First draft prepared by Dr J.C. Larsen,
    Institute of Toxicology, National Food Agency of Denmark.


         Benzo(a)pyrene (B(a)P) is a contaminant that occurs
    ubiquitously in the environment together with other polycyclic
    aromatic hydrocarbons (PAHs) as a product of incomplete combustion
    or pyrolysis of organic material containing carbon and hydrogen.  In
    addition to natural sources of PAHs (e.g., forest fires), there are
    numerous man-related combustion processes which result in
    contamination of air, water, food, soil and sediment.  Main sources
    of B(a)P and also PAHs in the environment are residential heating
    (coal and wood-burning stoves and fireplaces), industrial plants
    (refuse burning, smelting, coke production), vehicle exhausts and
    cigarette smoke.  (Fazio & Howard, 1983; Grimmer, 1979; Bjorseth,

         Humans may be exposed to PAHs from air, water, food and tobacco
    smoke.  While the majority of studies have concentrated on the
    determination of B(a)P, it is important to note that it constitutes
    1 to 20% of estimated total carcinogenic PAHs present and usually
    less than 5% of total PAHs (Fazio & Howard, 1983); Bjorseth, 1983).

         B(a)P may contaminate foods via deposition of airborne
    particulate matter, by direct drying with smoke, or by absorption
    during the  smoking process.  Also high temperature heat-processing
    of food may lead to contamination with B(a)P and other PAHs. 
    Benzo(a)pyrene has not previously been evaluated by the Joint
    FAO/WHO Expert Committee on Food Additives.

         B(a)P has been evaluated for carcinogenicity by IARC
    (International Agency for Research on Cancer) (IARC, 1973; 1982;
    1983).  The following evaluations were made for B(a)P:  Inadequate
    evidence for carcinogenicity to humans; sufficient evidence for
    carcinogenicity to animals; sufficient evidence for activity in
    short-term tests (IARC, 1982; 1983).

    1.1  Dietary exposure

         Sources of B(a)P contamination of food-stuffs are numerous,
    varied, and widespread.  They include contaminated air, water, soil
    and sediment, modes of cooking (i.e., charcoal grilling), food
    processing (e.g., smoke curing, flue-drying), and food additives
    (e.g., smoke flavorings).

         There are two major sources of the occurrence of B(a)P in
    foods.  The most important source is probably the deposition and

    uptake of B(a)P and other PAHs from polluted air on food crops. 
    This makes cereals, vegetables, fruits and vegetable oils important
    contributors to the intake of B(a)P by humans.  In particular,
    drying of cereals and plants used for production of crude vegetable
    oils using direct application of combustion gases can result in
    contamination of the product with PAHs.  Kale, lettuce, barley, rye,
    and wheat are examples of crops that can be contaminated with B(a)P
    from air pollution.  In kale 4.2 to 15.6 g B(a)P/kg has been found
    in various locations in Western Germany, and wheat samples from
    rural areas contained 0.19 to 0.34 g B(a)P/kg, whereas 0.72 to 3.52
    g B(a)P/kg was measured in wheat grown near industrial plants.  In
    crude vegetable oils 1.2 to 15.3 g B(a)P/kg has been reported.  The
    content of B(a)P in fruit depends on the site of growth.  In
    different sorts of fruit 0.2 to 0.5 g B(a)P/kg was found in
    residential areas, while 30 to 60 g B(a)P/kg was found near
    industrial plants (Grimmer & Pott, 1983).  Certain seafoods,
    especially filter feeders (e.g. clams, oysters) normally have higher
    levels than do finfish (Vaessen  et al., 1984).

