297 926
Polynuclear Aromatic Hydrocarbons
Ambient Water Quality Criteria
Criteria and Standards Division
Office of Water Planning and Standards
U.S. Environmental Protection Agency
•Washington, D.C.
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CRITERION DOCUMENT
POLYNUCLEAR AROMATIC HYDROCARBONS
CRITERIA
Aquatic Life
For freshwater aquatic life, no criterion for any polynuclear
aromatic hydrocarbon can be derived using the Guidelines, and
there are insufficient data to estimate a criterion using other
procedures.
'r For saltwater aquatic life, no criterion for any polynuclear
aromatic hydrocarbon can be derived using the Guidelines, and
there are insufficient data to estimate a criterion using other
procedures.
Human Health
For the maximum protection of human health from the potential
carcinogenic effects of exposure to polynuclear aromatic hydrocar-
bons (PAH) through ingestion of water and contaminated aquatic or-
ganisms, the ambient water concentration is zero. Concentrations
of PAH estimated to result in additional lifetime cancer risks
ranging from no additional risk to an additional risk of 1 in
100,000 are presented in the Criterion Formulation section of this
document. The Agency is considering setting criteria at an in-
terim target risk level in the range of 10~5, 10"^, or 10"? with
corresponding criteria of 9.7 ng/1, 0.97 ng/1 and 0.097 ng/1,
respectively.
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Introduction
Polynuclear aromatic hydrocarbons (PAH) are a diverse
class of compounds consisting of substituted and unsubsti-
tuted polycyclic and heterocyclic aromatic rings. PAH are
formed as a result of incomplete combustion of organic com-
pounds with insufficient oxygen. This leads to the formation
of C-H free radicals which can polymerize to form various
PAH. Among these PAH are compounds such as benzo[a]pyrene
and benz[a]anthracene, which are well-known for their ubiqui-
tous presence in nature and carcinogenic effects in experi-
mental animals.
Under the Consent Decree in NRDC v. Train maximum per-
missible concentration are to be recommended for the follow-
ing PAH: benzopyrene; benzanthracene; chrysenes; benzofluor-
antheses; indenopyrenes. In this report, criteria are recom-
mended for PAH as a class, derived using available data con-
cerning several of the most extensively studied individual
carcinogenic components in the class. There are no published
studies available which adequately compare the carcinogenic
activities of all ten of the specified PAH under similar ex-
perimental conditions. Likewise, there are no data available
concerning human responses to individual compounds in the PAH
class, since environmental exposures to PAH invariably in-
volve contact with complex, and usually undefined, PAH mix-
tures.
This report considers the various human health aspects
associated with exposure to environmental levels of PAH.
Particular attention is directed at the contribution of food,
A-l
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water, and air to the total human PAH exposure. Assessment
of anticipated health risks is directed specifically at the
development of PAH-induced cancers as being the endpoint of
greatest concern.
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AQUATIC LIFE TOXICOLOGY*
FRESHWATER ORGANISMS
Introduction
No standard toxicity tests have been reported for freshwater
organisms and any polynuclear aromatic hydrocarbon (PAH) not dis-
cussed in documents on specific compounds (e.g., fluoranthene and
acenaphthene). There are some data for bioconcentration during
tests with model ecosystems for short periods of time.
Residues
No measured steady-state bioconcentration factors (BCFs) are
available for acenaphthylene, anthracene, benzo[a]pyrene, 3-
methylcholanthene, and phenanthrene; bioconcentration factors can
be estimated using the octanol-water partition coefficients of
5,500, 28,000, 1,150,000, 9,300,000, and 28,000, respectively.
These coefficients are used to derive estimated BCFs of 410,
1,400, 24,000, 120,000, and 1,400 for acenaphthylene, anthracene,
benzofa]pyrene, 3-methylcholanthene, and phenanthrene for aquatic
*The reader is referred to the Guidelines for Deriving Water Qual-
ity Criteria for the Protection of Aquatic Life [43 FR 21506 (May
18, 1978) and 43 FR 29028 (July 5, 1978)] in order to better un-
derstand the following discussion and recommendation. The follow-
ing tables contain the appropriate data that were found in the
literature, and at the bottom of each table are the calculations
for deriving various measures of toxicity as described in the
Guidelines.
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organisms that contain about 8 percent lipids. If it is known
that the diet of the wildlife of concern contains a significantly
different lipid content, appropriate adjustments in the estimated
BCFs should be made.
Miscellaneous
Lu, et al. (1977) conducted studies with benzo[a]pyrene in a
terrestrial-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).
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CRITERION FORMULATION
Freshwater-Aquatic Life
Summary of Available Data
No freshwater criterion can be derived for any polynuclear
aromatic hydrocarbon using the Guidelines because no Final Chronic
Value for either fish or invertebrate species or a good substitute
for either value is available, and there are insufficient data to
estimate a criterion using other procedures.
B-3
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Table 1. Other freshwater data for polynuclear aromatic hydrocarbons
Test
Result
Organism
Benzo lajpyrgnti
Alga.
Oedogonium cardiacum
Cladoceran,
Daphnia pulex
Snail,
Phy_s_£ sp.
Mosquito,
Culex pipiens
quinqueTasciatus
1 Mosquitofish,
*> Gamluisia affinis
Protozoa,
Paramecium caudatum
Cladoceran,
Daphnia magna
Cladoceran ,
Dapjinia ma go a
Mayfly.
llc-xagenia sp.
3 days
3 days
3 days
3 days
3 days
60 min
1 hr
.-24 hrs
28 hrs
Model ecosystem,
bioconcentration
factor = 5,258
Model ecosystem,
bioconcentration
factor = 134,248
Model ecosystem,
bioconcentration
factor = 82.231
Model ecosystem,
bioconcentration
factor = 11.536
Model ecosystem,
bioconcentration
factor = 930
Anthracene
90% lethal
photodynamic response
Bioconcentration
factor = -200
Bioconcentration
factor = 760
Bioconcentration
factor = 3,500
Benzo- (a) -anthracene
0.1
Blut'gill,
I.epomis macrochrius
6 mos 87% mortality
1,000
Lu. et al. 1977
I.u, et al. 1977
Lu, et al. 1977
Lu. et al. 1977
Lu, et al. 1977
Epstein, 1963
Herbes, 1976
llerbes & Risi, 1978
llerbes, 1976
Brown, et al. 1975
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SALTWATER ORGANISMS
Introduction
As was true for freshwater organisms, no standard toxicity
tests with saltwater organisms have been conducted with any poly-
nuclear aromatic hydrocarbon. There are a variety of data for
bioconcentration during short exposures.
Residues
No measured steady-state bioconcentration factors (BCFs) are
available for acenaphthylene, anthracene, benzo[a]pyrene, 3-
methylcholanthene, and phenanthrene; bioconcentration factors can
be estimated using the octanol-water partition coefficients of
5,500, 28,000, 1,150,000, 9,300,000, and 28,000, respectively.
These coefficients are used to derive estimated BCFs of 410,
1,400, 24,000, 120,000, and 1,400 for acenaphthylene, anthracene,
benzofa]pyrene, 3-methylcholanthene, and phenanthrene, respec-
tively for aquatic organisms that contain about 8 percent lipids.
If it is known that the diet of the wildlife of concern contains a
significantly different lipid content, appropriate adjustments in
the estimated BCFs should be made.
Miscellaneous
The data (Table 2) on bioconcentration of polynuclear aro-
matic hydrocarbons are lower than those observed with freshwater
organisms (Table 1) but may be due to the short exposure periods.
A polychaete worm was exposed to various crude oil factions and
96-hour LC50 values were between 300 and 1,000 ug/1 (Neff, et al.
1976a).
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CRITERION FORMULATION
Saltwater-Aquatic Life
Summary of Available Data
No saltwater criterion can be derived for any polynuclear
aromatic hydrocarbon using the Guidelines because no Final Chronic
Value for either fish or invertebrate species or a good substitute
for either value is available, and there are insufficient data to
estimate a criterion using other procedures.
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Table 2. Other marine data for polynuclear aromatic hydrocarbons
Ttfst
Organise
Eastern oyster.
Crassostrea virginica
Clam,
Rangia cuneata
Clam.
Rangia cuneata
Clam.
Rangia cuneata
Result
tuq/il
Benzola1pyrene
14 days Bioconcentration
factor - 242
24 hrs Bioconcentration
factor =8.66
24 hrs Bioconcentration
factor = 236
Chryaene
24 hrs Bioconcentration
factor »8.2
Couch, et al..
Neff. et al. 1976a
Neff. et al. 1976b
Neff. et al. 1976a
Benzofa1pyrene Edible Tissue
Mudsucker, 96 hrs Bioconcentration
Gillichthys mirabilia factor = .048
Tidepool sculpin, 1 hr
OlioROCottus maculosus
Sand dab, 1 hr
Citharichthys stigmacus
Bioconcentration
factor - .13
Bioconcentration
factor » .02
Polychaete worm,
Neanthes arenaceodentata
Polychaete worm,
Neanthes arenaceodentata
Crude oil extract (fluorene)
96 hrs LC50 1,000
Crude oil fraction (phenanthrene)
96 hrs LCSO 600
Lee, et al. 1972
Lee. et al. 1972
Lee. et al. 1972
Neff, et al. 1976a
Neff. et al. 1976a
Polychaete worm,
Neanthes arenaceodentata
Crude oil fraction (1-roethylphenanthrene)
96 hrs LCSO 300 Neff. etal. 1976a
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POLYNUCLEAR AROMATIC HYDROCARBONS
REFERENCES
Brown, E.R., et al. 1975. Tumors in fish caught in polluted
waters: possible explanations. Comparative Leukemia Res.
1973, Leukemogenesis. Univ. Tokyo Press/Karger, Basel,
pp. 47-57.
Couch, J.A., et al. The American oyster as an indicator
of carcinogens in the aquatic environment. Iin Pathobiology
of Environmental Pollutants - Animal Models and Wildlife
as Monitors. Storrs, Conn. National Academy 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 poly-
cyclic aromatic hydrocarbons (PAH) in aquatic systems.
Ir\ Coal technology program quarterly progress report for
the period ending December 31, 1975, Oak Ridge National
Lab., Oak Ridge, TN. ORNL-5120. pp. 65-71.
Herbes, S.E., and G.F. Risi. 1978. Metabolic alteration
and excretion of anthracene by Daphnia pulex. Bull. Environ.
Contam. Toxicol. 19: 147.
B-8
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Lee, R.G., et al. 1972. Uptake, metabolism and discharge
of polycyclic aromatic hydrocarbons by marine fish. Mar.
Biol. 17: 201.
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.
Contam. 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 Hydrocarbons in the Aquatic Environment.
Proceedings of a symposium, American University, Washington,
D.C., American Institute of Biological Sciences, p. 520.
Neff, J.M., et al. 1976b. Accumulation and release of
petroleum-derived aromatic hydrocarbons by four species
of marine animals. Mar. Biol. 38: 279.
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Mammalian Toxicology and Human Health Effects
EXPOSURE
Ingestion from Water
The uptake of 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 run off, and leaching from
polluted soils, all of which contain substantial PAH concen-
trations (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 concentra-
tion 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 cleanup procedures and analyzed either by TLC-
spectrofluonmetry or by u.v.-spectrophotometry. These values
are presented in Table 2.
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TABLE 1
PAH Concentration in Ground Water
Source
G. Finthen,
Germany,
Mainz,
Germany
Unspecified
locations in
Germany
Average of 12
German ground
waters
Champaign,
111.3
Elkhart,
Ind.
Fairborn,
O.a
Concentration, ^
Carcinogenic
BaP PAH
0.002
0.005
0.0004 0.003
N.D.b 0.003
0.004 0.004
0.0003 0.0008
ig/i
Total
PAH Reference
Borneff, 1964
Borneff, 1964
0.04 Borneff & Kunte,
1964
0.06 Borneff & Kunte,
1969
0.007 Basu & Saxena,
1977-78
0.02 Basu & Saxena,
1977-78
0.003 Basu & Saxena,
1977-78
a
These are results of 6 specified PAH
5N.D.: not detected
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TABLE 2
Concentration of PAH in surface waters
Concentration,
Source
BaP
Carcinogenic Total
PAH PAH
Reference
Rhine River 0.08
at Mainz
River Main at 0.0024
Seligentadt
River Danube 0.0006
at Ulm
River Gersprenz 0.0096
at Munster
River Aach at 0.017
Stockach
River Schussen 0.01
River Plyussa;
at Shale-oil 12
effluent dis-
charge site
3500 m 1
downstream
at Narvy 0.1
water intake
A river;
15 m below 8-12
coke by-pro-
duct discharge
site
500 m down- 2-3
stream
Thames River
at Kew 0.13
bridge
at Albert 0.16
bridge
at Tower 0.35
bridge
0.49
0.155
0.067
0.047
0.95
0.20
0.18
0.27
0.56
1.12
0.48
0.24
0.14
2.5
1.0
0.50
0.69
1.33
Borneff & Kunte,
1964
Borneff & Kunte,
1964
Borneff & Kunte,
1964
Borneff & Kunte,
1964
Borneff & Kunte,
1965
Borneff & Kunte,
1965
Dikun &
Makhinenko, 1963
Dikun &
Makhinenko, 1963
Dikun &
Makhinenko, 1963
Fedorenko, 1964
Fedorenko, 1964
Harrison, et al«
1975
Harrison, et al,
1975
Harrison, et al,
1975
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Keegan (1971) analyzed the PAH content in three relatively
unpolluted U.S. river waters by removing the PAH from water
by solvent extraction. The extract was subjected to cleanup
and the PAH were analyzed by TLC-spectrofluonmetry. 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 (1971), 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 removed by sedimentation, flocculation
and filtration processes. The remaining one-third dissolved
PAH usually requires oxidation for partial removal/transforma-
tion. 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. ClO^ on the other hand, reduces BaP concen-
tration by 90 percent. But a't 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 03 requires intensified pre-
purification to prevent oxidation of other chemicals. Filtra-
tion with activated carbon has been suggested by Borneff
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TABLE 3
Concentrations of PAH in Raw and Treated Surface Water
used as Drinking Water Sources
o
i
Concentration, jug/1
Source
River Rhine
River Rhine
Lake Constance
Lake Constance
English River
English River
Monongahela River
at Pittsburgh
same as above
Ohio River at
Huntington, W. Va.
same as above
Ohio River at
Wheeling, W. Va.
same as above
Delawater River at
Philadelphia
same as above
Lake Winnebago at
Appleton, Wis.
same as above
Treatment
Untreated
Bank and act-
vated carbon
filtered
Untreated
Rapid sand
filtration
Chlorination
Untreated
Filtration &
Chlorination
Untreated
r\
Treated
Untreated
ri
Treated
Untreated
.j
Treated
Untreated
H
Treated
Untreated
,a
Treated"
BaP
(K082
0.0005
0.0013
0.0017
i
0.06°
0.009
0.04
0.0004
0.006
0.0005
0.21
0.002
0.04
0.0003
0.0006
0.0004
Carcinogenic
PAH
0.485
0.015
0.030
0.017
0.37C
0.051
0.14
0.002
0.020
0.002
0.57
0.011
0.16
0.002
0.002
0.002
Total
PAH
1.11
0.13
0.065
0.053
0.73b
0.24
0.60
0.003
0.058
0.007
1.59
0.14
0.35
0.015
0.007
0.006
Reference
Borneff & Kunte, 1964
Borneff & Kunte, 1964
Borneff & Kunte, 1964
Borneff & Kunte, 1964
Harrison, et al. 1976
Harrison, et al. 1976
Basu & Saxena,1978
Basu & Saxena, 1978
Basu & Saxena, 1978
Basu & Saxena, 1978
Basu & Saxena, 1977-78
Basu & Saxena, 1977-78
Basu & Saxena, 1978
Basu & Saxena, 1978
Basu & Saxena, 1977-78
Basu & Saxena, 1977-78
These are average of five determinations with the exclusion of a sixth high value
These values are estimates on the basis of average PAH adsorption in reservoir
These values may be a little higher due to the inability of separation of all the
,carcinogenic from non-carcinogenic PAH
The treatment included flocculation, activated carbon addition, filtration, pH
control, Chlorination and fluoridation.
