5 papillomas in 8 ani-
mals treated for 10-11 months
12 tumors (2 esophagus, 8 fore-
stomach, 2 intestinal) in 8
animals
Polyethylene glycol
*Source: IARC, 1973
-------
BaP has remarkable potency for the induction of skin tumors in
mice. Therefore, caution must be exercised in considering the car-
cinogenicity of PAH as a class, and in extrapolating data derived
from studies with BaP to the effects of PAH mixtures.
An examination of comparative carcinogenicities within the
same tumor model system can provide valuable insight concerning
relative risks of various PAH. By single intravenous injection of
about 0.25 mg of aqueous dispersions of PAH to mice, a direct com-
parison of carcinogenic potency was possible (Table 24). In this
test system, MCA displayed the greatest lung tumor-forming capabil-
ity; dibenz(a,h)anthracene followed closely in activity with BaP
being considerably less potent.
Intratracheal instillation of PAH to Syrian golden hamsters
has been widely utilized for the conduct of studies on pulmonary
carcinogenesis (Saffiotti, et al. 1968,1972; Henry, et al. 1975).
Several studies are summarized in Table 25 and indicate that:
(1) dose-response relationships are clearly evident, and (2) the
co-administration of carrier particles such as Fe-O., (i.e., with
BaP) can markedly increase tumor incidence, depending on the condi-
tions of the experiment and physical characteristics of the parti-
cle. Since environmental exposures to PAH occur in conjunction
with particulate material in air, this effect may be particularly
relevant to human situation.
In addition to the hamster model system, respiratory tract
tumors have been readily induced by PAH in rats and mice. The re-
sults of several representative studies are summarized in Table 26.
C-89
-------
TABLE 24
Comparative Carcinoqenicity of Polycylic Hydrocarbons and Related Compounds
Measured by Induction of Lung Tumors (LT)a'
o
1
>£>
O
Compound
3-Methylcholanthrene, 0.1 mg
3-Methylcholanthrene, 0.5 mg
Dibenz (a,h) anthracene
7H-Dibenzo(c,g)carbazole
Benzo(a) pyrene
Dibenz (a, j ) aceanthrylene
Dibenz (a, h) acr idine
S-Methylbenzo(c) phenanthrene
7-Methylbenzo(a) pyrene
5-Methoxy-7-propylbenz (a) anthracene
Benz (a) anthracene
Untreated controls
Dose,
ymoles/kg
15
74
36
38
40
33
36
42
38
33
44
—
Mice with LT/
No. of Mice
15/15
6/6
10/10
12/12
10/10
9/10
11/12
6/11
5/10
1/10
2/11
4/19
Mean No.
LT/mouse
11
47
31
5.7
3.7
2.7
2.0
0.7
0.6
0.1
0.2
0.2
yMoles/kg for
1 LT Response
0.9
1.0
6.0
9.5
14
18
--
—
--
__
--
Source: Shimkin and Stoner, 1975
Strain A mice, 8-12 weeks old, received single intravenous injection of 0.24 mg of methlchol-
anthrene in aqueous dispersion and were killed 20 weeks later.
-------
TABLE 25
Induction of Respiratory Tract Tumors in Syrian Golden Hamsters by Intratracheal Instillation of PAH
Compound
BaP
BaP
BaP
BaP
DaP
BaP
BaP and Fe2O3
RaP and Fe20,, coated
DaP and Fe2O,, ground
BaP and Fe2CK, mixed
BaP and gelatin
BaP and Fe2O3
BaP and Fe2O3
DaP and Fe-,0-.
BaP and Fe2O3
No. Animals
30
30
30
29
28
48
48
49
49
43
46
28 (male) , 29 (female)
33 (male) , 34 (female)
33 (male) , 30 (female)
47 (male) , 41 (female)
Total Dose,
mg
3.25a
6.5a
13
26a
52a
30b
30b
26. lc
27.4°
26. 3C
26. 4C
60d
30d
I5d
7.5d
Respiratory Tumor
Incidence,
Percent
10
13
30
86
93
15
71
73
84
12
17
60.7 (male), 58.6 (female)
66.7 (male), 58.8 (female)
30.3 (male), 30.0 (female)
12.8 (male), 9.8 (female)
Reference
Feron, et al. 1973
Feron, et al. 1973
Feron, et al. 1973
Feron, et al. 1973
Feron, et al. 1973
Sellakumar, et al. 1976
Sellakumar, et al. 1976
Henry, et al. 1975
Henry, et al. 1975
Henry, et al . 1975
Henry, et al. 1975
Saffioti, et al. 1972
Saffioti, et al. 1972
Saffioti, et al. 1972
Saffioti, et al. 1972
-------
TftBLE 25 (cont.)
O
VD
to
Compound
BaP
BaP
OB (a, i)P
nB(a,i)P
DMBA and Fe2O3
DMBA and Fe2Oj
No. Animals
32 (male)
28 (female)
48
48
46
28
Total Dose,
mg
30e
30e
12f
8.59
1.2h
0.851
Respiratory Tumor
Incidence,
Percent
42.3
57.7
75
64.6
43.5
46.4
Reference
Kobayashi
Kobayashi
Stenback
1974a
Stenback
1974a
Stenback
1974b
Stenback
1974b
, 1975
, 1975
and Sellakumar,
and Sellakumar,
and Sellakumar,
and Sellakumar,
Animals treated once weekly for 52 weeks with BaP suspended in 0.9* NaCl solution.
3 mg BaP administered once weekly for 10 weeks.
"Animals received 30 weekly intratracheal instillations.
-j
Animals received 30 weekly instillations of BaP mixed with equal amounts of Fe2O, and suspended in 0.2 ml saline.
"Animals received 30 weekly intratracheal instillations of BaP suspended in 0.9% Nad.
Animals received 12 weekly intratracheal instillations of 1 mg r>B(a,i)P suspended in distilled water.
^Animals received 17 weekly intratracheal instillations of 0.5 mg DB(a,i)P suspended in distilled water.
'Animals received 100 yig DMBA and 100 |ig Fe^O-, intratracheally once a week for 12 weeks in saline suspensions.
"Animals received 50 ii'J DMBA ami !50 \,q "e-O intratrachealJy once a week for 17 weeks in saline suspensions.
h
-------
TABLE 26
Induction of Respiratory Tract Tumors in Rats and Mice
Compound
DMBA and
Indian ink
DMDA and
Indian ink
OMBA and
_^ Indian ink
1
VD
U) DB(a,h)A
MCA
MCA
MCA
Organism
Rat
(Wistar and
random-bred)
Rat
{Wistar and
random-bred)
Rat
(Wistar and
random-bred)
Mouse
(DBA/2)
Rat
(Osborne-Mendel)
Rat
(Osborne-Mende] )
Rat
(Osborne-Mendel)
No.