         The other significant source is the formation and deposition of
    PAHs during heat processing using methods such as roasting, smoking,
    and grilling.  Lijinsky and Shubik (1964) identified B(a)P and other
    PAHs in charcoal-broiled meat at an average level of 8 g B(a)P/kg
    steak.  It is important to note that the formation of PAHs is only
    significant at higher temperatures, generally over 350-400 C, and
    that below this temperature the endogenous formation of B(a)P in the
    food is minimal.  Thus, cooking procedures using heat conduction,
    such as pan-frying, or radiation, as in electric broiling and
    baking, do not lead to significant formation of B(a)P in food (Toth
    & Potthast, 1984).  When meat was placed directly in contact with
    the flames of a log fire a significant amount of B(a)P was formed
    (6-212 g B(a)P/kg meat) (Larsson  et al., 1983).  The B(a)P
    content of grilled food primarily stems from the fuel used and from
    the pyrolysis of fat dripping down on the heat source.  Among the
    fuels normally used in grilling, charcoal yielded only small amounts
    of PAHs (0.1-1.0 g B(a)P/kg food), while smoldered spruce or pine
    cones yielded 2 to 31 g B(a)P/kg meat (Larsson  et al., 1983). 
    The fat content of the meat is also important.  The more fat that
    drips on the fuel the more PAHs may be formed and deposited on the
    meat (Lijinsky & Ross, 1967; Toth & Blaas, 1973; Doremire  et al.,
    1979).  Increasing the fat content of charcoal-grilled ground beef
    patties from 15% to 40% increased the B(a)P content from 16 to 121
    g/kg (Fretheim, 1983).

         Smoking of food may be another source of B(a)P. Curing smoke is
    normally produced from wood (sawdust).  In traditional smoking the
    smoke is generated at the bottom of the oven and the food is placed
    directly over the smoking wood.  In modern industrial smoking ovens
    the smoke is generated in a separate chamber and led into the
    smoking chamber where the products are placed.  This gives a better

    control of the smoking process.  In various investigations the
    average values ranged from 0.2 to 0.9 g B(a)P/kg product for smoked
    meat products, such as sausages, ham, bacon, etc.  Similar B(a)P
    concentrations have been found in the edible part of smoked fish. 
    Larsson (1982) found 0.4 to 2.7 g B(a)P/kg in a survey of Swedish
    smoked fish.  Very high values (23 to 55 g/kg) may be found in
    intensively smoked products (black-smoked) (Grimmer & Pott, 1983).

         Various estimates of the dietary intake of B(a)P have been
    made.  Based on an average life expectancy of 70 years an inhabitant
    of East Germany was estimated to ingest a total of 24 to 85 mg of
    B(a)P.  This corresponds to approximately 1.0 to 3.3 g/day.  The
    major part of B(a)P was estimated to be ingested from cereals and
    vegetable oils (Fritz, 1971).   Santodonato  et al., (1980; 1981)
    suggested on the basis of a total daily food consumption by man from
    all types of foods of 1600 g/day and an estimated typical range of
    concentrations for B(a)P of 0.1-1.0 ppb in foods that the possible
    dietary intake of B(a)P was 0.16-1.6 g/day.  In comparison, the
    intakes of B(a)P from air, water, and cigarette smoking were
    estimated at 0.0095-0.0435, 0.0011, and 0.44 g/day, respectively. 
    In a UK survey the average intake of B(a)P was estimated at 0.25 g
    B(a)P/person/day (total PAHs/intake: 3.7 g/person/day).  Cereals
    and oils/fats accounted for 80% of the intake (Dennis  et al.,
    1983). Very similar figures were estimated in a Swedish survey on
    the intake of PAHs with the diet (Larsson, 1986).

         Human exposure to B(a)P from inhalation and food was compared
    for 10 homes in Phillipsburg, New Jersey, a city that contains a
    metal pipe foundry, which is a suspected major source of B(a)P.  The
    mean outdoor concentration of B(a)P was 0.9 ng/m3, and the indoor
    concentrations ranged from 0.1-8.1 ng/m3.  Food samples were
    acquired from family meals each day. The range of B(a)P per gram of
    wet weight of food was between 0.004 and 1.2 ng/g.  Of the 20 weeks
    of exposure (10 x 2 weeks), 10 had higher food exposures and the
    other 10 had higher inhalation exposures.  Of the two groups, the
    higher food exposures usually had a greater number of ng of
    B(a)P/week.  The weekly ingestion of B(a)P with food ranged from 10
    to 4000 ng (Lioy  et al., 1988).