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(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 (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 (1978, 1977-
78), 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 compounds 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 (1978, 1977-78). Six representative
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PAH recommended by the World Health Organization (1970)
as the measure of PAH contamination in drinking water was
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 Organic Monitoring
Survey (NOMS, 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 concentration. 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 trans-
ported to the consumers through a variety of pipelines.
Borneff (1977) reported a tenfold 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 concentration was studied by Basu and Saxena (1977-
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78). Because of the intermixing of the pipes, it is difficult
to draw definite conclusions from their results. However,
it seems likely that in instances 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 concentrations,
possibly due to adsoprtion of PAH from the water.
There are very few epidemiclogical studies concerning
the correlation 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 communitites 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
morbidity 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 reduced 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 exposure (more effective than an equivalent
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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 Jjg/year. Assuming the PAH concentration value of 13.5
ng/1 in U.S. drinking water (Basu and Saxena, 1978; Basu
and Saxena, 1977-78), and a daily consumption of 2.5 liters
of drinking water, the yearly intake of PAH from U.S. drinking
would be 12.3 pg/ or 0.3 percent of the total food intake.
Nevertheless, the accumulation of PAH in edible aquatic
organisms through polluted surface waters can greatly increase
their amount in foods, including fish, some mollusks, and
edible algae (Andelman and Snodgrass, 1974). The use of
contaminated water for irrigation can also spread PAH into
other vegetable foodstuffs (Shabad and Il'nitskii, 1970).
Therefore, it is important to monitor the PAH levels in
surface waters not used as drinking water sources as well
as drinking waters, in order to estimate accurately the
human intake of PAH.
Ingestion from Foods
PAH formed through both natural and man made sources
can enter the food chain of man. There is considerable
disagreement, however, concerning the contribution of each
of these sources to the total PAH contamination in foods.
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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 to 10 to 20 times
more BaP than areas remote from the runway. The results
of Dunn and Stich (1976) indicated a correlation between
the PAH level in mussels with industrial, urban, and recreational
activity. The highest occurrence of BaP in marine organisms
in the areas adjacent to the sea lanes tends to support
the view that exogenous sources are the predominant factor
for PAH contamination in foods.
The primary routes of entry for PAH in foods are surface
adsorption and biological accumulation from the environment
(Binet and Malet, 1963). 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 demon-
strated 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 concentrate PAH through surface adsorption (Hetteche,
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1971 and Kolar, et al. 1975). Kolar, et al. (1975) have
shown that the concentration of BaP in vegetation is propor-
tional to the exposure time during the growing season (bio-
accumulation through adsorption) and the structure of the
surface of the plant (surface adsorption). The above-ground
parts of the vegetables contain move BaP than underground
parts. Only about ten 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 paraffin wax wrapping for food contained
PaA, CH, BeP, and BaP at levels of 29 ppb, 2 ppb, 0-48 ppb,
and 2 ppb, respectively. Certainly, some of these PAH in
the packing material can diffuse into the food. Hexane,
a commercial solvent used to extract edible vegetable oils,
is also a source of PAH contamination. PAH present in food-
grade carbon blacks used for food processing can be transported
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 contamination originates from the
cooking process itself.
In order to summarize the available data on PAH levels,
various foods have been categorized following the pattern
of USDA-FDA for total diet samples (Martin and Duggan, 1968) .
These are shown in Tables 5 through 11. It should be recog-
C-12
-------
nized 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 considerable 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 personal 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.
It has been claimed by Zitko (1975) that PAH are not
bioaccumulated along the food chain. However, Bj0rseth
(1978) demonstrated that both common and horse mussels bio-
accumulated PAH, although not to the same degree. Dunn
and Stitch (1976) have shown that mussles cannot metabolize
BaP upon their removal from water. In water, mussels released
79 percent of naphthalene in 3 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
C-13
-------
>£.
TABLE 5
PAH Concentrations (ppb) in a few Vegetable Oils and Margarine
Corn3
Coconut
Margarine
Q
Sunflower
Soybean
Olive3
Peanut3
A PA FL P BaA
3.1 0.8
36 51 18 15
1.4-
29.5
d
13°
1.3 1.6 0.9
3.2 2.6 1.0
3.3 2.9 1.1
BeP
0.7
2
0.5-
1.2
4
1.6
0.4
BaP PR BPR CH
0.7 0.6
2 12
0.2-
6.8
8 4
1.4 1.0
0.5 0.9
0.6 0.9
3Howard, et al. 1966c
bBeirnoth and Rost, 1967
cSwallow, 1976
This value represents concentration of BaA and CH
-------
TABLE 6
PAH Concentrations (ppb) in Smoked and Non-Smoked Fish
Fish F
Smoked3 Eel 9
Smoked3 Lumpfish 5
Smoked3 trout 67
Smoked, herring
Smoked herring
(dried)
Smoked!" salmon
Smoked, sturgeon
Smoked whitefish
Smoked0, whiting
0 Smoked*? redfish
i Smoked cod
£ Electric smoked
mackerel 2.6
Gas smoked.
mackerel 8.2
Non -smoked
haddock
Non-smoked
herring
(salted)
Non-smoked
salmon
A PA FL
4 37 4
t 10 2
26 52 12
3
1.8
3.2
2.4
4.6
1.5 4.1 4.0
1.9 9 5.2
2.3 11 2.6
1.6
0.8
1.8
P
6
1
5
2.2
1.8
2.0
4.4
4.0
<0.5
3.0
0.6
3.6
4.0
0.8
1.0
1.4
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
3Thorsteinsson, 1969; Dungal, 1961
bHoward, et al. 1966a
GMalanoski, et al. 1968
Masuda and Kratsune, 1971
-------
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 reported 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; Malanoski, et al.
1968) .
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. (1968b) 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 per year.
Fish and Other Marine Foods: Raw' fish from unpolluted
waters usually do not contain detectable amounts of PAH,
C-16
-------
but smoked or cooked fish contain varying levels of PAH.
In addition to the origin 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 migration of PAH into the body tissues. This was postulated
by Malanoski, 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 un.smoked fish
are shown in Table 6. In addition to the fishes presented
in this table, various 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, repsectively
(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. (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 chemicaJ in water to the concentration in aquatic orga-
C-17
-------
nisms, but BCF's are not available for the edible portions
of all four major groups of aquatic organisms consumed in
the United States. Since data indicate that the BCF for
lipid-soluble compounds is proportional to percent lipids,
BCF's can be adjusted to edible portions using data on percent
lipids and the amounts of various species consumed by Americans
A recent survey on fish and shellfish consumption in the
United States (Cordle, et al. 1978) found that the per capita
consumption is 18.7 g/day. From the data on the nineteen
major species identified in the survey and data on the fat
content of the edible portion of these species (Sidwell,
et al. 1974), the relative consumption of the four major
groups and the weighted average percent lipids for each
group can be calculated:
Consumption Weighted Average
Group (Percent) Percent Lipids
Freshwater fishes 12 4.8
Saltwater fishes 61 2.3
Saltwater molluscs 9 1.2
Saltwater decapods 18 1.2
Using the percentages for consumption and lipids for each
of these groups, the weighted average percent lipids is
2.3 for consumed fish and shellfish.
No measured ste'ady-state bioconcentration factor (BCF)
is available for any of the following compounds except BaP
(Lu, et al. 1977), but the equation "Log BCF = 0.76 Log
P - 0.23" is commonly used (Veith, et al. Manuscript) to
estimate the BCF for aquatic organisms that contain about
C-18
-------
eight percent lipid's from the octanol-water partition coeffi-
cient (P). An adjustment factor of 2.3/8.0) = 0.2875 can
be used to adjust the estimated BCF from the 8.0 percent
lipids on which the equation is based to the 2.3 percent
lipids that is the weighted average for consumed fish and
shellfish. Thus, the weighted average bioconcentration
factor for the edible portion of all aquatic organisms consumed
by Americans can be calculated (Table 6a):
TABLE 6a
Calculated Bioconcentration Factors of PAH
Based Upon the Octanol-water Partition Coefficient
Compound
Acenaphthylene
Anthracene
Benz (a) anthracene
Benzo(b) fluoranthene
Benzo(k) fluoranthene
1 , 12-benzoperylene
Benzo(a) pyrene
Chrysene
Dibenz (a,h) acr idine
Dibenz (a,h) anthracene
Dibenzofuran
Fluoranthene
Fluorene
1-methylphenanthrene
Phenanthrene
2,3-phenylene pyrene
Pyrene
P
5,500
28,000
410,000
1,100,000 '
1,100,000
3,200,000
1,150,000
410,000
540,000
5,900,000
13,000
79,000
15,000
100,000
28,000
3,200,000
76,000
BCF
410
1,400
11,000
24,000
24,000
52,000
24,000
11,000
13,000
82,000
800
3,100
880
3,700
1,400
52,000
3,000
Weighted BCF
120
410
3,100
6,800
6,800
15,000
6,800
3,100
3,800
24,000
230
900
250
1,100
410
15,000
870
C-19
-------
Caution must be excercised in application of common practice
in obtaining BCF described above, 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).
This observation is not unexpected, in light of convincing
evidence to show that PAH are rapidly and extensively metab-
olized. 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 avail-
able which suggest that adverse effects may occur.
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 7 shows the
concentration of PAH detected 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
temperature of combustion affect the composition and concentra-
tion of PAH in cooked meat (Howard, et al. 1966a) In addition
to the pyrolysis of fats, incomplete combustion of charcoal
-------
TABLE 7
PAH Concentrations (ppb) in a Few Smoked Meat and Meat Products
Meat A
Charcoal broiled
steaks3
Barbecued ribs3 7.1
Smoked beef .
(chipped)
Smoked ham
Smoked pork
(roll)0
Smoked frank-
furters
V Barbecued beef
£ Smoked hot
sausages0 ,
Smoked mutton 13.0
Smoked mutton
sausages 2.0
Smoked bologna6
f
Smoked salami 0.7
Smoked Morta-
della 2.6
Heavily smoked
bacon 20.0
PA FL
21.0 43
58.0 49
0.
14.
3.
6.
2.
104.0 18.
17.0 6.
D^ 5 .
D 22.
D 35.
.0
.0
6
0
1
4
0
0
0
6
0
0
P
35.
42.
0.
11.
2.
3.
3.
1.
8.
2.
5.
15.
27.
BaA
0
0
5
2
5
8
2
5
0
0
2
0
0
1
3
0
2
1
13
0
2
0
0
0
0
2
29
.4
.6
.4
.8
.5
.2
.5
.0
.5
.04-
.55
.6
.8
.0
BeP
5.5
7.5
1.2
1.7
5.0
t
5.0
0.2
1.8
D
BaP
5
10
3
2
3
0
0
0
2
0
0
3
.8
.5
.2
.0
.5
.4
t
t
.04-
.08
.0
.8
.7
.6
. PR BPR
0.9 6.7
1.5 4.7
1.4
4.3
0.04- 0.04-
0.07 0.20
3.2 D
0.1 0.4
0.9 3.0
CH
0.6
2.2
9.6
1.0
0.15-
1.201
1.2
3.4
D
3Lijinsky and Shubik, 1965a
Howard, et al. 1966a,b; Panalaks, 1976
^Malanoski, et al. 1968
Thorsteinsson, 1969
^Frethein, 1976; Panalaks, T., 1976
Lo and Sandi, 1978
PD: detected
h.
. t: trace
compound unseparated
-------
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 8.
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 Iceland 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 concent.ations of PAH
in Norwegian bologna sausages (see Table 8) are probably
indicative of the tradi tion of light smoking of food in
Norway (Fretheim, 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 low penetration has also been noted by Rhee
and Bratzler (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 frankfurters 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 variety of PAH. Lijinsky and Shubik (1965b)
have detected BaP, FL, P, BPR, BaA, and CH in liquid smoke
at concentrations of 1 ppb, 16 ppb, 7 ppb, 1 ppb, 12 ppb,
and 6 ppb, respectively. In hickory liquid smoke flavoring,
C-22
-------
TABLE 8
Effect of Different Cooking Variables on the Concentration of PAH (ppb) in Cooked Meat
Meat
Charcoal broiled
hamburger9 ,
Fate, hot
Lean , hot
No-drip pan
Charcoal broiled
hamburger
Lean , hot
Lean, cool
Broiled T-bone
steak3
Charcoal, hot
Flame, hot
Smoked ham
Light
Heavy
Effect
Fat Content
Heating
temperature
Heat
source
Degree of
Smoke
FL
13.3
0.3
0.2
0.3
1.3
19.8
19.0
4.0-
14.0
48.0-
156.0
P
7.7
1.6
0.1
1.6
0.6
19.1
20.0
2.0-
11.0
35.0-
161.0
BaA BeP BaP BPR
2.7 2.6 14.9
0.9
t
0.9
31.0 17.6 50.4 12.4
3.9 5.7 4.4 6.2
0.5- 0-2.0 3.0- 0-1.4
3.0 4.0
6.0- 4.0- 3.8- 2.5-
33.0 26.0 55.0 25.0
CH CR
1.7 1.0
t
0.3
25.4 8.0
2.0 9.0
0-3.0
12. fl-
ee. o
^Lijinsky & Ross, 1967
"Filipovic & Toth, 1971; Toth & Blass, 1972
°Fat: 21% fat
Hot: 7 cm. from heat source
fLean: 7% fat
cool: 25 cm from heat source
-------
Youngblood, and Blumer (1975) found the total concentration
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 Adjuncts, and Beverages: Various European and Japanese
workers have reported the presence of BaP and other PAH
in these products; their results are summarized in Tables
9 to 11. 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, 1963) . 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 reactions (Hetteche, 1971). In kale, Hetteche (1971)
found the concentration of PAH to be the following: PA,
70-586 ppb; A, 2.4-97.5 ppb; P, 36.2-510 ppb; PL, 53.6-
1196 ppb; BaA, 11.2-230 ppb; CH, 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
proportional 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 ten percent of the
BaP (Kolar, et al. 1975). Fruits grown in polluted environ-
C-24
-------
TABLE 9
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
Product
Wheat grain
Wheat sprouts
Cereals
Barley
Oats
Polished rice
Rye seedling
Lentil seedlings
Sesame seeds
Product
Charred biscuits
Caramel
Chocolate
Concentration
(ppb)
0.02
8.3
0.02
0.03
0.2
0.02
0.05
1.9
0.02
0.02
0.04
29.7
0.2 to 1.5
0.03
0.15
N.D.a
N.D. to trace
Grains &
Concentration
(ppb)
0.1
60.0
0.2 to 4.1
0.3
0.2
N.D.a
10.0 to 20.0
10.0 to 20.0
N.D.