Animals
34
56
61
14 (male)
13 (female)
100
100
100
Total Dose,
nig
2.5a
6b
10°
236 (male)d ,
179 (female)0
0.005f
0.05C
0.10f
Route of
Administration
Intratracheal
instillation
Intratracheal
instil lation
Intratracheal
instillation
Oral
Pulmonary
injection
Pulmonary
injection
Pulmonary
injection
Tumor
Incidence,
%
17.6
35.7
26.2
100 (male)6
77 (female)6
lq
13*
27g
Reference
Pylev, 1962
Pylev, 1962
Pylev, 1962
Snell atid Stewart,
1962
Ilirano, et al .
1974
Ilirano, ct al.
1974
Hirano, et al.
1974
-------
TABLE 26 (cont.)
O
I
ID
Compound
MCA
MCA
MCA
MCA
'""I" AnKil. Tot'iD0"'
Rat 100 0.20f
(Osborne-Mendel)
Rat 100 0.30£
(Osborne-Mendel)
Rat 100 0.40f
(Osborne-Mendel)
Rat 100 0.50E
(Osborne-Mendel)
«.BKrSllM, -=-
Pulmonary 47q
injection
Pulmonary 40^
injection
Pulmonary 51^
injection
Pulmonary 45g
injection
Reference
llirano, et al.
1974
llirano, et al .
1974
llirano, et al.
1974
llirano, et al.
1974
Administered as a single dose with 0.2 mg of Indian ink in 0.2 ml of a colloid protein solution.
3Administered as three 2 mg doses at monthly intervals with 0.2 mg of Indian ink in 0.2 ml of a colloid protein solution.
cAdministered as five 2 mg doses at monthly intervals with 0.2 mg of Indian ink in 0.2 ml of a colloid protein solution.
Administered as an aqueous-olive oil emulsion of DB(a,h)A given in place of drinking water for 237 to 279 days.
'Tumors were alveologenic carcinomas, a 100% incidence of pulmonary adenomatosIs was also observed.
Administered as a single MCA-containing beeswax pellet placed directly into the lower peripheral segment of tho left lung.
squamous cell carcinoma.
-------
The published literature regarding chemical carcinogenesis in
cell cultures is vast, despite the fact that systematic studies
were not begun until the early 1960's due to the lack of a reproduc-
ible transformation assay. Berwald and Sachs (1963) first demon-
strated that polycyclic hydrocarbons (MCA, BaP) could cause the
direct malignant transformation of hamster embryo cells in culture.
Transformed colonies have growth characteristics visually distinct
from normal colonies and are readily seen above a background of
normal cells. This assay can therefore be easily used as a screen
to compare carcinogenic activity of suspect compounds. A common
feature of these, and nearly all, transformed cells is that they
give rise to fibrosarcomas upon inoculation into immunosuppressed
animals. In addition to hamster embryo cells, malignant transfor-
mation has been demonstrated in organ cultures, liver cell cul-
tures, fibroblastic cells derived from mouse ventral prostate, 3TC
cell lines derived from mouse embryo cells, and various types of
epithelial cells from humans and other animals (Heidelberger, 1973,
1975; Heidelberger and Boshell, 1975).
Early reports by Berwald and Sachs (1965) and Dipaolo and
Donovan (1967) described alterations in hamster embryo cells in-
duced by BaP, DMBA, and MCA which could be used as indicators of a
change from normal to neoplastic state. The compounds were applied
to cells in culture either dissolved in paraffin and impregnated on
filter disks or as a colloidal suspension in growth medium. Fol-
lowing marked cytotoxicity, foci of transformed cells developed
which displayed chromosomal abnormalities and the ability to grow
indefinitely in culture. In addition, these transformed mass cul-
C-95
-------
tures, when transplanted to four- to six-week-old hamsters, contin-
ued to grow and form tumors. A good correlation was obtained be-
tween _in vitro carcinogenicity of a polycyclic hydrocarbon and the
number of transformed clones they produced. The maximum rate of
cell transformation in these studies was 25.6 percent in surviving
cells, obtained by treatment with 10 ug/ml of BaP for six days. BaP
treatment at 1 yg/ml for six days produced 19.9 percent transforma-
tion in surviving cells. Further data indicating the activity of
several polycyclic carcinogens and their derivatives are summarized
in Table 27. The K-region epoxides of DBahA and MCA are more active
in the production of malignant transformation in hamster embryo
cells than the parent hydrocarbons or the corresponding K-region
phenols (Grover, et al. 1971; Huberman, et al. 1972). Although
these results confirm the view that metabolism is necessary for
carcinogenic activity, they conflict with data generated in_ vivo
which indicate that K-region epoxides of polycyclic carcinogens are
less active than the parent compound in various species. A possi-
ble reason for the lack of correlation is the relative instability
of K-region epoxides as compared to the parent hydrocarbon when
applied to the skin. It is likely that ir\ vivo far less of the
reactive K-region epoxide can survive passage through the skin to
reach the basal cell layer. Furthermore, it has become apparent
that the non-K-region diol-epoxide is likely to be the ultimate
carcinogenic metabolite for most PAH. Several investigators have
also made it evident that the toxicity and transforming activity of
PAH are dissociable and occur by different processes (Landolph, et
al. 1976; DiPaolo, et al. 1971a,b), with the toxicity being due to
C-96
-------
o
I
TABLE 27
Hamster Embryo Cell Transformation Produced by Several Polycyclic Hydrocarbons and Their Derivatives
Concentration,
Mq/ml
DB(a,h)Aa
DB(a,h)Ab
DB ( a , h) R5 , 6-epox i dea
DB( a, h)A5, 6-epox ideb
MCAC
MCAd
MCA epoxide0
BaPd
2.5
5
10
2.5
5.0
10
2.5
5
7.5
10
2.5
5.0
7.5
10
2.5
5
7.5
2.5
3.5
5
7
1
5
Total No.
Colonies
760
690
790
1,341
1,363
1,365
590
601
395
350
895
(166
817
707
404
370
349
664
364
245
103
l,OJ6
394
Cloning
Efficiency,
%
4.2
3.B
4.4
13.4
14.0
14.5
3.3
3.3
2.5
1.9
10.1
9.3
9.3
7.7
10.1
9.2
8.7
9.6
2.4
1.5
0.7
8.46
7.17
No.