         In a "human diet", used for animal experiments, and prepared
    according to mean levels of consumption in The Netherlands, and
    containing fried meat, baked bread, cereals, fruits, and vegetables,
    the B(a)P content was determined to be 0.15 g/kg (Alink  et al.,


         The toxicological literature on B(a)P is overwhelming. 
    However, the majority of toxicological studies on B(a)P have
    addressed the question of B(a)P as an air pollutant, potentially
    implicated in lung cancer in man, and therefore have not used oral
    application.  B(a)P has been the model compound  per se in chemical
    carcinogenesis, and has been intensively tested for carcinogenicity
    in model systems such as the skin carcinogenesis models, for
    interactions with DNA  in vitro and  in vivo, for  in vitro
    activation in numerous cell types, and has been used as a positive
    control in a multitude of short-term  in vitro and  in vivo test
    systems.  This monograph is centered on studies using oral
    application of B(a)P, while studies using other routes are primarily
    included when they add to an understanding of B(a)P toxicology and
    are deemed valuable for the evaluation of B(a)P as a contaminant in

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         The absorption, distribution and excretion of B(a)P have been
    reviewed by IARC (1983).

         After subcutaneous and intravenous administrations of B(a)P to
    rats the main excretory route was the bile.  About 1% of the dose
    was recovered as unchanged B(a)P in the faeces (Chalmers & Kirby,

         When 14C-B(a)P (0.45 mg) was given intravenously to female
    rats a rapid uptake by the liver and subsequent elimination in the
    bile were seen.  In 24 hours 65% of the radioactivity had been
    excreted in the faeces and 18% in the urine, while 1.8% remained in
    the liver (Heidelberger & Weiss, 1951).

         The pattern of distribution of 14C-B(a)P was similar after
    subcutaneous, intravenous, and intratracheal administration to
    strain A mice and Wistar rats.  After intravenous administration (11
    g) radioactivity rapidly disappeared from the blood and peak levels
    were obtained in the liver.  Minimal localization was seen in
    spleen, kidney, lung, and stomach.  After 24 hours 50-60% of the
    radioactivity had been recovered from intestine and faeces and 8-13%
    from urine (Kotin  et al., 1959).

         Female Sprague-Dawley rats were given intravenously 10 g of
    14C-B(a)P.  After 15 minutes 7% of the dose had been excreted in
    the bile and after 600 minutes the cumulative excretion amounted to
    53% of the radioactivity.  Pretreatment of the rats with 20 mg

    B(a)P/kg body weight for 7 days enhanced the initial rate of biliary
    excretion of 14C-B(a)P but not the total excretion.  No
    accumulation of B(a)P was seen in body fat (Schlede  et al., 1970a;

         Bock and Dao (1961) showed relatively high localization of
    B(a)P in the mammary gland and general body fats after a single
    feeding of 10-30 mg B(a)P to rats.

         B(a)P (50-150 mg/kg body weight) was readily absorbed from the
    gastrointestinal tract of the female Sprague-Dawley rat and the
    concentration of B(a)P in adipose and mammary tissues increased
    exponentially with the dose.  In another experiment peak levels of
    B(a)P were found in the cannulated thoracic lymph duct 3-4 hours
    after treatment.  Ten to 20% of the dose was recovered in the lymph
    (Rees  et al., 1971).

         Radioactivity was excreted in the milk from lactating rabbits
    and sheep when 1 mg of 14C-B(a)P was given in their diet.  The
    amount excreted in 6 days was 0.003% of the dose in rabbits, and
    0.01% of the dose in the sheep (West & Horton, 1976).

         One hour after intravenous administration of 14C-B(a)P to
    lactating rats the average amount of radioactivity detected was
    0.21% of the administered dose per ml of milk as compared with 0.17%
    per ml of blood (LaVoie  et al., 1987).

         A large fraction (45%) of unchanged B(a)P in rat blood was
    associated with serum lipoproteins while only 8% of the metabolites
    was associated with this component.  Forty to forty-five percent of
    each was associated with red blood cells.  Clearance of B(a)P by the
    perfused rat liver greatly depended on the presence of serum
    lipoproteins and albumin in the medium perfusing the organ (Wiersma
     et al., 1984).