Sugar
Concentration
(ppb)
11.0-72.0
N.D.a
0.2-1.7
Comments
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Cereal Products
Comments
Polluted
environment
Polluted
environment
Polluted
environment
8 other PAH
identified
8 other PAH
identified
and Adjuncts
Comments
4 other PAH
quantified
References
Shiraishi, et al. 1975
Kolar, et al. 1975
Shiraishi, et al. 1975
Shiraishi, et al. 1975
Kolar, et al. 1975
Shiraishi, et al. 1975
Shiraishi, et al. 1975
Kolar, et al. 1975
Shiraishi, et al. 1975
Shiraishi, et ai. 1975
Shiraishi, et al. 1975
Kolar, et al. 1975
IARC, 1973
Shiraishi, et al. 19^5
Shiraishi, et al. 1975
Shiraishi, et al. 1975
Shiraishi, et al. 1974
Reference
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
Graf and Nowak, 1966
Shiraishi, et al. 197:;
Reference
Kuratsune, 1956
Shiraishi, 1973
Fabian, 1969
N.D.: not detected
C-25
-------
TABLE 10
Concentration (ppb) of a BaP in a Few Vegetables
Vegetable Concentration Comments
Parsley leaf and
stem
Red clover
Mushroom
Lettuce
Lettuce
Spinach
Spinach
Spinach
Radish leaves
Radish roots
Radish roots
Tomatoes
24
7
7
8
N.
6
1
7
5
1
N.
0
.3
.5
.0
.6
D.
.2
.3
.4
.3
.2
D.a
.1
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
environment
Polluted
References
Kolar
Kolar
Kolar
. Kolar
Shira
Kolar
Shira
IARC,
Kolar
Kolar
Shira
Kolar
, et
, et
, et
, et
ishi ,
, et
ishi ,
1973
, et
, et
ishi ,
, et
al.
al.
al.
al.
et
al.
et
al.
al.
et
al.
1975
1975
1975
1975
al.
1975
al.
1975
1975
al.
1975
1974
1973
1974
Tomatoes
Cabbage
Cabbage
Chinese cabbage
Potatoes
Potatoes
Sweet potatoes
Sweet pepper
Cauliflower
Bean paste
Kidney bean
Carrot
Cucumber
Eggplant
Onion bulb
Onion greens
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
environment
Polluted
environment
Polluted
environment
Polluted
environment
IARC, 1973
Kolar, et al. 1975
Shiraishi, et al. 1974
Shiraishi, et al. 1974
Shiraishi, et al. 1974
Kolar, et al. 1975
Shiraishi, et al. 1974
Shiraishi, et al. 1974
Kolar, et al. 1975
Shiraishi,
Shiraishi,
Shiraishi,
Shiraishi,
Shiraishi,
Shiraishi,
Shiraishi,
et al.
et al.
et al.
et al.
et al.
et al.
et al.
1973
1973
1973
1973
1973
1974
1974
N.D.: not detected
C-26
-------
TABLE 11
BaP Concentrations (ppb) in Beverages
Beverage
Dark rum
Whiskey
Concentration
1.0
0.04
Comments
3 quinolines
References
Swallow, 1976
IARC, 1973; Nishimura
Tea leaves
Black tea aroma'
Roasted coffee
(moderate dark)
Roasted coffee
(darkest)
Coffee soots
3.9 to 21.3
N.D.
N.D. to 4.0
200.0-440.0
detected and Masuda, 1971
IARC, 1973
7 quinolines Vitzthum, et al. 1975
detected
Kuratsune and Hueper,
1960
Kuratsune and Hueper,
1958, 1960
Kuratsune and Hueper,
1958
This is the volatile components of black tea.
3These are the soots generated during direct and indirect roasting
of coffee beans.
C-27
-------
merits show a high degree of PAH contamination mainly 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
(1966a). Grimmer (1974) analyzed baker's yeasts and determined
the level of PAH. The values are shown in Table 12.
Inhalation
A variety of PAH have been detected in ambient air
in the United States and elsewhere in the world. Because
of its carcinogenic 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, however, 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 concentration is lowest during the summer months and
highest during the winter, (Sawicki, et al. 1962) probably
due to commercial and residential heating during winter
(U.S. EPA, 1974). However, there are some exceptions.
Cleveland, for instance, docs 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).
C-28
-------
TABLE 12
PAH Concentrations (ppb) in a Variety of Baker's Yeast3'
PAH
PA
A
P
FL
BaA
CH
BeP
BaP
PR
f Grimmer
DThis is
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
, 1974
baker's yeas
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
t as opposed tc
Scottish
1620
567
327
93
203
50
40.4
6.2
16.7
3 dietary or brev
Russian
7.2
4.7
16.9
32.1
10.8
11.1
8.7
0.5
6.0
/er's yeast.
C-29
-------
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 relative 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
volatiles. Bartle, et al. (1974) also used a ?^H profiling
technique for the identification of air pollution sources,
such as coal burning, vehicular emissions, and oil and gas
burning.
Meteorological factors have a dominant effect on PAH
concentrations. 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 (Hoffmann and Wynder, 1977).
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 average BaP concentrations in U.S. urban and
rural areas obtained from this U.S. EPA study are shown
in Table 13.
C-30
-------
TABLE 13
Average BaP Concentrations (ng/m ) in U.S. Urban
and Rural Areas During 1966-763
Period
Urban
Rural
1966
3.2
0.4
1970
2.1
0.2
1976
0.5
O.lb
^Santodonato, et al. 1978
This value is the average of two rural locations.
An interesting trend has developed from the National
Air Surveillance Network (NASN) monitored BaP values listed
in Table 13. As can be seen, the average BaP concentrations
in urban areas decreased 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 concentration is
believed to be due primarily to decreases in coal consumption
for commercial and residential heating, improved disposal
of solid wastes, and restrictions on open burning (Faoro
and Manning, 1978). A further observation that can be made
from Table 13 is the five- to tenfold difference in BaP
concentration between urban and rural locations.
The NASN study did not include the determination of
concentrations 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 14.
TABLE 14
Summer-Winter Average of Ambient PAH Concentrations (ng/m )
in the Air of Selected Cities3
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.
15.
18.
2.
13.
3.
1.
5
7
5
9
2
1
3
BeP
3.1
8.0
14.2
4.4
7.6
4.8
1.7
BkFL
3.
8.
12.
3.
8.
2.
1.
7
8
5
1
0
9
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
aSawicki, et al. 1962
The average of total PAH concentrations for all cities
listed in Table 14 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 concentration is assumed (as in the
case of BaP), the present ambient PAH concentration in the
U.S. urban areas can be extrapolated as 9.3 ng/m . 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 15.
C-32
-------
Compound
TABLE 15
PAH Concentration Range in U.S. Cities Determined
by Various Authors in Recent Years
Concentration,
range, ng/m
Reference
NA
A
BaA
PA
FL
BbFL
BjFL
BkFL
P
BaP
BeP
IP
CH
PR
BPR
CR
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
912
6.4
Krstulovic, et al. 1977
Lunde and Bjjrfrseth, 1977
Fox and Staley, 1976; Gordon, 1976
Krstulovic, et al. 1977
Fox and Staley, 1976; Hoffman
and Wynder, 1977
Gordon and Bryan, 1973
Gordon and 3ryan, 1973
Gordon and Bvyan, 1973
Fox and Staley, 1976; Gordon and
Bryan, 1973
Colucci and Begeman, 1971; Fox
and Staley, 1976
Gordon, 1976; Fox and Staley, 1976
Gordon, 1976; Gordon and Bryan, 1973
Gordon, 1976; Fox and Staley, 1976
Gordon and Bryan, 1973
Gordon and Bryan, 1973
Gordon and Bryan, 1973
This Norwegian value is included because no recent U.S.
data are available.
C-33
-------
The exact amount of human PAH intake from all modes
is difficult 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 equivalent to NASN
data on national average BaP levels, the yearly average
data for Los Angeles are used for the derivation of PAH
intake due to inhalation. These values are given in Table 16.
TABLE 16
Average Ambient PAH Concentration in U.S. and
Daily Intake of PAH Through Inhalation
PAH
BaP
Ambient Cone. , ng/m
Inahaltion intake/day, ngc
0.
5.
5
0
Carcinogenic
PAHB
2.7
27.0
Total
10
109
PAH
.9
.0
aThese values are based on the study of Gordon, 1976.
Carcinogenicity of PAH are derived from Natl. Acad. Sci. 1972
c 3
These values are based on 10 m inhalation of air/day.
It can be seen from Table 16 that the yearly intake
of total PAH, carcinogenic PAH, and BaP through inhalation
is 39.8 ug, 9.9 ug, and 1.9 ug, 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 noteworthy, however, that
the total ambient PAH concentration of 10.9 ng/m derived
C-34
-------
from this work is very close to the earlier extrapolated
value of 9.3 ng/ra .
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 ambient levels of PAH, dermal absorption
is not likely to be a significant route of entry.
PHARMACOKINETICS
There are no data available concerning the pharmaco-
kinetics of PAH in humans. Nevertheless, it is possible
to make limited assumptions based on the results of animal
studies conducted with several PAH, particularly BaP. The
metabolism of PAH in human and animal tissues has been espe-
cially well-studied, and has contributed significantly to
an understanding of the mechanisms of PAH-induced cancer.
Absorption
The demonstrated toxicity of PAH by oral and dermal
administration (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), chrysene, 7, 12-dimethylbenz-
(a)anthracene (DMBA), benz(a)anthracene, and 3-methylcholan-
threne (MCA) confirmed that intestinal transport readily
occurs, primarily by passive diffusion (Rees, et al. 1971).
In addition, there is ample evidence to indicate that benzo(a)-
C-35
-------
pyrene, and presumably other PAH, are easily absorbed through
the lungs (Kotin, et al. 1969; Vainio, et al. 1976).
Distribution
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)anthra-
cene, 3-methylcholanthrene, 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
following a single intravenous injection was very rapid
(Schlede, et al. 1970a). The concentration of BaP in the
blood one minute after a 10 jug injection was 193 + 29 ng;
after five minutes concentration of BaP in the blood was
31 + ng. 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, disapperance of BaP from
the brain was slower than from blood or liver, and the concen-
tration of BaP in fat increased during the six-hour observation
period. Schlede and coworkers (1970a) concluded that a
rapid equilibrium-occurs for BaP between blood and liver,
and that rapid disapperance from the blood is due to both
metabolism and distribution into tissues. This contention
C-36
-------
is supported by data (Schlede, et al. 19705) showing that
pretreatment with BaP (which induces microsomal enzyme activity)
accelerates both the rate of BaP disappearance from all
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
intragastric or intravenous administration to pregnant rats
has been variable (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 biological effects. Early attempts to explain
the carcinogenicity of various PAH utilized physico-chemical
calculations (Pullman and Pullman, 1955). These early hypo-
theses were based on the assumption that those regions of
the molecule favoring substitution or addition reactions
would preferentially react with critical cellular target
sites to initiate a carcinogenic transformation. This concept,
however, did not prove successful for PAH.
More recently it was learned that PAH are metabolized
via enzyme-mediated oxidative mechanisms to form reactive
electrophiles (Lehr, et al. 1978). For many of the PAH,
certain "bioactivated" 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).
C-37
-------
The obligatory involvement of metabolic activation
for the expression of PAH-induced carcinogenesis has prompted
the investigation of PAH metabolism in numerous animal models
and human tissues. From these studies has emerged an under-
standing of the general mechanisms involved in PAH biotrans-
formation. It is now known that PAH are metabolized by
the cytochrome P-450-dependent microsomal 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 readily inducible by exposure to chemical and is found
in most mammalian 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, et al. 1973). The MFO system
is involved in the metabolism of endogenous substrates (e.g.,
steroids) and the detoxification of many xenobiotics. Para-
doxically, 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, 1975; 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. Figure 1 presents a
schematic representation of the various enzymes involved
in activation and detoxification pathways for BaP. At present
C-38
-------
iENDOPLAT/.IIC
RETICULUMI
CYTOCHRO'ME P--J50
.MIXED - FUNCTION OXIDASE : MFO»
E,-,r SO
GLUTATHIONE
«1 BiP OXIDES
(DETC,..fiCATION TRANSFERASE
PF..-._;i.;CTSI (CYTOSOLi
--> 3.iP PHENOLS
EPOXIDE
HYDRASE
iENOOPLASMIC
RETICULUMI
V
S.iP QUINONES
-•» sulface?
u'lucuronides
nioi, EHOXIDES
fOIItD UL i IMA IE
5..P DIHYDROOIOLti i PROPOSED PROXIMATE CAnCI\OG£\'S.'
UDP--GLUCUROi\OSYL TRATsSFERASE
S.MIC KETICULUM/
H,0-SOLU'JLE CONJUGATES
(UETOXIrlCAVIO.N1 PHODUCTSi
Figure 1. Enzymatic pathways involved in the
activation and detoxification of BaP.
C-39
-------
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 chemical fate of the most representative and
well-studied compound in the PAH class, namely BaP. The
metabolism of BaP has been extensively studied in rodents,
and the results of these investigations provide useful data
which can be directly compared to and contrasted wich 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 general, 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 mixture by an organic solvent.
This group consists of ring-hydroxylated products such as
phenols and dihydrodiols (Selkirk, et al. 1974; Sims, 1970),
and hydroxymethyl derivatives of those PAH having aliphatic
side chains, such as 7, 12-di-methylbenz(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 enzymaticallv by
microsomes and non-enzymatically by air oxidation of phenols.
Labile metabolic intermediates such as epoxides can also
C-40
-------
be found in this fraction (Selkirk, et al. 1971; Sims and
Grover, 1974; Selkirk, et al. 1975; 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 hydroxylated PAH metabolites with glutathione,
glucuronic acid, and sulfate. Enzyme systems involved in
the formation of water-soluble metabolites include glutauhione
S-transferase, UDP-glucuronosyl transferase, and sulfotrans-
ferases (Bend, et al. 1976; Jerina and Daly, 1974; Sims
and Grover, 1974). Conjugation reactions are believed 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-dihydro-
diol and 9,10-dihydrodiol as well. Considerable evidence
has recently become available which implicates the diol
epoxide, 7$,8
-------
n
i
*>
NJ
6,12-0
MO, - I' ''I01
I-.VK-I' >]
V" '9-OH
Hljnf6]nj ?
i-oi-
— [2,3-epox
w L
, -JUI&J
9,10-cpox 9. 10-diol
7,8,9,10-tclroi]
?
3-OH
BENZO(a)PYRENE
/I
6-OH-Me
IQTOl j:n ~~~~ I'Ol1.-^
11 i'"\ ' f
Oli
4,5-epox 4,5-diol
x?,8-epox
9,10-epox
IfiNOXY
RADICAL
,JM
6 -OH
I "loVor' —M.J "l^^Jul' — p.S-diol-S.IO-epox]
)-»r " "^ ,;OH -
/i8-epox 7,8-diol
7-OH
CONJUGATES
BOUND MACROMOLECULES
DNA
i\'NA
Figure 2. Metabolites of Benzo(a)pyrene
-------
Since the resonance properties of PAH make ring openings
difficult, enzymatic attack in the microsomes funcfions
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 formation
of a transient carbonium ion, would be greatly enhanced.
Arylations of this type are common to many classes of carcino-
genic 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 cytrochrome P-450
can be isolated from animals treated with different enzyme
inducers (Wiebel, e't al. 1973; Nebert and Felton, 1976;
Conney, et al. 1977; Lu, et al. 1978). Moreover, the meta-
bolite profiles of BaP can be qualitatively altered depending
on the type of cytochrome P-450 present in the incubation
mixture (Lu, et al. 1976; Weibel, et al. 1975). This observa-
tion 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
C-43
-------
among animals 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
important consideration in evaluating the health hazards
of PAH is whether metabolism in various animal tissues and
species is indicative 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 the qualitative and quantita-
tive differences exist in the metabolism of BaP by different
tissues and animal species (Sims, 1976; Leber, et al. 1976;
Wang, et 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 practice. 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 difference
may also exist in human tissues (Conney, et al. 1976).