Transformed
Colonies
4
4
7
3
11
7
3
12
31
14
7
20
22
30
9
10
15
20
13
8
17
25
21
Transformation, Reference
0.5
0.7
0.9
0.2
0.8
0.5
0.5
2.0
7.8
4.0
0.8
2.3
2.7
4.2
2.2
2.7
4.3
3.46
3.6
3.3
16.5
2.46
5.33
Huberman, et al. 1972
Huberman, et al. 1972
Huberman, et al. 1972
Grover, et al. 1971
Grover, et al. 1971
Grover, et al. 1971
Huberman, et al . 1972
Huberman, et al. 1972
Huberman, ot al. 1972
Huberman, et al. 1972
Grover, et al . 1971
Grover, et al. 1971
Grover, et al. 1971
Grover, et al. 1971
Huberman, et al. 1972
Huberman, et al. 1972
Huberman, et al. 1972
DiPaolo, et al . 1971a,b
Huberman, et al. 1972
Huberman, et al. 1972
Huberman, et al. 1972
DiPaolo, et al . 1971a
DiPaolo, et al . 1971a
a7-day treatment of cells seeded on a feeder layer.
7-8 day treatment of cells.
c
4-hour treatment of cells seeded in conditioned medium.
8-day treatment of cells.
-------
random alkylation of nucleophilic regions within the cell. How-
ever, when hamster embryo cells are pretreated with weak chemical
carcinogens which can induce microsomal enzyme activity [e.g.,
benz(a)anthracene, methyl methanesulfonate, ethyl methanesulfo-
nate] before the addition of a potent carcinogen (e.g., MCA, BaP,
DMBA), transformation may be considerably enhanced (DiPaolo, et al.
1971a,b, 1974) .
As a prescreen for chemical carcinogens, cell transformation
in vitro may be one of the most sensitive techniques available.
Pienta and coworkers (1977) reported that 90 percent (54/60) of the
carcinogens they tested transformed hamster embryo cells iri vitro,
whereas none of the noncarcinogens tested showed any activity.
Moreover, many of the carcinogens which have not been shown to be
mutagenic toward S._ tymphimurium _in vitro (e.g., chrysene) were
capable of transforming the hamster cells. It is noteworthy, how-
ever, that large differences exist in dosage requirements for
transformation among those various test systems. Calculations have
been made which show that a battery of tests using S]_ typhimurium
(Ames assay) , polymerase A-def icient E_._ coli, and hamster embryo
cell transformation is capable of detecting nearly all carcinogens
tested, both PAH and non-PAH types.
The alteration of microsomal enzyme activity either _ini vitro
or in, vivo is known to have a marked effect on the carcinogenic re-
sponse to PAH. Nesnow and Heidelberger (1976) reported that in
10T1/2CL8 cells' a line of contact-sensitive C3H mouse embryo
fibroblasts, transformation in culture was altered by chemical mod-
ifiers of microsomal enzymes. Pretreatment of 10T,/2C18 cells with
C-98
-------
benz(a)anthracene, a microsomal enzyme inducer, caused a doubling
in MCA-mediated transformation. Similarly, treatment with inhibi-
tors of epoxide hydrase (e.g., cyclohexene oxide; styrene oxide;
l,2,3,4-tetra-hydronaphthalene-l,2-oxide)caused an increase in
transformation over that obtained with MCA treatment alone. Thus,
treatments which can induce epoxide-forming enzymes and/or lower
the activity of epoxide-degrading enzymes seemed to enhance the
degree of transformation in cultured cells by altering steady-state
levels of oncogenic epoxides.
Chen and Heidelberger (1969a,b) developed a system using C3H
mouse ventral prostate cells to examine transformation by carcino-
genic hydrocarbons under conditions in which no spontaneous malig-
nant transformation occurred. Cells treated with MCA (1 yg/ml) for
six days in culture produced fibrosarcomas in 100 percent of mice
into which they were subcutaneously injected. When treated for
only one day with MCA at the single cell stage, transformed foci
were found in all clones grown to confluency. A good quantitative
correlation was obtained between the in vivo oncogenic activity of
eight hydrocarbons (including BaP, MCA, DMBA, and DBahA) and the
number of transformed colonies produced in this system. In con-
trast to the enhanced transforming ability of K-region epoxides
relative to the parent hydrocarbon in hamster embryo cells, the
K-region epoxide derived from DMBA was less active and the K-region
epoxides from MCA, DBahA, and benz(a)anthracene were more active
than the parent compound in mouse prostrate cells (Marquardt, et
al. 1972, 1974). Moreover, the epoxide derived from DMBA was more
toxic than DMBA itself. The anomalous behavior of DMBA may have
C-99
-------
been due, however, to a decreased intracellular half-life of the
epoxide because of its greater chemical reactivity.
Attempts to transform human cells in culture with PAH (e.g.,
BaP, MCA, DMBA) have generally met with failure (Leith and Hay-
flick, 1974). However, Rhim and coworkers (1975) reported that a
human osteosarcoma clonal cell line could be further transformed _in_
vitro with DMBA. Morphologic alterations and abnormal growth pat-
terns became evident in cells treated with DMBA at 2.5 and 1.0
ug/ml in the fifth subculture 52 to 57 days after exposure. One of
the altered cell lines obtained from the 1 yg/ml treatment was
tumorigenic in nude mice by subcutaneous and intracerebral injec-
tion. Interpretation of the significance of these results is made
difficult by the fact that an aneuploid sarcomatous cell line had
to be employed in order to demonstrate successful transformation.
The use of organ cultures for the assessment of chemical car-
cinogenicity suffers from the lack of reliable biochemical and mor-
phological parameters for measuring early neoplastic changes.
Nevertheless, pioneering work in the application of organ culture
to chemical carcinogenesis was performed by Lasnitzki (1963).
Microgram quantities of MCA added to organ cultures of rat and
mouse prostate fragments caused extensive hyperplasia and squamous
metaplasia. However, these preneoplastic morphological effects are
generally not associated with subsequent tumor development when
carcinogen-treated pieces of tissue are implanted into host animals
(Heidelberger, 1973). Limited success has been achieved with organ
cultures of rat tracheas, which showed characteristic morphologic
alterations when treated with DMBA, BaP, and MCA (Heidelberger,
C-100
-------
1973). In addition, Crocker (1970) has exposed respiratory epithe-
lia from the hamster, rat, dog, and monkey to BaP at 7 to 15 pg/ml
and observed occasional squamous metaplasia. More commonly, pleo-
morphic cells in a dysplastic epithelium were evident as a result
of the treatment. Rat tracheas maintained in organ culture have
been suggested as a useful system for the predictive screening of
potential carcinogens (Lindsay, et al. 1974).
A unique organ culture technique has recently been reported in
which BaP (4 or 12 mg) was administered to pregnant mice (strain A
and C57B1), and lung tissue of their 19- to 20-day-old embryos was
subsequently explanted in culture (Shabad, et al. 1974). A trans-
placental influence of BaP was manifested as a proliferative stimu-
lus in embryonic lung tissue. Hyperplasia arising in the bronchial
epithelium led to the development of adenomas in a large percentage
of the explants.
In the environment, man is unlikely to come in contact with
only a single PAH, regardless of the route of exposure. Instead,
PAH occur as complex mixtures in all environmental media. Despite
this generally accepted fact, very few studies have been conducted
on the carcinogenicity of defined PAH mixtures.