Freudenthal and coworkers (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 chromatography 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 inter-
species variation also existed (Table 17). Nevertheless,
the qualitative differences between man and the other animal
species were by no means dramatic, and probably do not com-
promise the validity of extrapolations concerning PAH metabolism,
The metabolite pattern obtainted for BaP in human lympho-
cytes is similar to that obtained with human liver microsomes
(Selkirk, et al. 1975), and human lymphocytes (Booth, et
al. 1974). However, 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 relative abundance of
the trans-7,8-diol metabolite of BaP was demonstrated.
This result is noteworthy in light of the possiblity that
the 7,8-diol is capable of further oxidative metabolism
to an ultimate carcinogenic form of BaP. It is not known
whether a longer incubation period would have changed the
pattern of metabolite formation.
Excretion
There is no direct information available concerning
the excretion 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
C-45
-------
TABLE 17
Metabolite Percentages of BP Metabolites from Rat, Rhesus,
and Human Lung Microsomal Assays (Freudenthal, et al. 1978)
I
>
Tl
Metabolite percentages
(pmoles metabolite/pmoles total metabolites x
Metabolite
Pre-9,10
9,10-Diol
A
U (B)
4,5-Diol
7,8-Diol
1 , 6-Dione
3,6-Dione
6 ,12-Dione
9 -OH
3 -OH
1
9.7
4.4
8.3
5.3
4.4
7.8
6.8
12.6
40.8
Rata
2
6.3
3.4
9.2
5.2
7.5
8.0
8.6
11.5
40.2
3
9.6
2.9
8.3
8.0
8.3
9.9
8.6
3.5
41.1
1
2.7
1.5
6.9
9.0
4.2
11.4
14.5
11.8
7.3
30.8
Rhesus
2
3.0
4.6
9.2
8.6
14.8
16.0
8.0
35.9
3
5.3
2.6
7.7
7.7
5.1
12.8
20.5
15.3
23.1
1
8.9
4.1
24.9
22.5
22.5
5.7
11.4
100)
Man
2
7.1
3.9
15.0
11.6
13.8
18.3
6.2
24.0
b,c
3
6.0
7.5
13.3
12.6
19.2
27.4
13.9
4
30.0
9.9
4.4
8.5
15.7
8.5
22.9
^Lungs of 5 rats pooled for each group.
Determinations made on lung samples from separate individuals.
GWith the exception of subject 4, activity determinations were
,made using microsomes which had been stored at -84 C.
The structural characteristics of unknown, U, may differ between
species.
-------
both by structure of the parent compound and the route of
administration (Heidelberger and Weiss, 1951; Aitio, 1974).
Moreover, the rate of disappearance of a PAH (i.e., benzo(a)-
pyrene) from body tissues can be markedly stimulated by
prior treatment with inducers of microsomal enzymes (e.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 parathion and paraoxon, can decrease the rate of
BaP metabolism in certain animal tissues (Weber, et al.
1974). From the available evidence concerning excretion
of PAH in animals, it is apparent that extensive bioaccumulation
is not likely to occur.
EFFECTS
Acute, Sub-acute, and Chronic Toxicity
The potential for PAH to induce malignant transformation
dominates the consideration given to health hazards resulting
from exposure. This is because toxic effects are not usually
produced by many PAH until doses are well into the carcinogenic
range. Although the emphasis on carcinogenicity is certainly
justified when dealing with public health issues concerning
PAH, one must recognize that non-neoplastic 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.
As long ago as 1937, investigators knew that carcinogenic
PAH, produced systemic toxicity as manifested by an inhibition
C-47
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of body growth in rats and mice (Haddow, et al. 1973).
Tissue damage resulting from the administration of various
PAH to experimental animals is often widespread and severe,
although selective organ destruction may occur (e.g., adrenal
necrosis, lymphoid tissue damage). Few investigators, however,
have attempted to ascertain the molecular mechanism of PAH-
induced cytotoxicity. Nevertheless, current opinion favors
the concept that normally proliferating tissues (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 exposure 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 selective 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 CF1 Swiss and C57BL mice given a single intra-
peritoneal injection of MCA (0.3 to 1.0 mg) between 12 hours
and 9 days after birth. Degeneration of young cells in
tha bone marrow and retardation of thyroid gland development
C-48
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were also noted. Newborn mice were highly susceptible to
the toxic effects of MCA, with many animals dying from acute
or chronic wasting disease following treatment. Among surviving
CF1 mice, numerous thymomas 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 lymphoid tissues. With single feedings (112 or 133
mg/kg B.W.) to female Sprague-Dawley rats, age 50 days,
DMBA induced pancytopenia 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 inhibition of DNA replication may be involved in the
toxicologic response.
Philips and coworkers (1973) provided strong support
for the argument that DMBA-induced cytotoxicity is mediated
via an interaction with DNA. Female Sprague-Dawley rats
receiving 300 mg/kg B.W. DMBA orally and male rats receiving
an intravenous injection of 50 mg/kg B.W. DMBA displayed
injury to the intestinal epithelium, extreme atrophy of
the hematopoietic elements, shrinkage of lymphoid organs,
agranulocytosis, lymphopenia, and progressive anemia. Mortality
among rats receiving DMBA by gastric intubation (females)
was about 65 percent. In rats given 50 mg/kg B.W. DMBA
14
intravenously, incorporation of C-labeled thymidine into
C-49
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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 in
several organs at six hours, and 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 degenerative 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 intravenously
(5 mg). Lesions of the testes were highly specific and
involved destruction of spermatogonia and resting spermatocytes,
both of which are the only testicular cells actively syn-
thesizing DNA. Neither the remaining germinal cells nor
the interstitial cells were damaged by DMBA. Surprisingly,
no testicular damage was produced by single feedings of
BaP (100 mg), MCA (105 mg), or 2-acetoaminophenanthrene
(40 mg).
For many years researchers have known that the application
of carcinogenic polycyclic hydrocarbons to mouse skin leads
to the destruction of sebaceous glands, hyperplasia, hyper-
keratosis, and even ulceration (Bock, 1964). Sebaceous
C-50
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glands are the skin structures most sensitive to polycyclic
hydrocarbons, and assay methods for detection of carcinogens
have been based on this effect. Although a good correlation
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 carcinogenesis. However, workers exposed
to PAH-containing materials such as coal tar, mineral oil,
and petroleum waxes are known to show chronic dermatitis,
hyperkeratoses, etc. (Hueper, 1963; Natl. Acad. Sci. 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 destruction 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
and are under genetic control.
A toxic reaction which is apparently unique to DMBA
is the selective destruction of the adrenal cortex and induc-
tion of adrenal apoplexy in rats (Boyland, et al. 1965).
Adrenal apoplexy, increased adrenal gland weight, and increased
adrenal hemoglobin content were induced in female Sprague-
C-51
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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 phenomenon.
Repeated injections of benz(a)anthracene derivatives
to mice and rats have produced gross changes in the lymphoid
tissues. Early investigators administered DBA, benz(a)anthra-
cene, and anthracene to mice in weekly subcutaneous injections
for 40 weeks (Hoch-Ligeti, 1941). Analysis of lymph glands
removed at weekly intervals showed an increase of reticulum
(stem) cells and an accumulation of iron in all treatment
groups. 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 controls
and those animals receiving benz(a)anthracene or anthracene.
A more detailed study on the effects of repeated adminis-
tration of DBA on lymph nodes of male rats was reported
in 1944 (Lasnitzki 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 presence 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 non-carcinogen, anthracene, on the
C-52
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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
alterations and pre-neoplastic lesions in this tissue has
particular significance.
In a study conducted by Reznik-Schuller and Mohr (1974),
BaP-induced damage to the bronchial epithelium of Syrian
golden hamsters was examined in detail using semithin (1
jum) tissue sections. Animals were treated intratracheally
with 0.63 mg BaP (total dose) dispersed in a solution of
saline, dodecylsulfate, Tris-HCl, and EDTA once weekly for
life. Animals were serially sacrificed at weekly intervals
following the first month of treatment, and semithin sections
of the bronchi were examined microscopically. In the first
animals sacrificed, minimal focal cell proliferation in
the area of the basement membrane was evident in the bronchial
epithelium. By 7 weeks, cytoplasmic vacuolization of both
goblet and ciliated cells had occurred. Epithelial and
basal cell proliferation continued for several weeks and
led to the formation of three- to four-layered hyperplastic
regions by the llth week. Epithelial cells began to penetrate
through the basement membrane by the 12th week, and within
2 or more weeks the bronchial epithelium began to continuously
grow into the surrounding lung tissues. Microscopic broncho-
genie 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
C-53
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amount of basal cell proliferation may have been the reason
why squamous metaplasia was not observed by the time the
experiment had ended after 21 weeks. Squamous 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
macrophages, 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).
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 carcinogenic 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 esta-
blished that single carcinogenic doses of MCA, DMBA, and
BaP caused a prolonged depression of the immune response
to sheep red blood cells (Stjernsward, 1966, 1969). Noncarcino-
genie hydrocarbons such as benzo(e)pyrene and anthracene
reportedly had no immunosuppressive activity. In a recent
review on irnmunosuppression and chemical carcinogenesis,
C-54
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substantial 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) components in the diet, (2) inducers and inhibitors
of microsomal enzymes, (3) other co-administered noncarcinogenic
or weakly carcinogenic chemicals, and (4) the vehicle used
to deliver a carcinogenic PAH to experimental animals.
These factors tend to complicate the extrapolation of animal
dose-response data to human situations. 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 simul-
taneous administration of various noncarcinogenic PAH.
Similarly, they showed that neutral 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 injection of mixtures containing increasing amounts
of the components. This effect, however, was less dramatic
C-55
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than if BaP were administered alone, and it paralleled the
dose-response curve for DBA acting singly.
Many studies on cocarcinogenesis have been concerned
with the identification of tumor accelerating substances
present in cigarette smoke. These compounds are generally
tested for cocarcinogenic 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,
pyrene, BeP, and benzo(g,h,i)perylene. Doses of 12, 15,
21, and 2,000 jug of these compounds, respectively, were
applied three times a week for 52 weeks to female ICR/Ha
Swiss mice. Each animal also received 5 jug of BaP in 0.1
ml acetone with each dose of test substance. Although phenol
has been regarded as a tumor-promotor in the two-stage carcino-
genesis sytem (Van Duuren, 1976), this compound 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 inter-
changeably. Other PAH (e.g., fluoranthene, pyrene, pyrogallol)
also possess 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 have an inhibitory effect on skin
C-56
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tumorigenesis (250 >ug, three times a week) as induced by
BaP (3 /jg, 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 effective inhibitors of PAH-induced tumor development.
This action has been demonstrated with selenium (Shamberger,
1970; Shamberger and Rudolph, 1966; Riley, 1969), dl-«-toco-
pherol (Vitamin E) (Shamberger, 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 tocopherol-rich diets in rats and mice
(Jaffe, 1946; Haber and Wissler, 1962). The antioxidant
food additives butylated hydroxytoluene (BHT), ethoxyquin,
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; Wattenberg, et al. 1976). The sulfur-containing
antioxidants disulfuram, 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 forestomach 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) .
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Rahimtula and coworkers (1977) examined the abilities
of several antioxidants to affect BaP hydroxylation by rat
liver microsomal mixed-function oxidases. Their results
indicated that antioxidants can markedly inhibit BaP hydroxyla-
tion 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 ir\ vivo
by inhibiting the formation of carcinogenic intermediates
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 upon how and where they are used. These
investigators found that several 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 resulting
from oral administration of BaP was totally prevented, and
skin tumors initiated by BaP application to mice were signifi-
cantly reduced (?50 percent) by treatment with the synthetic
flavone, ^-naphthoflavone. Pulmonary tumor formation was
also reduced 50 percent by incorporation of the naturally
occurring flavone, quercetin pentamethyl ether, into the
diet. Sullivan and coworkers (1978) recently demonstrated
C-58
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that BHA, BHT, phenothiazine, phenothiazine methosulfate,
and ethoxyquin can all reduce the quantitative yield of
BaP metabolites in incubations with rat liver microsomes.
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 formation. Vitamin A has clearly been shown
to play a role in reducing carcinogen-induced tumors (Nettesheim,
et al. 1975; Cone and Nettesheim, 1973; Chu and Malmgren,
1965; Smith, et al. 1975). Nettesheim 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 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 given intragastrically
provided no additional protection, however.
Further studies on naturally occurring antineoplastic
compounds were recently reported by Wattenberg (1977).
Benzyl isothiocyanate and phenethyl isothiocyanate, both
found in cruciferous plants such as cabbage, brussel sprouts,
cauliflower, etc., inhibited DMBA-induced mammary cancer
C-59
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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 interesting speculation regarding the role and importance
of diet in human susceptibility to environmental carcinogens.
In cases where dietary consituents can alter the metabolism
of xenobiotics such as PAH, then the anticarcinogenic effect
may result from an alteration of steady state levels of
activated versus detoxified metabolites.
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 content in the total liver. Diets
deficient in both protein and choline produced even further
reductions in liver weight and cytochrome P-450 content..
Liver microsomes isolated from these animals 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.
Conversely, the inactivation of the direct-acting (ultimate)
carcinogen N-methyl-N'-nitro-N-nitrosoguanidine was reduced
in liver microsomes from mice receiving a protein-deficient
diet.
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In humans fed charcoal-broiled beef, the metabolism
of the drug phenacetin was enhanced; in pregnant rats a
similar diet stimulated the activity of AHH in the placenta
and liver (Conney, et al. 1977a,b). Further studies showed
that high-protein diets enhanced 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 carbonyl,
and nickel, tin, cobalt, and other metals (Conney, et al.
1977a,b). It is not known whether exposure of humans to
these agents may affect susceptiblity to cancer formation
(see Criterion Formulation section).
Teratogenicity
No information is available concerning the possible
teratogenic 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 non-mammalian 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 generally regarded as an
environmental contaminant.
C-61
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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 altera-
tion 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 typhimurium assay, support
the belief that most carcinogenic chemicals are mutagenic
as well. For PAH, the Ames assay has been very effective
in detecting those parent structures and thei" 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.
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 18.
C-62
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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 testing of PAH.
Using this system, Huberman and Sachs (1974, 1976)
demonstrated that a number of carcinogenic PAH produced
forward mutations involving three genetic markers: (1)
ouabain resistance; (2) temperature sensitivity; and (3)
8-azaguanine resistance. Noncarcinogenic PAH such as BeP,
phenanthrene, and pyrene were not mutagenic. In addition,
studies by Huberman indicated that a correlation could be
shown between the degree of carcinogenicity and the frequency
of induced somatic mutations (Huberman, et al. 1977). The
demonstration that covalent binding of carcinogenic PAH
with DNA of V79 cells was the same as occurs ir± vivo further
strengthed 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 Ch-inese hamster V79 cells to test the muta-
genicity of BaP metabolites has contributed significantly
C-63
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TABLE 18
Comparison of Inherent Mutagenic Activity of Thirty
in Salmonella typhimurium TA98 and in Chinese Hamster V79 Cells
Derivatives
(Jerina, et al. 1976)
o
i
cr>
Relative % activity
Compound
Strain TA98
V79
Diol epoxide-1
Diol epoxide-2
H. 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
BaP 1,6-, 3,6-, 6,12-, 4,5-, 11, 12-quinone
BaP 4,5-, 7,8-, 9,10-, 11,12-dihydrodiol
100
35
95
10
20
1
1
0.5
5
1.5
0.5
0.5
< 0. 1
< 0. 1
< 0.1
40
100
40
0.2
1
< 0.1
^ 0.1
1
0.3
^0.1
0.1
< 0.1
^0.1
< 0. 1
<0.1
BaP
The relative percent mutagenic activities are approximations since the data were compiled
from several separate studies conducted at different times. In some experiments, BaP 7,8-
dihydrodiol was 0.1 to 0.4% as active as diol epoxide-2 in V79 cells.