Among the most relevant studies conducted on the effects of
PAH mixtures were those concerned with the carcinogenic components
of automotive engine exhaust. Pfeiffer (1973, 1977) treated grouos
of 100 female NMRI mice with single subcutaneous injections of a
mixture containing 10 noncarcinogenic PAH, in addition to BaP
and/or dibenz(a,h)anthracene. The treatment combinations and dos-
ages are summarized in Table 28. As the results deoicted in
C-101
-------
o
I
TABLE 28
Classification of Test Groups*
A Dose
A , .
(U9)
A, 3.12
A, 6.25
A^ 12.5
A^ 25.0
A^ 50.0
Ag 100.0
C
Substance
benzo(e) pyrene
benzo(a) anthracene
phenanthrene
anthracene
pyrene
f luoranthene
chrysene
perylene
ben zo(ghi) perylene
coronene
D
Substance
benzo (a) pyrene
Cl
dose
(ug)
2.15
3.125
125.0
31.25
65.1
28.1
3.125
0.2
12.8
3.125
C2
dose
(ug)
4.3
6.25
250.0
62.5
131.2
56.25
6.25
0.4
25.6
6.25
B
Bl
B2
B3
B*
^
B6
C3
dose
(M9)
8.75
12.5
500.0
125.0
262.5
112.5
12.5
0.87
51.25
12.5
E
Dose
(ug)
2.35 d
4.7
9.3
18.7
37.5
75.0
C4
dose
(ug)
17.5
25.0
1,000.0
250.0
525.0
225.0
25.0
1.75
102.5
25.0
Substance
ibenz (a,h) anthracene
C5
dose
(ug)
35.5
50.0
2,000.0
500.0
1,050.0
450.0
50.0
3.5
205.0
50.0
C6
dose
(ug)
70.0
100.0
4,000.0
1,000.0
2,100.0
900.0
100.0
7.0
410.0
100.0
Si
D<
Dc
D;
A
B/
BC
B;
E.
E;
Ec
E;
Si
D;
*Source: Pfeiffer, 1977
-------
Table 29 indicate, increases in tumor incidence could be attributed
to the presence of increased amounts of BaP and of dibenz(a,h)an-
thracene. It is noteworthy that, at the lower dosages, dibenz-
(a,h) anthracene was more effective in producing tumors at the in-
jection site than was BaP. Moreover, no effect of the 10 noncarci-
nogens on tumorigenic response was evident. Probit analysis of tu-
mor incidence data indicated that the tumorigenic response from ap-
plication of all 12 PAH was attributable solely to dibenz(a,h)an-
thracene.
Similar studies intended to reveal carcinogenic interactions
among PAH found in automobile exhaust were conducted by Schmahl, et
al. (1977) . Eleven PAH were selected for their experiments, and
various combinations were applied to the skin of NMRI mice in a
proportion based on their respective weights in automobile exhaust
(Table 30). Animals received twice weekly treatments for life (or
until a carcinoma developed). Their results (Table 31) indicated
that a mixture of carcinogenic PAH was more effective than BaP
alone, and that the whole mixture (carcinogenic plus noncarcinogen-
ic PAH) was not significantly more effective than the carcinogenic
PAH group alone. Thus, the carcinogenic effects observed were
solely attributable to the carcinogenic components of the mixture.
Human data: Although exposure to PAH occurs predominantly by
direct ingestion (i.e., in food and in drinking water) there are no
studies to document the possible carcinogenic risk to humans by
this route of exposure. It is known only that significant quanti-
ties of PAH can be ingested by humans, and that in animals such ex-
posures are known to cause cancers at various sites in the body.
C-103
-------
TABLE 29
Tumor Incidence Resulting, by the End of the 114th Week,
from a Single Subcutaneous Application of Test Substances*
BaP Group (A)
n
i
o
Dose
3
6
12
25
50
100
(yg)
.12
.25
.5
.0
.0
.0
No. of
Tumors
9
35
51
57
77
83
DBA
Dose
2
4
9
18
37
75
Group (B) BaP + DAB Group (D)
(M9)
.35
.7
.3
.7
.5
.0
No. of
Tumors
37
39
44
56
65
69
No. of
Tumors
48
44
61
68
69
79
10 PAH Group (C)
No. of
Tumors
6
8
6
4
13
5
12 PAH Group (E)
No. of
Tumors
41
55
61
72
68
82
*Source: Pfeiffer, 1977
-------
TABLE 30
Doses (yg) Applied in Dermal Administration
Experiments, in Relation to Benzo(a)pyrene*
Controls
Acetone
Benzo(a) pyrene
C PAH
Benzo(a) pyrene
Dibenz (a,h) anthracene
Benzo(a) anthracene
Benzo (b) fluoranthene
total
NC PAH
(Benzo (a) pyrene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Chrysene
Benzo(e) pyrene
Benzo (ghi ) perylene
total
C PAH + NC PAH
(Benzo (a) pyrene
Total C PAH
Total NC PAH
Total C PAH + NC PAH
Relation of C PAH-.NC PAH
as solvent
1.0
1.0
0.7
1.4
0.9
4.0
1.0
27.0
8.5
10.8
13.8
1.2
0.6
3.1
65.0
1.0
4.0
65.0
69.0
is constantly
1.7
1.7
1.2
2.4
1.5
6.8
3.0
81.0
25.5
32.4
41.4
3.6
1.8
9.3
195.0
1.7
6.8
110.5
117.3
1:16.25
3.0
3.0
2.1
4.2
2.7
12.0
9.0 27.0)
243.0 729.0
76.5 229.5
97.2 291.6
124.2 372.6
10.8 32.4
5.4 16.2
27.9 83.7
585.0 1,755.0
3.0)
12.0
195.0
207.0
*Source: Schmahl, et al. 1977
C-105
-------
TABLE 31
Findings at the Site of Application of PAH to Mouse Skin*
Histological
o
i
0
CTi
Application
Solvent
BaP
BaP
BaP
C PAH
C PAH
C PAH
NC PAH
NC PAH
NC PAH
NC PAH
C PAH +
NC PAH
C PAH +
NC PAH
C PAH 4-
NC PAH
Single
Dose
yg
-
1.0
1.7
3.0
4.0
6.8
12.0
65.0
195.0
585.0
1,755.0
69.0
117.3
207.0
Initial
No. of
Animals
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Effective
No. of
Animals
81
77
88
81
81
88
90
85
84
88
86
89
93
93
Negative
Abs. %
80
66
63
36
52
31
25
84
84
87
70
43
36
28
99
86
72
44
64
35
28
99
100
99
81
48
39
30
Diagnosis at the Site of
Papilloma Carcinoma
Abs. % Abs. %
_
11 10
25
23 43
45 25
33 53
11 63
- - 1
_ _
- - 1
- - 15
11 44
22 54
11 64
-
13
28
53
31
60
70
1
-
1
17
49
58
69
Application
Sarcoma
Abs. %
1 1
-
-
-
-
1 1
1 1
-
-
-
1 1
1 1
1 1
- -
aThe decimal points have been rounded off; therefore, the sum of % values will not always be
equivalent to 100%.