Abbreviations used: BaP, benzo(a)pyrene; 1-HOBaP, 1-hydroxybenzo(a)pyrene; 2- to 12-HOBaP,
other BaP phenols; BaP 1,6-quinone, benzo(a)pyrene 1,6-quinone; BaP 3,6-quinone, BaP 4,5-
quinone, BaP 6,12-quinone, and BaP 11,12-quinone, other BaP quinones; BaP 4,5-dihydrodiol,
trans-4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene; BaP 7,8-, 9,10- and 11,12-dihydrodiol, other
dihydrodiols of BaP; BaP 4,5-oxide, benzo(a)pyrene 4,5-oxide; BaP 7,8-, 9,10-, and 11,12-
oxide, other BaP oxides; diol epoxide-1 (+)-7^,8^..,^J^ ~l n »_«.... -ion tn • Z i j
oxide, 9,10-epoxy-7,8,9,10-tetrahydro BaP;
BaP.
H
7,8-epoxide, 7,9-epoxy-7,8,9,10-tetrahydro
-------
to an understanding of the molecular action of PAH (Huberman,
et al. 1977, 1976a,b; Maleveille, et al. 1975; Newbold and
Brookes, 1976; Jerina, et al. 1976). Comparison of the
mutagenic activities of the optically pure (+) and (-)-en-
antiomers 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 ultimate mutagen/carcino-
gen. Studies by Wood, et al. (1977) 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 V70 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 convincing by the recent studies of Huberman and
coworkers (1976b). They demonstrated for the first time
that BaP and BaP 7,8-dihydrodiol can induce both neoplastic
C-65
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transformation and mutagenesis (ouabain resistance) in the
same culture of normal diploid hamster embryo cells.
In further adaptation of the cell-mediated mutagenesis
system, V79 cells are metabolically activated by rat liver
homogenates containing 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 carcinogenic potencies.
It should be noted, however, that the selection of a particular
activating system (i.e., microsomes vs. feeder cells) may
have a significant influence on the test results.
The analysis of chromosomal aber-rations and sister
chromatid exchanges (SCE's) is often recommended as a screening
technique for potential mutagens and carcinogens. Several
investigators have examined the effects of PAH on the chromo-
somes 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 specific for the carcinogenic
agent nor are they a prerequisite for neoplastic growth.
Moreover, an increased rate of SCE's can be produced by
BaP in cultured human lymphocytes (Rudiger, et al. 1976;
C-66
-------
Schonwald, et al. 1977) but this increase is not correlated
with different rates of BaP metabolism (Rudiger, et al.
1976), a surprising result in light of the known importance
of metabolic activation for BaP mutagenicity. BaP-induced
SCE's rates did not differ between lymphocytes taken from
normal humans and those from patients with lung cancer (Schonwald,
et al. 1977). In recent studies with cultured Chinese hamster
cells exposed to DMBA, BaP, and MCA, none of the chemicals
produced chromosome breaks and only DMBA could successfully
induce SCE's (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 substi-
tution) , there is no reason to believe that detectable chromo-
some damage should occur.
In recent comparisons of three cytogenetic tests, (1)
induction of chromosome aberrations, (2) induction of micro-
nuclei, and (3) in vivo induction of sister chromatid exchanges,
the last test proved to be the most sensitive with carcinogenic
polycyclic hydrocarbons (Bayer, 1978). Since positive results
were also obtained with phenanthrene, however, 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
C-67
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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 theoretically also be detected by examining DNA repair
(Stich and Laishes, 1973). The suggestion that DNA repair
is applicable as a 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 confirmed 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 hydrocarbons 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
C-68
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cells (Swenberg, et al. 1976) has been successfully used
for the detection of chemical carcinogens, including numerous
PAH. However, the percentage of carcinogens giving positive
results for DNA repair is considerably less than in the
cell transformation or microbial mutagenesis assays. Neverthe-
less, 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 repair system (Regan, et al. 1978). These results
support the concept 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 primary
rat leukemias (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 chromosome alterations in PAH-induced tumors in vivo
are not consistent either in frequency or in pattern. DMBA-
induced tumors (fibrosarcoma, squamous carcinoma, lymphosarcoma)
of the uterine cervix in ICR mice revealed various karyotypic
C-69
-------
compositions (Joneja and Coulson, 1973; Joneja, et al.
1971) . These tumors displayed diploid, aneuploid, 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;
Hellstrom, 1959) . Mice treated with 30 /ag 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 jug DMBA) , the incidence of abnormal
chromosomes did not significantly differ frcm controls.
Subcutaneous tumors in Syrian hamsters induced by single
injections of BaP (0.1 ^jg) or DMBA (0.1 mg) , and cultured
cell populations derived from these tumors, failed to reveal
common karyotypic changes (DiPaolo, et al. 1971). Tumor
cells had subdiploid, diploid, and hypotetraploid chromosome
constitutions; further karyotype rearrangements occurred
with subsequent growth i_n vitro.
In humans, the presence of the "Philadelphia" chromosome
in myeloid leukemia appears to be the only example of a
human chromosome abnormality which is tumor-specific (Nowell
Jf
and Hungerford, 1960) . In PAH-induced experimental tumors,
lymphatic leukemia in mice produced by DMBA also displays
consistent chromosome abnormalities (Joneja and Coulson,
1973) . Beyond this common feature, convincing 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 trans-
C-70
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formed 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) .
Evidence has not been encountered in the published
literature concerning the likelihood of PAH-induced somatic
mutation in the absence of neoplastic transformation.
Carcinogenicity
Animal data: Numerous polycyclic aromatic compounds
are distinctive in their ability to produce tumors in skin
and most epthelial 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 histori-
cally involved primarily effects on the skin or lungs. In
addition, subcutaneous or intramuscular injections are fre-
quently employed to produce sarcomas at the injection site.
Ingestion has not been a preferred route of administration
for the bioassay of PAH.
Concern over potential human cancer risk posed by PAH
present in the atmosphere stems from studies demonstrating
that crude extracts of airborne particulate matter can be
carcinogenic to animals (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 subcutaneous injection.
Both the aromatic and oxygenated neutral subfractions were
C-71
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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 polycyclic carcinogens
were painted on the skin of mice has emerged the two-stage
theory of skin carcinogenesis (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 following 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)anthra-
cene) . The second stage, promotion, is a prolonged process
which does not necessarily require the presence of a carcinogen,
but nevertheless a chemical stimulus must be supplied (e.g.,
by croton oil). A complete carcinogen is one which, if
applied in sufficient quantity, can supply both initiating
and promoting stimuli (e.g., DMBA, BaP). The formation
of skin tumors by polycyclic hydrocarbons may also be in-
fluenced by inhibitors and accelerators (cocarcinogens),
thus complicating the interpretation of experimental 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
C-72
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at a method for comparing the carcinogenic potencies of
various polycyclic aromatic chemicals. His results, presented
in Table 19, 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 relative
activity of carcinogens. An important data compilation
on agents tested for carcinogenicity has more recently been
published 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 tracheobronchial 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 carcino-
genesis to lung cancer has not been clearly established.
Many of the bioassay data on PAH-induced lung cancer have
been derived from animal model systems employing various
modes of administration (inhalation, intratracheal instillation,
intravenous injection), 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 co.itmonly employed method for the study of PAH-induced
C-73
-------
n
TABLE 19
Carcinogenic Compounds in Descending Order of Potency (Iball, 1939)
Number
of mice
alive when
first tumor
Compound appears
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
J 1.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
7, 12-Di me thylbenz (a) anthracene
3-Methylcholanthrene (a)
3-Methylcholanthrene (b)
3-Methylcholanthrene
(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
10-Me thy l-benz (a) anthracene
5 , 6- Dime thy l-benz (a) anthracene
6-isoPropyl-benz (a) anthracene
Dibenzo(c,g)carbdzole
Dibenzo(a ,h) pyrene
5-Methy l-benz (a) anthracene
5-Ethy l-benz (a (anthracene
Dibenz (a, h) anthracene
Benzo (c) phenanthrene
Dibenzo (
-------
lung cancer involves intratracheal instillation of test
material in the Syrian golden hamster.
Following the identification of the first carcinogenic
hydrocarbon from soot (BaP) an intensive effort was mounted
to isolate the various active components of carcinogenic
tars (Int. Agency Res. Cancer, 1973). From the earliest
studies conducted, the realization emerged that carcinogenic
PAH are structurally derived from the simple angular phenan-
threne nucleus (Arcos and Argus, 1974). However, unsubstituted
PAH with less than four condensed rings that have been tested
have not shown tumorigenic activity. Furthermore, of the
six possible arrangements with four benzene rings, only
two of these compounds 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, di-
benzo(a,i)pyrene, dibenzo(a,1)pyrene, dibenzo(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')-
pyrene, peropyrene, and dibenzo(h,rst)pentaphene. Beyond
seven unsubstituted aromatic rings, there are very few known
carcinogenic hydrocarbons. However, many physico-chemical
and enzymatic parameters must be dealt with in respect
to carcinogenic PAH. Factors such as solubility and intracell-
ular localization to achieve metabolic activation are likely
to be important determinants of the true carcinogenicity
of a particular PAH.
C-75
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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-cyclopentano-
benz(a)anthracene, 6,7-ace-benz(a)anthracene, acenaphthanthra-
cene, 1,2,5,6-tetrahydrobenzo(j)cyclopent(f,g)aceanthrylene,
and "angular" steranthrene. All of these compounds retain
an intact conjugated phananthrene segment.
The addition of alkyl substituents in certain positions
in the ring system of a fully aromatic hydrocarbon will
often confer carcinogenic activity or dramatically enhance
existing carcinogenic potency. In this regard, Arcos and
Argus (1974) noted that monomethyl substitution of benz(a)an-
thracene can lead to strong carcinogenicity in mice, with
potency depending on the position of substitution in the
decreasing order, 7>6>8=OL2>9. A further enhancement of
carcinogenic activity is produced by appropriate dimethyl
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 POM carcinogens known,
although it has not been shown as a product of fossil fuel
pyrolysis. Methyl substitution in the angular ring of benz(a)an-
thracene, however, tends to deactivate the molecule, although
4,5-dimethylbenz(a)anthracene may be an exception. Carcinogenic
trimethyl- and tetramethylbenz(a)anthracenes are known,
and their relative potencies are comparable to the parent
7,12-DMBA. In general, free radical synthesis of polycyclic
076
-------
hydrocarbons by pyrolysis does not favor alkyl side chain
formation.
Alkyl substitution of partially aromatic condensed
ring systems may also add considerable carcinogenic activity.
The best example of this type of activation is 3-methylcholan-
threne, a highly potent carcinogen.
With alkyl substituents longer than methyl, carcino-
genicity tends to decrease, possibly due to a decrease in
transport through cell membranes. However, different positions
in the benz(a)anthracene molecule will vary with respect
to the effect of n-alkyl substitution on carcinogenicity.
Benz(a)anthracene is especially sensitive to decreased carcino-
genicity 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.
The substitution of highly polar groups (e.g., -OH, -COOH)
in the 7-position of benz(a)anthracene abolishes tumorigenic
activity whereas a wide variety of less-polar substituents
can enhance activity in position 7 (e.g., -CH2OH, -CH2CH2OH,
-CH2COONa, -CH2COOCH3, -CH2OOCCH3, -CN, -CH2CN, -CHO, -NH2/
-SH, -COCC13, -OCH3).
Recent studies have indicated that methylation of the
angular "bay region" (see Effects section) of the benzene
ring, not only in benz(a)anthracene but also in other four,
five, and six-ring aromatic hydrocarbons, leads to a significant
C-77
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decrease, or even to elimination, of the carcinogenic activity
of the molecule. Methylation in other postions does not
diminish, but frequently increases, carcinogenicity. For
example, 7- and 8-methyl-BaP are inactive, whereas 2-,3-,
4-,5-,6-,11-, and 12-methyl-BaP are strong carcinogens.
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)anthracene, 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 extensive resonance capability
is preserved. Moreover, 5,6-dihydro-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 transport
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 cellular constituents. A basic assump-
tion arising from the work of the Pullmans and other (Pullman
and Pullman, 1955) was that a meso-phenanthrenic region
("K-region") of highTT-electron density and with a propensity
for addition reactions was a critical structural feature for
polycyclic carcinogens. In expanding this hypothesis, further
C-78
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biological significance was attributed to the concomitant
presence of a rather unreactive meso-anthracenic region
("L-region") for high carcinogenicity. In addition, a region
of comparatively low reactivity which characteristically
undergoes metabolic perhydroxylation (corresponding to the
3,4-positions of benz(a)anthracene) has been designated
the M-region. According to the theory, only binding of
the K-region to critical cellular sites would cause tumor
formation; protein binding at the L-region causes no tumori-
genic effect, while inactivation is produced by metabolic
perhydroxylation in the M-region. The three regions of
reactivity are readily distinguished in the benz(a)antrhacene
skeleton:
~~' '•'-region of metacol ic
perhydroxylat ion
K-region
The electronic K-L theory of carcinogenic reactivity has
encountered numerous inconsistencies, primarily because
these relationships were derived from physical-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 potential 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
C-79
-------
agents to produce .carcinogenesis and mutagenesis, the "bay
region" theory has been proposed (Lehr, et al. 1978; Wood,
et al. 1977). Examples of a "bay region" in a polycyclic
hydrocarbon are the regions between the 10 and 11 positions
of BaP and the 1 and 12 positions of benz(a)anthracene:
,3ay region Say region
3en:oCa]pyrene 3enZ[a]anchracene
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 epoxides in which the oxirane oxygen is not situated
in a "bay region." Experimentally, the "bay region" diol
epoxides of benz(a)anthracene, BaP, and chrysene were more
mutagenic in vitro and/or tumorigenic than other diol epoxide
metabolites, their precursor dihydrodiols, the parent hydro-
carbons, 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 re-
activity in this manner may be useful in screening PAH as
potential carcinogens (Smith, et al. 1978). Among several
indices of theoretical reactivity examined, the presence
of a bay region for a series of PAH displayed a high degree
of correlation with positive carcinogenic activity (Table 20).
C-80
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TABLE 20
Reactivity Indicies for Polycyclic Hydrocarbons (Smith, et al. 1978)
Carcinogenicity index
K- L- Bay Arcos and Jerina, et al.
Compound region? region? region Argus (1974) (1977)
Naphthalene 0
Anthracene - + - 0
Tetracene - + - 0
Pentacene - + - 0
Hexacene - + - ?
BA + •+ + 5 +
Benzo(a)tetracene + + +
Phenanthrene + - + 0
Benzo(c)phenanthrene + - + 4 +
Chrysene + - + 3 +
Benzo(b)chrysene + + +
Picene + - + 0
Triphenylene + 0
Benzo(g)chrysene + - + 17 ++
Dibenz(a,c)anthracene - + + 3 +
Dibenz(a,j)anthracene + + + 4 +
Dibenz(a,h)anthracene + + + 26 ++
Naphtho(2,3-b)pyrene + + a 27 ++
Benzo(a)pyrene + - + 73
Benzo (e) pyrene + - •*• 2
Dibenzo(a,l)pyrene + - + 33
Dibenzo(a,i)pyrene + - + 74
Dibenzo(a,e)pyrene + - + 50
Dibenzo(a,h)pyrene + - + 70
Triben£o(a,e,i)pyrene - - + 16
aThis compound does not strictly possess a bay region but does contain a "pseudo"
.bay .egion.
Jerina, et al. (1977) have assigned this as ++++.
-------
The carcinogenic activity- of BaP has been studied ex-
tensively 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. 1977,
1976a,b; Slaga, et al. 1977, 1976; Kapitulnik, 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 21) and as
tumor initiators (Table 22} revealed that marked differences
in tumorigenic potency exist. The apparent lack of activity
for the BaP 7,8-diol-9,10-epoxides, despite their exceptional
mutagenicity, may be due to poor skin penetration of adult
mouse skin because of high chemical reactivity. 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. 1977, 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
activity of BaP by subcutaneous injection to rats and mice
is summarized in Table 23.