*Source: Schmahl, et al. 1977
-------
Convincing evidence from air pollution studies indicates an
excess of lung cancer mortality among workers exposed to large
amounts of PAH-containing materials such as coal gas, tars, soot,
and coke-oven emissions (Kennaway, 1925; Kennaway and Kennaway,
1936, 1947; Henry, et al. 1931; Kuroda, 1937; Reid and Buck, 1956;
Doll, 1952; Doll, et al. 1965, 1972; Redmond, et al. 1972, 1976;
Mazumdar, et al. 1975; Hammond, et al. 1976; Kawai, et al. 1967).
However, no definite proof exists that the PAH present in these
materials are responsible for the cancers observed. Nevertheless,
our understanding of the characteristics of PAH-induced tumors in
animals, and their close resemblance to human carcinomas of the
same target organs, strongly suggests that PAH pose a carcinogenic
threat to man, regardless of the route of exposure (Santodonato, et
al. 1980).
The magnitude of the carcinogenic risk of PAH to man remains
obscure in the community setting. Ambient levels of PAH in air are
much lower than are encountered in occupational situations, and
populations exposed are much more heterogeneous with regard to age,
sex, and health status. However, the current state of knowledge
regarding chemical carcinogenesis would lead to the conclusion that
the number of cancers produced is directly proportional to the dose
received by any route. One must assume, therefore, that the small
amounts of PAH present in the environment (air, food, and water)
under ambient conditions contribute in some degree to the observed
incidence of lung cancer in most populations.
C-107
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CRITERION FORMULATION
Existing Guidelines and Standards
There have been few attempts to develop exposure standards for
PAHs, either individually or as a class. In the occupational set-
ting, a Federal standard has been promulgated for coke oven emis-
sions, based primarily on the presumed effects of the carcinogenic
PAH contained in the mixture as measured by the benzene soluble
fraction of total particulate matter. Similarly, the American Con-
ference of Governmental Industrial Hygienists recommends a work-
place exposure limit for coal tar pitch volatiles, based on the
benzene-soluble fraction containing carcinogenic PAH. The National
Institute for Occupational Safety and Health has also recommended a
workplace standard for coal tar products (coal tar, creosote, and
coal tar pitch), based on measurements of the cyclohexane extract-
able fraction. These standards are summarized below:
Substance Exposure Limit Agency
Coke Oven 150 ug/m, 8-hr. U.S. Occupational Safety
Emissions time-weighted and Health Administration
average
Coal Tar Pro- 0.1 mg/m , 10-hr. U.S. National Institute
ducts time-weighted for Occupational Safety
average and Health
Coal Tar Pitch 0.2 mg/m (benzene American Conference of
of Volatiles soluble fraction) Governmental Industrial
8-hr, time- Hygienists
weighted average
A drinking water standard for PAH as a class has been devel-
oped. The 1970 World Health Organization European Standards for
Drinking Water recommends a concentration of PAH not to exceed 0.2
ug/1. This recommended standard is based on the composite analysis
C-108
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of six PAH in drinking water: 1) fluoranthene, (2) benzo(a)pyrene,
(3) benzo(g,h,i) perylene, (4) benzo(b)fluoranthene, (5) benzo(k)-
fluoranthene, and (6) indeno(l,3,-cd)pyrene.
The designation of these six PAH for analytical monitoring of
drinking water was not made on the basis of potential health ef-
fects or bioassay data on these compounds (Borneff and Kunte,
1969) . Thus, it should not be assumed that these six compounds
have special significance in determining the likelihood of adverse
health effects resulting from absorption of any particular PAH.
They are, instead, considered to be useful indicators for the pre-
sence of PAH pollutants. Borneff and Kunte (1969) found that PAH
were present in ground water at concentrations up to 50 ng/1, and
in drinking water at concentrations up to 100 ng/1. Based on these
data they suggested that water containing more than 200 ng/1 should
be rejected. However, as data from a number of U.S. cities indi-
cate (see Exposure section), levels of PAH in raw and finished
waters are typically much less than the 0.2 yg/1 criterion.
Current Levels of Exposure
This report presents considerable data which may be used to
calculate an estimate of human exposure to PAH by all routes of
entry to the body. However, quantitative estimates of human expo-
sure to PAH require numerous assumptions concerning principal
routes of exposure, extent of absorption, conformity of human life-
style, and lack of geographic-, sex-, and age-specific variables.
Nevertheless, by working with estimates developed for PAH as a
class, it is possible through certain extrapolations to arrive at
an admittedly crude estimate of PAH exposure.
C-109
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Unfortunately, there are no environmental monitoring data
available for most of the PAH which are specified under the Consent
Decree in NRDC v. Train. By far the most widely monitored PAH in
the environment is BaP; data on BaP levels in food, air, and water
are often used as a measure of total PAH. Among the PAH routinely
monitored in water, four compounds are included in the Consent
Decree list: BaP, IP, BbFL, and BjFL. In addition, levels of PL
and BPR have been routinely determined in water, as recommended by
the World Health Organization.
The reported estimated average concentrations of BaP, carcino-
genic PAH (BaP, BjFL, and IP) , and total PAH in drinking water are
0.55 ng/1, 2.1 ng/1, and 13.5 ng/1, respectively (see Exposure sec-
tion; Basu and Saxena, 1977) . Thus, assuming that a human consumes
2 liters of water per day, the daily intake of PAH via drinking
water would be:
0.55 ng/1 x 2 liters/day =1.1 ng/day (BaP)
2.1 ng/1 x 2 liters/day =4.2 ng/day (carcinogenic PAH)
13.5 ng/1 x 2 liters/day = 27.0 ng/day (total PAH)
Borneff (1977) estimates that the daily dietary intake of PAH
is about 8 to 11 yg/day. As a check on this estimate, PAH intake
may be calculated based on reported concentrations in vairious foods
(see Exposure section) and the per capita estimates of food con-
sumption by the International Commission on Radiological Protection
(1974). Taking a range of 1.0 to 10.0 ppb as a typical concentra-
tion for PAH in various foods, and 1,600 g/day as the total daily
food consumption by man from all types of foods (i.e., fruits,
vegetables, cereals, dairy products, etc.), the intake of PAH from
C-110
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the diet would be in the range of 1.6 to 16.0 yg/day. An estimate
of BaP ingestion from the diet may be similarly derived. Using 0.1
to 1.0 ppb as the range of BaP concentration in various foods,
total daily BaP intake would be 0.16 to 1.6 pg/day.
Ambient air is reported to contain average levels of 0.5
33 3
ng/m , 2.0 ng/m , and 10.9 ng/m for BaP/ carcinogenic PAH, and
total PAH, respectively (see Exposure section, Table 15). Taking
the range of 15 m to 23 m as the average amount of air inhaled by
a human each day results in an estimated intake of 0.005 to 0.0115
ng/day, 0.03 to 0.046 ng/day, and 0.164 to 0.251 ng/day for BaP,
carcinogenic PAH, and total PAH, respectively.
In summary, a crude estimate of total daily exposure to PAH
would be as shown in Table 32.
Two important factors are not taken into account in this esti-
mate. First, it is known that tobacco smoking can contribute
greatly to PAH exposure in man. Exposure to BaP from smoking one
pack of cigarettes per day was shown to be 0.4 ug/day (WAS, 1972).
Second, the possibility for dermal absorption of PAH is assumed to
contribute only a negligible amount to the total exposure. Only in
certain occupational situations is dermal exposure expected to be
quantitatively important.
Special Groups at Risk
An area of considerable uncertainty with regard to the carci-
nogenic hazard of PAH to man involves the relationship between aryl
hydrocarbon hydroxylase (AHH) activity and cancer risk. Genetic
variation in AHH inducibility has been implicated as a determining
factor for susceptibility to lung and laryngeal cancer (Kellerman,
C-lll
-------
TABLE 32
Estimate of Human Exposure to PAH from Various Media
Source
Water
Food
o Air
1
*° Total
Estimated Exposure
BaP Carcinogenic PAHa Total PAH
0.0011 Mg/day 0.0042 ug/day 0.027 ug/day
0.160-1.6 ug/day 1.600-0.251 ug/day
0.005-0.0115 ug/day 0.03-0.046 ug/day 0.164-0.251 ug/day
0.166-1.6 ug/day 1.6-16 ug/day
aTotal of BaP, BjFL, and IP; no data are available for food.
-------
et al. 1973a,b). It was suggested that the extent of AHH induci-
bility in lymphocytes was correlated with increasing susceptibility
to lung cancer formation.
Paigen, et al. (1978) have examined the question of genetic
susceptibility to cancer, and concluded that epidemiologic evidence
supports this hypothesis. Moreover, they were able to show that
AHH inducibility in lymphocytes segregates in the human population
as a genetic trait. However, their studies failed to find a corre-
lation between this inducibility and presumed cancer susceptibil-
ity, either among healthy relatives of cancer patients or in pa-
tients who had their cancer surgically removed. It is noteworthy
that previous investigations on AHH inducibility were conducted in
persons with active cancer.
Recent studies with other human tissues (liver and placenta)
have provided important new data concerning the carcinogen-metabo-
lizing capacity of man and its implications for cancer susceptibil-
ity. Conney, et al. (1976) examined individual differences in the
metabolism of drugs and carcinogens in human tissues, and have
identified drugs which may serve as model substrates to provide an
indirect index of carcinogen metabolism for man. The rates for
antipyrene, hexobarbital, and zoxazolamine hydroxylation in human
autopsy livers were highly, but not perfectly, correlated with the
rates of BaP metabolism. In human placenta, an almost perfect cor-
relation was found between zoxazolamine hydroxylase activity and
BaP hydroxylase activity (Kapitulnik, et al. 1976a). Thus, metab-
olism of BaP and zoxazolamine by human placenta occurs by the same
enzyme system(s) or by different enzyme systems under the same reg-
C-113
-------
ulatory control (Kapitulnik, et al. 1977a) . BaP and 2;oxazolamine
hydroxylase activities were also shown to be significantly enhanced
in placentas obtained from women who smoked cigarettes.
The lack of perfect correlations for the hepatic metabolism of
BaP and certain drugs in many subjects indicated the presence of
several monooxygenases in human liver which catalyze the oxidative
metabolism of these compounds. Furthermore, large inter-individual
differences exist in the capacity of humans to metabolize foreign
chemicals both in_ vitro and rn vivo. Further studies showed that
7,8-benzoflavone markedly stimulated the hydroxylation of BaP,
antipyrene, and zoxazolamine in human liver samples, but with a
wide variation in magnitude among different samples. These results
suggested the presence of multiple monooxygenases or cytochrome
P-450 in the different liver samples (Kapitulnik, et al. 1977b).
Moreover, 7,8-benzoflavone did not affect the hydroxylation of
coumarin or hexobarbital, thereby indicating the existence of dif-
ferent monooxygenases for metabolism of these substrates.
Multiple forms of cytochrome P-450 have been shown in the
livers of rats, rabbits, and mice, but not thus fair in humans
(Kapitulnik, et al. 1977a). More important, however, MCA is a
potent inducer of BaP hydroxylase activity in rats but does not
stimulate antipyrene hydroxylase, clearly suggesting that metabo-
lism of PAH in rodents may be regulated by different enzyme systems
than in humans (Kapitulnik, et al. 1977a).
In contrast to the apparent multiplicity of cytochrome P-450
dependent enzyme systems for the oxidative metabolism of PAH in
man, a single epoxide hydrase with broad substrate soecificity may
C-114
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be present in human liver (Conney, et al. 1976? Kapitulnik, et al.
1977c). Because the hydration of arene oxides may lead to the for-
mation of dihydrodiol carcinogen precursors, the capacity of dif-
ferent humans to metabolize epoxides may affect cancer susceptibil-
ity. It is not known, however, if enhanced dihydrodiol formation
would increase cancer risk or decrease cancer risk.
Thomson and Slaga (1976) did not obtain a correlation of AHH
induction with skin-tumor-inducing ability in mice for a series of
unsubstituted hydrocarbons. Nevertheless, the highest AHH enzyme
activity was found in the epidermal layer of the skin, which is the
major point of contact with many environmental chemicals. These
results may be interpreted to indicate that a chemical carcinogen
may not necessarily induce its own bioactivation, but instead can
be transformed into a reactive intermediate by virtue of increased
AHH activity stimulated by other noncarcinogenic compounds.
Due consideration must also be given to the fact that, in
addition to the initiation of resting cells by a chemical carcino-
gen, a promotion phase involving cell proliferation is also in-
volved in skin carcinogenesis (Yuspa, et al. 1976). Therefore,
although certain aromatic hydrocarbons are effective enzyme induc-
ers, their bioactivated metabolites may function onlv as initiators
having no promoting ability. A potent complete carcinogen, how-
ever, will be transformed not only into a powerful tumor initiator
but will also be able to interact with cellular membranes, alter
genetic expression, and ultimately cause irreversible cell prolif-
eration. These observations raise certain doubts concerning the
validity and/or reliability of equating enzyme inducibility with
C-115
-------
carcinogenic potential for chemical agents. Further reinforcement
of this opinion has been provided by Shulte-Hermann (1977) who
showed that cell proliferation is not a direct result of enzyme
induction, even though both processes are normally coupled.