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 shortening of the latency period for carcinoma
C-82
-------
TABLE 21
Skin Tumors in Mice Treated with Benzo(a)pyrene and Derivatives
Treatment
BaP
BaP
BaP.
BaP
BaP
BaP
BaP
BaP
BaP
BaP
? BaP
£ Bap
1-HOBaP
2-HOBaP
3-HOBaP
4-HOBaP
5-HOBaP^
6-HOBaP
7-HOBaPc
8-HOBaPc
9-HOBaPc
10-HOBaPC
11-HOBaP
12-HOBaP
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
Dose,
jumoles
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
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
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
Reference
Wislocki, et al. 1977
Wislocki, et al. 1977
Albert, et al. 1978
Levin, et al. 1976
Wislocki, et al. 1977
Levin, et al. 1977a
Levin, et al. 1977a
Levin, et al. 1977a
Levin, et al. 1977a
Levin, et al. 1977a
Levin, et al. 1977a
Levin, et al. 1977a
Wislocki, et al. 1977
Wislocki, et al. 1977
Wislocki, et al. 1977
Albert, et al. 1978
Albert, et al. 1978
Albert, et al. 1978
Albert, et al. 1978
Albert, et al. 1978
Albert, et al. 1978
Albert, et al. 1978
Wislocki, et al. 1977
Wislocki, et al. 1977
Female C57BL/6J mice were treated with BaP or BaP derivatives (0.02-0.4 jumole) once every
2 weeks for 60 weeks by topical application to the shaved skin of the back.
Skin tumors consisted mostly of squamous cell carcinomas; other skin tumors were fibro-
sarcomas, papillomas, and keratocanthomas.
Mice were treated once every 2 weeks for 56 weeks.
-------
TABLE 21 (cont'd)
Skin Tumors in Mice Treated with Benzo(a)pyrene and Derivatives
Total no.
Treatment3 animals
BaP 4,
BaP 4,
BaP 7,
BaP 7,
BaP 7,
BaP 7,
BaP 9,
5-oxide
5-oxide
8-oxide
8-oxide
8-oxide
8-oxide
10-oxide
BaP 11,12-oxide
BaP 11
BaP 7,
i diol
£ BaP 7,
diol
BaP 7,
diol
BaP 7,
diol
BaP 7,
diol
,12-oxide
8-dihydro-
8-dihydro-
8-dihydro-
8-dihydro-
8-dihydro-
30-39
30-39
30-39
30
30
30-39
30-39
28
17
30
30
30
30
30
Dose,
/amole s
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
4
1
4
3
15
1
4
4
1
3
15
1
05
025
Mice with
tumors, %
4
6
94
53
18
9
0
0
0
100
100
92
76
7
Total no. .
skin tumors Reference
1
2
37
16
5
3
0
0
0
42
40
28
24
2
Levin
Levin
Levin
Levin
Levin
Levin
Levin
i
i
i
i
t
i
>
et
et
et
et
et
et
et
Wislocki,
Wislocki ,
Levin
Levin
Levin
Levin
Levin
i
i
i
i
i
et
et
et
et
et
al.
al.
al.
al.
al.
al.
al.
et
et
al.
al.
al.
al.
al.
1976a
1976a
1976a
1976a
1976a
1976a
1976a
al. 1977
al. 1977
1976b
1976b
1976a
1976a
1976a
hydroxy-9/3,104-
eopxy-7,8,9,10-
tetrahydrobenzo
(a)pyrene
(diol epoxide 1)
diol epoxide 1
diol epoxide 1
30
30
30
0.4
0.1
0.02
0
0
0
0
0
0
Levin, et al. 1976a
Levin, et al. 1976a
Levin, et al. 1976a
aFemale C57BL/6J mice were treated with BaP or BaP derivatives (0.02-0.4 /amole) once every
.2 weeks for 60 weeks by topical application to the shaved skin of the back
Skin tumors consisted mostly of squamous cell carcinomas; other skin tumors were fibro-
sarcomas, papillomas, and keratocanthomas.
-------
TABLE 21 (cont'd)
Skin Tumors in Mice Treated with Benzo(a)pyrene and Derivatives
Treatment
Total no.
animals
Dose,
/amoles
Mice with
tumors, %
Total no. .
skin tumors
Reference
n
i
00
hydroxy-9<»,10c*-
epoxy-7,8,9,10-
tetrahydrobenzo
(a)pyrene
{diol epoxide 2)
diol epoxide 2
diol epoxide 2
30
30
30
0.4
0.1
0.02
13
7
0
3
2
0
Levin, et al. 1976a
Levin, et al. 1976a
Levin, et al. 1976a
Female C57BL/6J mice were treated with BaP or BaP derivatives (0.02-0.4 jjmole) once every
.2 weeks for 60 weeks by topical application to the shaved skin of the back.
Skin tumors consisted mostly of squamous cell carcinoas; other skin tumors were fibro-
sarcomas, papillomas, and keratocathomas.
-------
TABLE 22
Summary of the Skin Tumor Initiation Activities of Benzo(a)pyrene and its Metabolites
n
i
OD
a\
Initiator
BaP
BaP
BaP
BaP 4,5-epoxide
BaP 7,8-epoxide
BaP 9,10-epoxide
BaP 11,12-epoxide
BaP 7^, 8<^-diol-9o(, 10^-epoxide
BaP 7^, 8d-diol-9$, 10^-epoxide
BaP 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
Reference
Slaga,
Slaga,
Levin,
Slaga,
Slaga,
Slaga,
Slaga,
Slaga,
Slaga,
Slaga,
Levin,
Levin,
et
et
et
et
et
et
et
et
et
et
et
et
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
1976
1977
1977b
1976
1976
1977
1977
1977
1977
1977
1977b
1977b
aFemale CD-I mice were treated with a single dose of initiator dissolved in '^etone, acetone: NH.OH (1000;!),
or dimethyl sulfoxide:acetone (1;3) and followed 1 week later by twice-weekly applications of 10 jug of TPA.
Promotion was by twice-weekly applications of 16 hmoles of TPA beginning 11 days after treatment with
initiator.
-------
TABLE 23
Induction of Sarcoma by Benzo(a)pyrene
Total dose Animals with
Species
Rat
(Sprague-Dawley)
Mouse
Mouse
Mouse
i Mouse
00
~j
Mouse
Mouse
aAdministeced as 0.
.30 doses beginning
No. and (sex)
13 (female)
14 (male)
16 (female)
9 (?)
10 (?)
12 (?)
15 (?)
2 jumole dissolved
jumoles sarcoma, %
6.0a
7.1b
7.1b
15. 9C
5.0C
0.5C
0.002C
in 0.1 ml sesame
100
93
50
66.6
70
66.6
0
Average latency
days
101 -•- 2.7
129
160
112
122
155
N.A.d
oil by subcutaneous injection
i
Reference
Flesher, et al. 1976
Buu-Hoi, 1964
Buu-Hoi, 1964
Gottschalk, 1942
Gottschalk, 1942
Gottschalk, 1942
Gottschalk, 1942
on alternate days for
at 30 days of age.
Administered as three injections of
^Administered as a
single injection
2.4 jjmoles each,
under the skin of
given at 1
the abdomen
month intervals.
, dissolved in 0.5
ml of neutral olive oil.
-------
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 jug and 64 jag
per week) . At the lower dose levels (8 >ug and 16 jug per
week), carcinomas appeared d£ 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 24).
With oral, intratracheal, and intravenous routes of
administration, BaP is less effective than other PAH (e.,g.,
DMBA, MCA, dibenz(a,h)anthracene) in producing carcinomas.
On the other hand, BaP has remarkable potency for the induction
of skin tumors in mice. Therefore, caution must be exercised
in considering the carcinogenicity 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 intra-
venous injection of about 0.25 mg of aqueous dispersions
of PAH to mice, a direct comparison of carcinogenic potency
was possible (Table 25). In this test system, MCA displayed
the greatest lung tumor-forming capability; dibenz(a,h)anthra-
cene followed closely in activity with BaP being considerably
less potent.
C-88
-------
LE 24
Carcinogenicity of Benzo(a)pyrene by Oral Administration
to Various Mammals (IARC, 1973)
Compound
Species
Dose
Route of
administration
Effects
BaP
Mouse
0.2 mg in PEGC
Mouse 50-250 ppm
(age 17-116 days)
Mouse
250 ppm
o
i
oo
Mouse
(age 18-30 days)
Rat
(Sprague-Dawley;
age 105 days)
Hamster
250 ppm
2.5 mg per day
2-5 mg bi-weekly
Hamster
500 ppm
Intragastric
Dietary
(110-197 days)
Dietary
14 tumors of the forestomach
in 5 animals out of 11
79% 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 incid-
ence 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 forestom-
ach in 3 out of 40 animals
5 stomach papillomas in
67 animals treated for 1-
5 months; 7 papillomas and
2 carcinomas in 18 animals
treated for 6-9 months;
5 papillomas in 8 animals
treated for 10-11 months
Dietary 12 tumors (2 esophagus,
(4 days per week 8 forestomach, 2 intestinal)
fc up to 14 months)in 8 animals
Dietary
(140 days)
Oral
Intragastric
Polyethylene glycol
-------
TABLE 25
Comparative Carcinogenicity of Polycylic Hydrocarbons and Related Compounds
Measured by Induction of Lung Tumors (LT) (Shimkin and Stoner, 1975)
o
I
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)acridine
8-Methylbenzo(c) phenanthrene
7-Methylbenzo(a) pyrene
5-Methoxy-7-propylbenz (a) anthracene
Benz (a) anthracene
Untreated controls
Dose,
;umoles/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
>uMoles/kg for
1 LT response
0.9
1.0
6.0
9.5
14
18
--
--
--
--
--
Strain A mice, 8-12 weeks old, received single intravenous injection of 0.24 mg of
methlcholanthrene in aqueous dispersion and were killed 20 weeks later.
-------
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 26 and indicate that: (1) dose-response relationships
are clearly evident, and (2) the co-administration of carrier
particles such as f^O^ (i.e., with BaP) can markedly increase
tumor incidence, depending on the conditions of the experiment
and physical characteristics of the particle. 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 results of several representative studies are
summarized in Table 27.
The published literature regarding chemical carcino-
genesis 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 reproducible transformation assay.
Berwald and Sachs (1963) first demonstrated 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, trans-
C-91
-------
TABLE 26
Induction of Respiratory Tract Tumors in Syrian Golden Hamsters
by Intratracheal Instillation of PAH
Respiratory tumor
Total dose, incidence.
Compound
BaP
BaP
BaP
BaP
BaP
BaP
BaP and
BaP and
BaP and
BaP and
BaP and
BaP and
BaP and
BaP and
BaP and
BaP
BaP
DB(a, i)P
DB(a, i)P
DMBA and
DMBA and
^Animals
Fe2O3
No.
Fe20,, coated
Fe2O-,, ground
Fe2O3/ mixed
gelatin
Fe,0,
Fe203
Fe,0,
Fe2°3
£. J
Fe_O,
2 J
Fe0O,
i J
28
33
33
47
treated once weekly
3 mg BaP administered once
^Animals
Animals
, Animals
Animals
^Animals
An • m;a 1 c
received 30
received 30
received 30
received 12
received 17
r £*r*£i i \* f*f\ 1 fir
weekly
weekly
weekly
weekly
weekly
I nn nium
(male) ,
(male) ,
(male) ,
(male) ,
for 52
weekly
animals
30
30
30
29
28
48
48
49
49
43
46
29
34
30
41
32
28
48
48
46
28
mg
percent
3.25a 10
(female)
(female)
(female)
(female)
(male)
(female)
6.5°
13
26
__a
30b
30b
26. lc
^>
27'4c
26. 3C
26 4C
60d
30d
15 d
V
30*
30*
12£
8.59
1.2h
0.85
13
30
86
93
15
71
73
84
12
17
60.7 (male) , 58.
66.7 (male), 58.
30.3 (male) , 30.
12.8 (male), 9.
42.
57.
75
64.
43.
46.
6 (female)
8 (female)
0 (female)
8 (female)
3
7
Reference
Feron, et
Feron, et
Feron, et
Feron, et
Feron, et
Sellakumar
Sellakumar
Henry, et
Henry, et
Henry, et
Henry, et
Saffioti,
Saffioti,
Saffioti,
Saffioti,
Kobayashi,
Kobayashi,
al
al
al
al
al
t
g
al
al
al
al
et
et
et
et
. 1973
. 1973
. 1973
. 1973
. 1973
et al. 1976
et al. 1976
. 1975
. 1975
. 1975
. 1975
al. 1972
al. 1972
al. 1972
al. 1972
1975
1975
Stenback and
6
5
4
1974a
Stenback and
1974a
Stenback and
1974b
Stenback and
1974b
Sellakumar,
Sellakumar,
Sellakumar,
Sellakumar,
weeks with BaP suspended in 0.9% NaCl solution.
for
10 weeks.
intratracheal instillations.
instillations of BaP
mixed with
intratracheal instillations of
intratracheal instillations of
intratracheal instillations of
& Anri TOO nn F**_ O_ i nt- r Ah r;mh«*A
equal amounts of Fe-jCK
BaP suspended in 0.9% R
1 mg DB(a,i)P suspended
and suspended
aCl.
in 0.2 ml
saline.
in distilled water.
0.5 mg DB(a,i)P suspended in distilled water.
llu nncp a week for 12 weeks in saline suspensions.
. v 2
1Animals received 50 /jg DMBA and 50 /jg Fe2O3 intratracheally once a week for 17 weeks in saline suspensions.
-------
TABLE 27
Induction of Respiratory Tract Tumors in Rats and Mice
Compound
DMBA and
Indian ink
DMBA and
Indian ink
0 DMBA and
^ Indian ink
UJ
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-Mendel)
Rat
(Osbocne-Mendel)
No.
animals
34
56
61
14 (male)
13 (female)
100
100
100
Total dose,
mg
2.5a
6b
10C
236(male)d
179 (female)a
0.005f
0.05f
0.10E
Tumor
Route of incidence,
administration %
Intratracheal 17.6
instillation
Intratracheal 35.7
insti llation
Intratracheal 26.2
instillation
Oral 100 (male)6
77 (female)6
Pulmonary 1-*
injection
Pulmonary is"
injection
Pulmonary 27"
injection
Reference
Pylev, 1962
Pylev, 1962
Pylev, 1962
Snell and Stewart,
1962
Hirano, et al.
1974
Hirano, et al.
1974
Hirano, et al.
1974
-------
TABLE 27 (contd)
Induction of Respiratory Tract Tumors in Rats and Mice
Compound
MCA
MCA
MCA
MCA
Organism
Rat
(Osborne-Mendel)
Rat
(Osborne-Mendel)
Rat
(Osborne-Mendel)
Rat
(Osborne-Mendel)
No.
animals
100
100
100
100
Total dose,
mg
0.20f
0.30f
0.40f
0.50f
Route of
administration
Pulmonary
injection
Pulmonary
injection
Pulmonary
injection
Pulmonary
injection
Tumor
incidence.
47g
40g
51g
45g
Reference
Hirano,
1974
Hirano,
1974
Hirano,
1974
Hirano,
1974
et al.
et al.
et al.
et al.
^Administered as a single dose with 0.2 mg of Indian ink in 0.2 ml of a colloid protein solution.
Administered 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 the left lung.
gOvert sguamous cell carcinoma.
-------
formed cells is that they give rise to fibrosarcomas upon
inoculation into immunosuppressed animals. In addition
to hamster embryo cells, malignant transformation has been
demonstrated in organ cultures, liver cell cultures, fibro-
blastic 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, 1975a,b).