The further possibility that the genetics of AHH inducibility
is organ-dependent rather than strain-dependent in animals has
important implications for evaluating susceptibility to PAH-
induced cancers (Kouri, et al, 1976). Most significant is the
demonstration that pulmonary AHH may be inducible in all strains of
mice, regardless of the inducibility of hepatic AHH. Since the
respiratory epithelium represents a primary portal of entry for
PAH, AHH activity which is induced in this tissue may bear impor-
tantly on susceptibility to malignancy.
Enzyme induction by PAH is not limited to AHH. Owens (1977)
recently demonstrated that MCA can induce hepatic UDP-glucuronosyl-
transferase activity in certain inbred strains of mice. This en-
zyme catalyzes the conjugation and excretion of PAH substrates
after they have first been oxygenated by AHH. The induction of
this transferase activity and that of AHH was apparently regulated
by a single genetic locus. However, transferase inducibility does
not depend on AHH levels, but rather is stoichiometrically related
to the concentration of a specific and common cytosolic receptor
regulating both enzyme induction processes. Owens further demon-
strated that AHH activity can be fully induced in certain mouse
strains (e.g., by 2,3,7,8-tetrachlorodibenzo-p-dioxin) without
greatly enhancing the transferase activity. Earlier studies had
established that chrysene and chlorpromazine were potent inducers
C-116
-------
of AHH activity while having little effect on transferase activity
(Aitio, 1974a,b). Subsequent exposure to carcinogenic PAH (i.e.,
MCA) could lead to maximal oxidative metabolism but little trans-
ferase-catalyzed removal of metabolites by glucuronic acid coniuga-
tion. This situation would be exacerbated by the fact that metabo-
lites of MCA are incapable of further inducing the transferase
activity. This effect may have considerable toxicologic signifi-
cance in that the highly reactive epoxides of PAH formed by the
action of AHH under these circumstances may not be adequately re-
moved by glucuronidation. Thus, one must consider the total expo-
sure of all environmental agents and their possible effect on crit-
ical enzymatic processes before attempting to assess the toxicolog-
ic impact of exposure to a specific PAH. Tn summary, there is a
need to further explore the relative effects of enzyme induction on
the metabolic activation of chemicals to toxic products, versus
metabolism of chemicals via detoxification pathways, when consider-
ing the possibility of special groups at risk.
Basis and Derivation of Criterion
The presently available data base is inadequate to support the
derivation of individual criteria for each of the PAH as specified
under the Consent Decree. This problem arises primarily from the
diversity of test systems and bioasssay conditions employed for
determining carcinogenic potential of individual PAH in experiment-
al animals. Furthermore, it is not possible to estimate the intake
via water of individual PAH, except for those compounds which have
been selected by the World Health Organization for environmental
monitoring. Therefore, an approach to criterion development is
C-117
-------
adopted in this report with the objective of deriving criteria for
individual carcinogenic PAH, which will lead to effective control
of PAH as a class. This approach is attractive in that it recog-
nizes the fact that environmental exposures to PAH invariably occur
by contact with complex, undefined, PAH mixtures.
The attempt to develop a drinking water criterion for PAH as a
class is hindered by several gaps in the scientific data base:
(1) The PAH class is composed of numerous compounds having
diverse biological effects and varying carcinogenic
potential. A "representative" PAH mixture, has not been
defined.
(2) The common practice of using data derived from studies
with BaP to make generalizations concerning the effects
of environmental PAH may not be scientifically sound.
(3) No chronic animal toxicity studies involving oral expo-
sure to PAH mixtures exist.
(4) No direct human data concerning the effects of exposure
to defined PAH mixtures exist.
However, assuming that the development of a criterion must
proceed despite these obstacles, certain approaches may be taken to
circumvent deficiencies in the data base. The choice of an appro-
priate animal bioassay from which to derive data for application to
the human cancer risk assessment should be guided by several con-
siderations. Primary emphasis must be placed on appropriate animal
studies which: (1) include sufficient numbers of animals for sta-
tistically reliable results; (2) involve long-term low-level expo-
sures to PAH; (3) include a prooer control grout>; and (4) achieve
positive dose-related carcinogenic response.
C-118
-------
Because there are no studies available regarding chronic oral
exposure to PAH mixtures, it is necessary to derive a criterion
based upon data involving exposure to a single compound. Two stud-
ies can be selected, one involving BaP ingestion (Neal and Rigdon,
1967) and one involving DBA ingestion (Snell and Stewart, 1962).
Both compounds are recognized as animal carcinogens, and both are
known to be environmental contaminants to which humans are exposed.
Presently, there is no way to quantitate the potential human
health risks incurred by the interaction of PAH, either among them-
selves or with other agents (e.g., tumor initiators, promoters,
inhibitors) in the environment. In addition, it is known that PAH
commonly produce tumors at the site of contact (i.e., forestomach
tumors by oral exposure to BaP; lung tumors by intratracheal admin-
istration; skin tumors be dermal application). Thus, consideration
of the extent of absorption may not always be necessary in the case
of carcinogenic PAH, and will in fact result in underestimation of
actual risk if only distant target sites are considered. Calcula-
tion of the water quality criterion based upon bioassay data for
BaP is presented in the Appendix.
The water quality criterion for BaP derived using the linear-
ized multistage model, as described in the Human Health Methodology
Appendices to the October 1980 Federal Register notice which an-
nounced the availability of this document, is 28 ng/1. For the
sake of comparison, a water quality criterion for DBA was calculat-
ed using the procedure developed by Mantel and Bryan (1961) . As
opposed to the linearized multistage model, which is logistic and
defines acceptable risk as 1/100,000, the Mantel and Bryan (1961)
C-119
-------
model is probablistic and defines acceptable risk as 1/100,000,000.
Furthermore, the Mantel and Bryan model (1961) is concerned with
the maximum tumor incidence in treated animals at the 99 percent
confidence level versus the 95 percent confidence level in the lin-
earized multistage model. Using the Mantel and Brvan (1961) ap-
proach with DBA, the resultant water quality criterion is
13.3 ng/1.
Under the Consent Decree in NRDC v. Train, criteria are to
state "recommended maximum permissible concentrations (including
where appropriate, zero) consistent with the protection of aquatic
organisms, human health, and recreational activities." BaP is a
known animal carcinogen. Because there is no recognized safe con-
centration for a human carcinogen, the recommended concentration in
water for maximum protection of human health is zero.
Because attaining a zero concentration level may be infeasible
in some cases and in order to assist the Agency and states in the
possible future development of water quality regulations, the con-
centrations of BaP corresponding to several incremental lifetime
cancer risk levels have been estimated. A cancer risk level pro-
vides an estimate of the additional incidence of cancer that may be
expected in an exposed population. A risk of 10 for example,
indicates a probability of one additional case of cancer for every
100,000 people exposed, a risk of 10" indicates one additional
case of cancer for every million people exposed, and so forth.
PAH are widely distributed in the environment as evidenced by
their detection in sediments, soils, air, surface waters, and plant
and animal tissues. The ecological impact of these chemicals, how-
0120
-------
ever, is uncertain. Numerous studies show that despite their high
lipid solubility, PAH show little tendency for bioconcentration in
the fatty tissues of animals or man. This observation is not unex-
pected, in light of convincing evidence to show that PAH are rapid-
ly and extensively metabolized.
Lu, et al. (1977) have published the only available study re-
garding the bioconcentration and biomagnification of a PAH in model
ecosystem environments. They reported that the bioconcentration of
BaP, expressed as concentration in mosquitofish/concentration in
water was zero. This was apparently due to the fact that the fish
metabolized the BaP about as rapidly as it was absorbed. On the
other hand, in a 33-day terrestrial-aquatic model ecosystem studv,
BaP showed a small degree of biomagnification which probably re-
sulted from food chain transfer. In this case the biomagnification
factor for mosquitofish was 30. Based on the results of Lu, et al.
(1977) a bioconcentration (BCF) factor of 30 was employed for the
purpose of calculating a water quality criterion.
In the Federal Register notice of availability of draft ambi-
ent water quality criteria, EPA stated that it is considering set-
ting criteria for BaP at an interim target risk level of 10~5,
10 , or 10 as shown in the following table.
C-121
-------
BaP
Exposure Assumptions Risk Levels and Corresponding Criteria (1)
(per day) ng/1
0. 10"7 10"6 10"5
2 liters of drinking
water and consumption g 0.28 2.8 28.0
of 6.5 grams fish
and shellfish (2)
Consumption of fish 3>11 31>1 311>0
and shellfish only.
(1) Calculated by applying a linearized multistage model as pre-
viously discussed. Appropriate bioassay data used in the cal-
culation of the model are presented in the Appendix. Since
the extrapolation model is linear at low doses, the additional
lifetime risk is directly proportional to the water concentra-
tion. Therefore, water concentrations corresponding to other
risk levels can be derived by multiplying or dividing one of
the risk levels and corresponding water concentrations shown
in the table by factors such as 10, 100, 1,000, arid so forth.
(2) Approximately 9 percent of the PAH exposure, assumed to be
BaP, results from the consumption of aquatic organisms which
exhibit an average bioconcentration potential of 30-fold based
on the work of Lu, et al. (1977). The remaining 91 oercent of
PAH exposure results from drinking water.
Concentration levels were derived assuming a lifetime exposure
to various amounts of PAH (1) occurring from the consumption of
both drinking water and aquatic life grown in water containing the
corresponding PAH concentrations and, (2) occurring solely from the
consumption of aquatic life grown in the waters containing the cor-
C-122
-------
responding PAH concentrations. Because data indicating other
sources of exposure and the concentration to total body burden are
inadequate for quantitative use, the criterion reflects the incre-
ment to risks associated with ambient water exposure only.
C-123
-------
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APPENDIX
Summary and Conclusion Regarding the Carcinogenicity
of Polynuclear Aromatic Hydrocarbons (PAH)
Polynuclear aromatic hydrocarbons (PAH) comprise a diverse
class of compounds consisting of substituted and unsubstituted
polycyclic and heterocyclic aromatic rings. They are formed as a
result of incomplete combustion of organic compounds arid appear in
food as well as ambient air and water.
Numerous studies of workers exposed to coal gas, coal tars,
and coke oven emissions, all of which have large amounts of PAH,
have demonstrated a positive association between the exposures and
lung cancer.
Several PAH are well-known animal carcinogens, others are not
carcinogenic alone but can enhance or inhibit the response of the
carcinogenic PAH and some induce no tumors in experimental animals.
Most of the information about the combined carcinogenic effects of
several PAH come from skin painting and subcutaneous injection
experiments in mice whereas oral administration, intratrachael
instillation, and inhalation have been shown to induce carcinogenic
responses to single compounds. In one subcutaneous injection study
in mice it was shown that a combination of several noncarcinogenic
PAH compounds, mixed according to the proportion occurring in auto
exhaust, does not enhance or inhibit the action of two potent PAH
carcinogens, benzo(a)pyrene (BaP) and dibenz(a,h)anthracene.
The mutagenicity of PAH in the Salmonella tvphimurium assay
correlates well with their carcinogenicity in animal systems. PAH
compounds have damaged chromosomes in cytogenetic tests, have
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induced mutations in mammalian cell culture systems and have in-
duced DNA repair synthesis in human fibroblast cultures.
The water quality criterion for carcinogenic PAH compounds is
based_°n the assumPtion that each compound is as potent as BaP and
that the carcinogenic effect of the compounds is orooortional to
the sum of their concentrations. Based on an oral feeding study of
BaP in mice, the concentration of BaP estimated to result in a
lifetime risk of 10~5 is 28 ng/1. Therefore, with the assumption
above, the sum of the concentrations of all carcinogenic PAH com-
pounds should be less than 28 ng/1 in order to keep the lifetime
cancer risk below 10.
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Summary of Pertinent Data
The water quality criterion for BaP is based on the experiment
reported by Neal and Rigdon (1967), in which benzq(a)pyrene at
doses ranging between 1 and 250 ppm in the diet was fed to strain
CFW mice for approximately 110 days. Stomach tumors, which were
mostly squamous cell papillomas but some carcinomas, appeared with
an incidence statistically higher than controls at several doses.
The extrapolation was based on the following parameters:
Dose Incidence3 ' -
(mg/kg/day) (No. responding/No.' tested)
0.0 0/289
0.13 0/25
1.3 0/24
2.6 1/23
3.9 0/37
5.2 1/40
5.85 4/40
6.5 24/34
13.0 19/23
32.5 66/73
le = 110 days w = 0.034 kg
Le = 183 days R = 30 I/kg
L = 630 days
With these parameters, the carcinogenic potency factor for humans,
q*, is 11.53 (mg/kg/day)"1. The result is that the water concen-
tration of BaP should be less than 28 ng/1 in order to keep the
individual lifetime risk below 10~5. It is recognized that numer-
ous carcinogenic PAH other than BaP are found in water. However,
there is probably little need to derive criteria for all such PAH,
since efforts to reduce BaP levels to within acceptable limits will
result in the reduction of all PAH.
aThe incidences at the highest three doses were not used in the
extrapolation due to lack of fit of the multistage model. See the
Human Health Methodology Appendices to the October 1980 Federal
Register notice which announced the availability of this document
for a discussion on the fit of data to the multistage model.
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t, U. S. GOVERNMENT PRINTING OFFICE • 1980 720-016/4395
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