Early reports by Berwald and Sachs (1965) and Dipaolo
and Donovan (1967) described alterations in hamster embryo
cells induced 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. Following marked cytotoxicity,
foci of transformed cells developed which displayed continuous
proliferation jui vitro, chromosomal abnormalities, and the
ability to grow indefinitely in culture. In addition, these
transformed mass cultures, when transplanted to four- to
six-week old hamsters, continued to grow and form tumors.
A good correlation was obtained between ir\ vitro carcino-
genicity of a polycyclic hydrocarbon and the number of trans-
formed clones they produced. The maximum rate of cell trans-
formation in these studies was 25.6 percent in surviving
cells, obtained by treatment with 10 jug/ml of BaP for six
days. BaP treatment at 1 ;ug/ml for six days produced 19.9
percent transformation in surviving cells. Further data
indicating the activity of several polycyclic carcinogens
>•« r» c
-------
and their derivatives are summarized in Table 28. 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 i£ vivo (see Effects section) which indicate
that K-region epoxides of polycyclic carcinogens are less
active than the parent compound in various species. A possible
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 dissoci-
able and occur by different processes (Landolph, et al.
1976; DiPaolo, et al. 1971), with the toxicity being due
to random alkylation of nucleophilic regions within the
cell. However, when hamster embryo cells are pretreated
with weak chemical carcinogens which can induce microsomal
enzyme activity (e.g., benz(a)anthracene, methyl methane-
sulfonate, ethyl methanesulfonate) before the addition of
C-96
-------
a potent carcinogen (e.g., MCA, BaP, DMBA), transformation
may be considerably enhanced (DiPaolo, et al. 1971, 1974).
As a prescreen for chemical carcinogens, cell transforma-
tion _iri 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 ir\ vitro, whereas none of the nonc-aj.cino-
gens 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, however,
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-deficient 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
i_n vitro or iji vivo is known to have a marked effect on
the carcinogenic response 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 modifiers of microsomal
enzymes. Pretreatment of 10T1/2C18 cells with benz(a)anthra-
cene, a microsomal enzyme inducer, caused a doubling in
MCA-mediated transformation. Similarly, treatment with
inhibitors of epoxide hydrase (e.g., cyclohexene oxide;
-------
TABLE 28
Hamster Embryo Cell Transformation Produced by
Several Polycyclic Hydrocarbons and Their Derivatives
Concentration,
Compound ug/ml
DB(a,h)Aa
DB(a,h) Ab
DB ( a , h) A5 , 6-epox idea
DB(a,h)A5,6-epoxideh
MCAC
MCAd
MCA opoxidec
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
1341
1363
1365
598
601
395
350
895
866
817
707
404
370
349
664
364
245
103
1016
394
Cloning
efficiency,
4.2
3.8
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. I
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
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
1.6.5
2.46
5.33
Reference
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, et al. 1972
Huberman, et al. 1972
Grover, et al. 1971
Grover, et al. 1971
Grover, et al. 197.1
Grover, et al. 1971
Huberman, et al. 1972
Huberman, et al. 1972
Huberman, et al. 1972
DiPaolo, et al. 1971
Huberman, et al. 1972
(luberman, et al. 1972
huberman, et al. 1972
DiPaolo, et al. 1971
DiPciolo, et al. 1971
.7-day treatment of cells seeded on a feeder layer.
.7-8 day treatment of cells.
,4-hour treatment of cells seeded in conditioned medium.
8-day treatment of cells.
-------
styrene oxide; 1,2,3,4-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 carcinogenic hydrocarbons under conditions in which no
spontaneous malignant transformation occurred. Cells treated
with MCA (1 ;ig/ml) for six days in culture produced malignant
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 J.n_ vivo oncogenic activity
of eight hydrocarbons (including BaP, MCA, DMBA, and DBahA)
and the number of transformed colonies produced in this
system. In contrast 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 been due, however, to a' decreased intracellular
C-99
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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 Hayflick, 1974). However, Rhim and coworkers (1975)
reported that a human osteosarcoma clonal cell line could
be further transformed iin vitro with DMBA. Morphologic
alterations and abnormal growth patterns became evident
in cells treated with DMBA at 2.5 and 1.0 jjg/ml in the fifth
subculture 52 to 57 days after exposure. One of the altered
cell lines obtained from the 1 jug/ml treatment was tumorigenic
in nude mice by subcutaneous and intracerebral injection.
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
carcinogenicity suffers from the lack of reliable biochemical
and morphological 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 carcino-
gen-treated pieces of tissue are implanted into host animals
(Heidelberger, 1973) . Limited success has been achieved
C-100
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with organ cultures of rat tracheas, which showed characteristic
morphologic alterations when treated with DMBA, BaP, and
MCA (Heidelberger, 1973). In addition, Crocker (1970) has
exposed respiratory epithelia from the hamster, rat, dog,
and monkey to BaP at 7 to 15 ug/ml and observed occasional,,
squamous metaplasia. More commonly, pleomorphic cells in
a dysplastic epithelium were evident as a result of the
treatment. Using this system, it was also possible to demon-
strate a protective effect of vitamin A against BaP-induced
abnormal differentiation. Rat tracheas maintained in organ
culture have been suggested as a useful system for the predic-
tive 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 C57 Bl), and lung tissue of their 19-
to 20-day-old embryos was subsequently explanted in culture
(Shabad, et al. 1974). A transplacental influence of BaP
was manifested as a proliferative stimulus 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.
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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 groups of 100 female NMRI mice with single subcutaneous
injections of a mixture containing 10 non-carcinogenic PAH,
in addition to BaP and/or dibenz(a,h)anthracene. The treatment
combinations and dosages are summarized in Table 29. As
the results depicted in Table 30 indicate, increases in
tumor incidence could be attributed to the presence of increased
amounts of BaP and of dibenz(a,h)anthracene. It is noteworthy
that, at the lower dosages, dibenz(a,h) anthracene was more
effective in producing tumors at the injection site than
was BaP. Moreover, no effect of the 10 non-carcinogens on
tumorigenic response was evident. Probit analysis of tumor
incidence data indicated that the tumorigenic response from
application of all 12 PAH was attributable solely to dibenz(a,h)-
anthracene.
Similar studies intended to reveal carcinogenic interac-
tions 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 31). Animals
recieved twice weekly treatments for life (or until a carcinoma
developed). Their results (Table 32) indicated that a mixture
of carcinogenic PAH was more effective than BaP alone, and
that the whole mixture (carcinogenic plus non-carcinogenic
PAH) was not significantly more effective than the carcinogenic
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TABLE 29
Classification of Test Groups
(Pfeiffer, 1977)
A
B
dose substance
(ug)
A, 3.12 benzo(a) pyrene
A, 6.25
A^ 12.5
AT 25.0
Ac 50.0
Ag 100.0
C
Substance C-,
benzo(e) pyrene
benzo (a) anthracene
phenanthrene
anthracene
pyrene
f luoranthene
chrysene
perylene
benzo(ghi) perylene
coronene
D
Dl Al + Bl
D2 A2 + B2
D3 A3 + B3
D4 A4 + B4
DC Ac + Be
555
D6 A6 + B6
dose
(/ig)
2.
3.
125.
31.
65.
28.
3.
0.
12.
3.
C-j
Bl
B3
B6
dose
15
125
0
25
1
1
125
2
8
125
4
6
250
62
131
56
6
0
25
6
.3
.25
.0
.5
.2
.25
.25
.4
.6
.25
E
El
E2
E3
E4
EC
5
E6
dose
(ug)
2.35
4.7
9.3
18.7
37.5
75.0
C3
dose
8.75
12.5
500.0
125.0
262.5
112.5
12.5
0.87
51.25
12.5
C,
C2
C3
£4
C6
substance
dibenz (
dose
(/ig)
17.
25.
1000.
250.
525.
225.
25.
1.
102.
25.
+ D,
+ D2
+ Dj
+ D4
^. jj
+ D6
5
0
0
0
0
0
0
75
5
0
.a/hjanthracene
C5 Cc
dose
35.
50.
2000.
500.
1050.
450.
50.
3.
205.
50.
5
0
0
0
0
0
0
5
0
0
dose
70
100
4000
1000
2100
900
100
7
410
100
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
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TABLE 30
Tumor Incidence Resulting, by the End of the 114th Week,
from a Single Subcutaneous Application of Test Substances (Pfeiffer, 1977)
n
i
BAP group
dose (ug)
3.12
6.25
12.5
25.0
50.0
100.0
(A)
no. of
tumors
9
35
51
57
TI
83
DBA group
dose (ug)
2.35
4.7
9.3
18.7
37.5
75.0
(B)
no. or
tumors
37
39
44
56
65
69
BaP + DAB group (D)
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
no. of
tumors
41
55
61
72
68
82
(E)
-------
TABLE 31
Doses (ug) Applied in Dermal Administration Experiments,
in Relation to Benzo(a)pyrene (Schmahl, et al. 1977)
Controls
Acetone
Benzo (a) pyrene
C PAH
Benzo (a) pyrene
Dibenz (a,h) anthracene
Benzo (a) anthracene
Benzo (b) f luoranthene
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 PAHrNC 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
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
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 1755.0
3.0)
12.0
195.0
207.0
is constantly 1:16.25
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TABLE 32
Findings at the Site of Application
of PAH to Mouse Skin3 (Schmahl, et al. 1977)
o
1
I-1
o
<3\
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 +
NC PAH
Single
dose
ug
-
1.0
1.7
3.0
4.0
6.8
12.0
65.0
195.0
585.0
1755.0
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 Histological diagnosis at the
no. of
animals
81
77
88
81
81
88
90
85
84
88
86
89
93
93
site of application
negative papilloma carcinoma sarcoma
abs. % abs. % abs. % abs. %
80
66
63
36
52
31
25
84
84
87
70
43
36
28
99
86 1
72
44 2
64 4
35 3
28 1
99
100
99
81
48 1
39 2
30 1
-
1 10
25
3 43
5 25
3 53
1 63
1
-
1
15
1 44
2 54
1 64
1
13
28
53
31
60 1
70 1
1
-
1
17 1
49 1
58 1
69
1
-
-
-
-
1
1
-
-
-
1
1
1
_
The decimal points have been rounded off; therefore, the sum of % values will not always be
equivalent to 100%
-------
PAH group alone. Thus, the carcinogenic effects observed
were solely attributable to the carcinogenic components
of the misture.
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 quantities of PAH can be ingested by humans,
and that in animals such exposures are known to cause cancers
at various sites in the body.
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.
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 occupa-
C-107
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tional 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.
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CRITERION FORMULATION
Existing Guidelines and Standards
There have been few attempts to develop exposure stand-
ards for PAHs, either individually or as a class. In the
occupational setting, a Federal standard has been promulgated
for coke oven emissions, based primarily on the presumed
effects of the carcinogenic PAH contained in the mixture
as measured by the benzene soluble fraction of total particu-
late matter. Similarly, the American Conference of Governmental
Industrial Hygienists recommends a workplace 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
extractable fraction. These standards are summarized below:
Substance Exposure Limit Agency
Coke Oven 150 jug/m , 8-hr. U.S. Occupational Safety
Emissions time-weighted and Health Administration
average
Coal Tar Pro- 0.1 mg/nr , 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
developed. The 1970 World Health Organization European
Standards for Drinking Water recommends a concentration
C-109
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of PAH not to exceed 0.2 jug/1. This recommended standard
is based on the composite analysis 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(1,3,-cd)pyrene.
The designation of these six PAH for analytical monitor-
ing of drinking water was not made on the basis of potential
health effects 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 presence 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 indicate (see Exposure section), levels of
PAH in raw and finished waters are typically much less than
the 0.2/jg/l criterion.
Current Levels of Exposure
Section I of 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, quantita-
tive estimates of human exposure to PAH require numerous
assumptions concerning principal routes of exposure, extent
C-HO
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of absorption, conformity of human lifestyle, and lack of
geographic-, sex-, and age-specific variables. Nevertheless,
by working with estimates developed for PAH as a class,
it is possible through certian extrapolations to arrive
at an admittedly crude estimate of PAH exposure.
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 for 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 FL and BPR have been routinely
determined in water, as recommended by the World Health
Organization.
The reported estimated average concentrations of BaP,
carcinogenic PAH (BaP, BjFL, and IP), and total PAH in drink-
ing water are 0.55 ng/1, 2.1 ng/1, and 13.5 ng/1, respective-
ly (see Exposure section; Basu and Saxena, 1977-78) . 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 pg/day. As a check on this estimate,
PAH intake may be calculated based on reported concentrations
in various foods (see Exposure section) and the per capita
C-lll
-------
estimates of food consumption by the International Commission
on Radiological Protection (1974). Taking a range of 1.0
to 10.0 ppb as a typical concentration for PAH in various
foods, and 1,600 g/day as the toal daily food consumption
by man from all types of foods (i.e., fruits, vegetables,
cereals, dairy products, etc.), the intkae of PAH from the
diet would be in the range of 1.6 to 16.0 ;ug/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 .16 to 1.6
>ig/day.
Ambient air is reported to contain average levels of
0.5 ng/m , 2.0 ng/m , and 10.9 ng/m for BaP, carcinogenic
PAH, and total PAH, respectively (see Exposure section,
Table 16). 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 follows:
C-112
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Table 33
Estimate of Human Exposure to PAH from Various Media
Source
Water
Food
Air
BaP
0.0011 jjg/day
. 16-1. 6 /jg/day
0.005-.0115 jug/day
Estimated Exposure
Carcinogenic PAH—
0.0042 pg/day
0.03-0.046 >ug/day
Total PAH
0.027 jug/day
1.6-16. jug/day
0.164-0.251 ;ag/day
Total
.166-1.6 jug/day
1.6-16. ;ug/day
aTotal of BaP, BjFL and IP; no data are available for food
Two important factors are not taken into account in
this estimate. 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 /ig/day (Natl. Acad. Sci. 1972). Second, the possi-
bility 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 carcinogenic hazard of PAH to man involves the relation-
ship 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, et al. 1973a,b).
It was suggested that the extent of AHH inducibility in
0113
-------
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 epidemio-
logic evidence supports this hypothesis. Moreover, they
were able to show that AHH inducibility in lymphocytes segre-
gates in the human population as a genetic trait. However,
their studies failed to find a correlation between this
inducibility and presumed cancer susceptibility, either
among healthy relatives of cancer patients or in patients
who had their cancer surgically removed. It is noteworthy
that previous investigations on AHH inducibility were conduct-
ed in persons with active cancer.
Recent studies with other human tissues (liver and
placenta) have provided important new data concerning the
carcinogen-metabolizing capacity of man and its implications
for cancer susceptibility. Conney, et al. (1976) examined
individual differences in the metabolism of drugs and carcino-
gens 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
correlation was found between zoxazolamine hydroxylase activ-
ity and BaP hydroxylase activity. (Kapitulnik, et al. 1976) .
Thus, metabolism of BaP and zoxazolamine by human placenta
occurs by the same enzyme systems(s) or by different enzyme
C-114
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systems under the same regulatory control (Kapitulnik, et
al. 1977a). BaP and zoxazolamine 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 metabo-
lism 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. Fur-
thermore, large inter-individual differences exist in the
capacity of humans to metabolize foreign chemicals both
i£ vitrP an<3 in vivo« Further studies showed -that 7,8-benzo-
flavone markedly stimulated the hydroxylation of BaP, antipy-
rene, and zoxazolamine in human liver samples, but with
a wide variation in magnitude among different samples.
These results suggested the presence of multiple monooxygen-
ases 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 different 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 far
in humans (Kapitulnik, et al. 1977a). More important, how-
ever, MCA is a potent inducer of BaP hydroxylase activity
in rats but does not stimulate antipyrene hydroxylase, clear-
ly suggesting that.metabolism of PAH in rodents may be regu-
lated by different enzyme systems than in humans (Kapitulnik,
et al. 1977a).
C-115
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In contrast to the apparent multiplicity of cytochrome
P-450 dependent enzyme systems for the oxidative meatbolism
of PAH in man, a single epoxide hydrase with broad substrate
specificity may be present in human liver (Conney, et al.
1976; Kapitulnik, et al. 1977c). Because the hydration
of arene oxides may lead to the formation of dihydrodiol
carcinogen precursors, the capacity of different humans
to metabolize epoxides may affect cancer susceptibility.
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 interpret-
ed 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
carcinogen, a promotion phase involving cell proliferation
is also involved in skin carcinogenesis (Yuspa, et al. 1976).
Therefore, although certain aromatic hydrocarbons are effec-
tive enzyme inducers, their bioactivated metabolites may
function only as an initiator having no promoting ability.
A potent complete carcinogen, however, will be transformed
C-116
-------
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 prolifera-
tion. These observations raise certain doubts concerning
the validity and/or reliability of equating enzyme inducibil-
ity with carcinogenic potential for chemical agents. Further
reinforcement of this opinion has been provided by Shulte-
Hermann (1971) 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 genet'.cs of AHH induci-
bility is organ-dependent rather than strain-dependent in
animals has important implications for evaluating susceptibil-
ity to PAH-induced cancers (Kouri, et al. 1976). Most signif-
icant 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 importantly on susceptibility
t:o malignancy.
Enzyme induction by PAH is not limited to AHH. Owens
(1977) recently demonstrated that MCA can induce hepatic
UDP-glucuronosyltransferase activity in certain inbred strains
of mice. This enzyme 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
C-117
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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 demonstrated 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 chlorproma-
zine were potent inducers 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 transferase-catalyzed removal
of metabolites by glucuronic acid conjugation. This situation
would be exacerbated by the fact that metabolites of MCA
are incapable of further inducing the transferase activity.
This effect may have considerable toxicologic significance
in that highly reactive epoxides of PAH formed by the action
of AHH under these circumstances may not be adequately removed
by glucuronidation. Thus, one must consider the total expo-
sure of all environmental agents and their possible effect
on critical enzymatic processes before attempting to assess
the toxicologic impact of exposure to a specific PAH. in
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 considering the possibility
of special groups at risk.
C-118
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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 experimental animals. Further-
more, 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 environ-
mental monitoring. Therefore, an approach to criterion
development is adopted in this report with the objective
of deriving a single criterion to encompass the entire PAH
class. This approach is attractive in that it recognizes
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 scientif-
ically sound.
C-119
-------
(3) No chronic animal toxicity studies exist involving
oral exposure to PAH mixtures.
(4) No direct human data exist concerning the effects
of exposure to defined PAH mixtures.
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 appropriate animal bioassay from which
to derive data for application to the linear non-threshold
model for human cancer risk assessment (see Appendix I)
should be guided by several considerations. Primary emphasis
must be placed on appropriate animal studies which: (1)
include sufficient numbers of animals for statistically
reliable results; (2) involve long-term low-level exposures
to PAH; (3) include a proper control group; and (4) achieve
positive dose-related carcinogenic response.
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. Even when considering single chemicals, almost
no studies are available which involved oral exposure at
more than one dose level to a reasonable number of animals.
Two studies have been selected, one involving BaP ingestion
(Rigdon and Neal, 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.
C-120
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In the strictest sense it can be argued that a criterion
for a chemical class derived from experiments involving
a single component of that class is invalid. On the other
hand, selection of those components (e.g., BaP and DBA)
which are among the more potent carcinogens in the PAH class
should lead to a conservative criterion approach. It must
be assumed that interactions among the various PAH components
resulting in either an enhancement or inhibition of biological
effect (see Effects section) will cancel each other out
in the environment. Presently, there is no way to quantitate
the potential human health risks incurred by the interaction
of PAH, either anomg themselves 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 administration;
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. Calculations of water quality criteria
for PAH based upon bioassay data for BaP and DBA are presented
in Appendix I.
The water quality criteria for BaP and DBA derived
using the linear non-threshold model as described in the
Appendix are 9.7 ng/1 and 43 ng/1, respectively. For the
sake of comparison, a water quality criterion for DBA was
calculated using the procedure developed by Mantel and Bryan
C-121
-------
(1961). As opposed to the linear non-threshold model, which
is logistic and defines acceptable risk as 1/100,000, the
Mantel and Bryan (1961) model is probablistic and defines
acceptable risk as 1/100,000,000. Furthermore, the Mantel
and Bryan (1961) is concerned with the maximum tumor incidence
in treated animals at the 99 percent assurance level. Using
•
the Mantel and Bryan (1961) approach with DBA, the resu ^ant
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) consister : with the
protection of aquatic organisms, human health, and recreation-
al activities." BaP and DBA are known animal carcinogens.
Because there is no recognized safe concentration 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 cases and in order to assist the Agency and
States in the possible future development of water quality
regulations, the concentrations of BaP and DBA corresponding
to several incremental lifetime cancer risk levels have
been estimated. A cancer risk level provides an estimate
of the additional incidence of cancer that may be expected
in an exposed population. A risk of 10~ for example, indi-
cates 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 evi-
denced by their detection in sediments, soils/ air/ surface
waters, and plant and animal tissues. The ecological impact
of these chemicals, however, is uncertain. Numerous studies
show that despite their high lipid solubility, PAHs show
little tendency for bioaccumulation in the fatty tissues
of animals or man. This observation is not unexpected,
in light of convincing evidence to show that PAH are rapidly
and extensively metabolized.
Lu, et al. (1977) have published the only available
study regarding 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 study, BaP
showed a small degree of biomagnification which probably
resulted from food chain transfer. In this case the biomagni-
fication 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 contrast/ as can be noted in Table 6a, the
BCF derived from octanol-water partition coefficients for
BaP is 6800.
In the Federal Register notice of availability of draft
ambient water .quality criteria, EPA stated that it is consid-
C-123
-------
ering setting criteria for BaP and DBA at an interim target
risk level of 10~ , 10~ or 10" as shown in the table below.
BaP
Exposure Assumptions Risk Levels and Corresponding Criteria (1)
(per day) ng/1
0 1£~7 1£~6 1£~5
2 liters of drinking water 0 0.097 0.97 9.7
and consumption of 18.7
grams fish and shellfish. (2)
Consumption of fish and 0.44 4.45 44.46
shellfish only.
DBA
Exposure Assumptions Risk Levels and Corresponding Criteria
(per day)
2 liters of drinking water
and consumption of 18.7
grams fish and shellfish. (2)
Consumption of fish and 0.196 1.96 19.63
shellfish only.
0
0
ID'7
0.043
ng/1
io-6
0.43
io-5
4.30
C-124
-------
(1) Calculated by applying a modified "one hit" extrapolation
model described in the FR 15926, 1979. Appropriate bioassay
data used in the calculation of the model are presented
in Appendix I. Since the extrapolation model is linear
to low doses, the additional lifetime risk is directly propor-
tional to the water concentration. ..Therefore, water concen-
trations corresponding to other risk levels can be derived
by multiplying or dividing one of the risk levels and corres-
ponding water concentrations shown in the table by factors
such as 10, 100, 1,000, and so forth.
(2) Approximately 22 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. The remaining 78 percent 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 corresponding 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 increment to risks associated with ambient water exposure
only.
C-124a
-------
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APPENDIX I
Carcinogenicity Risk Assessment by Extrapolation
from Laboratory Animal Toxicity Tests
An assessment of health risks associated with exposures
of a general environmental nature requires prediction of
effects from low level exposures of lifetime duration.
Carcinogenic risks effects from environmental exposures
must normally be estimated from animal data obtained at
much higher levels because of the difficulty in detecting
a small increase in tumor induction resulting from long-
term low level exposure. Because the carcinogenic process
is generally believed to be irreversible, self-replicating,
and often originating from a single somatic cell mutation,
assumptions of threshold levels of effect are believed to
be invalid for many, if not all, cancer-causative compounds.
Although many models have been proposed for extrapolation
from animal data to human risk assessment, the one utilized
here was chosen to facilitate uniform treatment of the variety
of chemical compounds that are discussed in the development
of those water criterion documents which deal with animal
carcinogens.
It is recognized that the process of evaluating existing
studies and resultant data in preparation for application
of mathematical methods involves a high level of professional
judgment. Many questions will necessarily arise due to
the unique characteristics of the specific compounds under
discussion and the tremendous variability in completeness
and comparability among the available studies.
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A general explanation of the evaluation and extrapolation
procedures to be used are as follows:
1. Since the compounds discussed are known, or suspect,
carcinogens, emphasis was placed on those studies
with carcinogenic or mutagenic endpoints. In
particular, those studies dealing with mammalian
species.
2. The extrapolation method employed is a mathematical
procedure which uses a single dose and observed
response of a toxicological experiment to estimate
a dose level for humans that will not increase
the risk of tumors by more than a specified level
(1 in 100,000) (Personal communication. Dr. Todd
Thorsland, CAG, U.S.EPA, Washington, D.C.). Clearly
this method is predicated on sound toxicologic
test procedures. Hence, each included study was
evaluated for adherence to sound toxicological
and statistical principles.
3. Judgment was exercised in prioritizing the signif-
icance of toxicologic studies that use different
routes of administration. In general, the preferred
route of exposure is oral (food, water, or gavage)
followed by intraperitonea.l, intravenous, inhalation,
or dermal routes of administration for the same
species. However, in some instances consideration
of absorption rates required that other routes
be evaluated.
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The NCI's Ad Hoc Committee on the Evaluation of
Low Levels of Environmental Chemical Carcinogens
outlined two conditions that would render the
extrapolation of animal carcinogenesis to man
inappropriate. This committee reported to the
Surgeon General as follows:
"Any substance which is shown conclusively
to cause tumors in animals should be considered
carcinogenic and, therefore, a potential hazard
for man. Exceptions should be considered
only where the carcinogenic effect is clearly
shown the results from physical rather than
chemical induction or where the route of
administration is shown to be grossly inappro-
priate in terms of conceivable human exposure."
After selection of the sound toxicologic studies
that form the basis for development if a recommended
criteria, a single dose and observed response
was selected for the most "sensitive" sex (if
both males and females were tested) according
to the following method: Select the lowest dose
which yields a tumor response rate that is greater
than the control rate. If the standard controls
and media control response rates are not significantly
different ( ^0.05), a combined rate was calculated
from controls.
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5. The extrapolation methods were applied independently
to each selected dose and response pair. The
lowest projected dose was selected as the "safe
level" based on the available toxicologic studies,
if judgement indicated equal confidence in the
various dose-response pairs.
6. The calculated safe dose was evaluated along with
the results from human studies to delvelop a
recommended criteria.
Calculation of Estimated Safe Levels for Humans;
The specific data analyses performed along with required
input data are described following in Mathematical Description
of Extrapolation Method. This model provides the additional
risk associated with ingestion of 2 liters of water per
day and contaminated aquatic foods. Any other risks associated
with air, food, or other exposure are not addressed by this
model. A copy of the working data sheet is also included.
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Mathematical Description of Extrapolation Method
A. Necessary information:
Nt = No. of animals (males or females) exposed to selected
dose that developed tumors (all sites combined
unless tumors appear to be related to route of
administration, e.g., peritoneal tumors would
not be included if interperitoneal injection method
is used).
NT = Total number of animals (male or females) exposed
to selected dose level.
nc = Number of control animals (males or females)
with tumors.
NC = Total number of control animals (males or females).
Le = Actual maximum lifespan for test animals.
le = Length of exposure (no. of hours, days, weeks, etc.)
d = Average dose per unit of time (mg/kg).
w = Average weight of test animals (kg).
B. Necessary information from .general literature:
70 kg = Average weight of man.
L = Theoretical average length of life for test
species, unless specified in article. (See
attached table for appropriate values)
F = Average weight of fish consumed per day,
assumed 18.7 grams.
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C. Necessary ecological information:
R = Bioaccumulation factor for edible portions of fish
(Supplied by Environmental Research Laboratory,
Duluth)
(Note: If a bioacculumation factor is provided for
the total fish or for some part other than the total
edible portion (such as the fat) an attempt should
be made to estimate factor for edible portion).
D. Mathematical Model
Pt = PC + (1-Pc)
Where:
Pt = nt -t- NT = Proportion of test animals with tumors,
PC = nc •*• NC = Proportion of control animals with
tumors.
d x le
D = — = Lifespan weighted average dose
e level (mg/kg)/(unit of Time).
i ' "" r
B =t In
[*rf W
MB- £ *
,_ lifespan fo
. *~ . _ lifespan for test animals _ Le
length of life for species L
(Note: It is assumed that average
weight of man = 70 kg.)
If and only if s'^Q.l then
SL = X? = Safe level (mg/1) for man
If BX7p.l then
SL = +BM2 = RxF) } x 70 = Safe level (mg/D for man
(Note: It is assumed average daily consumption
is 2 liter/day)
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APPENDIX II
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 and 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 non-carcino-
genic 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.
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The mutagenicity of PAH in the Salmonella typhimur ium
assay correlates well with their carcinogenicity in animal
systems. PAH compounds have damaged chromosomes in cytogenetic
tests, have induced mutations in mammalian cell culture
systems and have induced DNA repair synthesis in human fibro-
blast cultures.
The water quality criterion for carcinogenic PAH compounds
is based on the assumption that each compound is as potent
as BaP and that the carcinogenic effect of the compounds
is proportional 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" is
9.7 nanograms per liter. Therefore, with the assumption
above, the sum of the concentrations of all carcinogenic
PAH compounds should be less than 9.7 nanograms per liter
in order to keep the lifetime cancer risk below IQ~^.
Roy E. Albert, M.D,
Chairman
PARTICIPATING MEMBERS
Elizabeth L. Anderson, Ph.D.
Jacqueline V. Carr, M.S.
Chao W. Chen, Ph.D.
John R. Fowle III, B.S.
Bernard H. Haberman, D.V.M., M.S.
charalingayya B. Hiremath, Ph.D.
David A. Mann, B.A.
Robert McGaughy, Ph.D.
Barbara Shelton, B.S.
Dharm V. Singh, D.V.M., Ph.D.
Nancy A Tanchel, B.A.
Todd Thorslund, Sc.D.
Adrienne J. Zahner, Ph.D.
C-183
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Summary of Pertinent Data
The water quality criterion for PAH is based on the
experiment reported by Neal and Rigdon (1967) in which benzo(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 doses of 45 ppm and above. At 45
ppm the incidence in controls and treated groups was 0/289
and 4/40, respectively. The one-hit model has the following
parameters:
nt = 4 d = 45 ppm X 0.13 = 5.85 mg/kg/day
Nfc = 40 w = 0.034 kg
n = 0 L = 78 weeks X 7 days/wk = 546 days
NC =289 R = 30
Le = 110 days F = .0187 kg/day
le = 110 days
With these values, the one-hit slope parameter is BH = 28.020
(mg/kg/day)~ .
The result is that the water concentration of BaP should
be less than 9.7 nanograms per liter in order to keep the
individual lifetime risk below 10~ . On the conservative
assumptions that all carcinogenic PAH compounds are as potent
as BaP, that the effect of a mixture of carcinogenic PAH
compounds depends on the sum of their concentrations, and
that the non-carcinogenic PAH compounds have no effect on
the response of the carcinogenic PAH, it follows that the
sum of the concentration of all carcinogenic PAH compounds
should bo less than 9.7 nanograms per liter in order to
keep the lifetime risk less than 10 .
C-184
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