         Male Sprague-Dawley rats with biliary and mesenteric lymphatic
    catheters received intraduodenally a dose of 0.4 mol (100 g) 3H-
    B(a)P in different amounts of olive oil.  Cumulative radioactivity
    recovered in 24 hours was 20% of the dose, regardless of the amount
    of oil used as vehicle.  Eighty percent of the material was found in
    bile.  It was concluded that the lymphatics play a limited role in
    the systemic entry of orally administered B(a)P (Laher  et al.,

         Conscious rats with bile duct and duodenal catheters were given
    1 mg of 3H-B(a)P intraduodenally in corn oil with or without
    exogenous bile.  Cumulative recovery of radiolabel in bile and urine
    over 24 hours showed that in the presence of bile 30% of the
    radiolabel was recovered.  In the absence of bile only 7% was found
    (Rahman  et al., 1986).

         B(a)P was found distributed to all regions of male Wistar rat
    brain after an intraperitoneal injection of 15 nmole.  The highest
    level was found in liver, followed by kidney, lung, brain (Das  et
     al., 1985a).

         3H-B(a)P was administered by intratracheal instillation (1
    g/kg body weight) to male Sprague-Dawley rats, and the amount of
    radioactivity in various organs was determined at timed intervals
    between 5 and 360 min.  Radioactivity in liver increased rapidly,
    reaching a maximum of 21% of the dose within 10 minutes after
    instillation.  The carcass accounted for 15-30% of the dose within
    the time intervals investigated.  B(a)P disposition indicated that
    enterohepatic circulation of metabolites was occurring (Weyand &
    Bevan, 1986).

         Intestinal absorption, bioavailability, hepatic and pulmonary
    extraction, and elimination of low doses of 3H-B(a)P (0.7-4.4 nmol)
    were studied in the male Sprague-Dawley rat.  The hepatic extraction
    ratio was 0.4 both in a liver perfusion model and  in vivo as
    determined by comparison of intravenous and intraportal infusion
    experiments in anaesthetized rats.  The pulmonary extraction ratio
     in vivo was 0.11.  Analysis of B(a)P concentrations in atrial
    blood and in the bile after continuous B(a)P infusion into the
    duodenum of anaesthetized rats indicated that at least 30% of the
    dose was absorbed from the gut.  When B(a)P was given by gavage
    about 10% of the dose escaped the liver and appeared in the blood
    (Foth  et al., 1988).

    2.1.2  Biotransformation

         The biotransformation of B(a)P and other polycyclic aromatic
    hydrocarbons has been reviewed extensively (Sims & Grover, 1974;
    Gelboin, 1980; Levin  et al., 1982; Pelkonen & Nebert, 1982;
    Conney, 1982; IARC, 1983; Cooper  et al., 1983; Grover, 1986).

         B(a)P is initially oxidized, primarily by the microsomal NADPH-
    dependent cytochrome P-450 monooxygenase system, to several arene
    oxides.  The arene oxides may rearrange spontaneously to phenols (3-
    OH-, 7-OH-, 9-OH-, and 6-OH-B(a)P), undergo hydration to the
    corresponding trans-dihydrodiols (catalyzed by microsomal epoxide
    hydrolase), or may react covalently with glutathione, either
    spontaneously or catalyzed by cytosolic glutathione-S-transferases. 
    6-OH-B(a)P is further oxidized to the 1,6-, 3,6-, or 6,12-quinones;
    3-OH-B(a)P can be oxidized to the 3,6-quinone, and 9-OH-B(a)P can be
    oxidized to the 4,5-oxide, which is hydrated to the corresponding
    4,5-dihydrodiol.  The phenols, quinones and dihydrodiols can all be
    conjugated to glucuronides and sulfate esters, and the quinones also
    form glutathione conjugates (IARC, 1983).

         The dihydrodiols may undergo further oxidative metabolism.  A
    number of unidentified metabolites are formed from the 4,5-
    dihydrodiol, and the 9,10-dihydrodiol can be oxidized to its 1-
    and/or 3-phenol derivatives.  The principal route of oxidative
    metabolism of the B(a)P-7,8-dihydrodiol is to the B(a)P-7,8-
    dihydrodiol-9,10 epoxide, which has been implicated as the most
    important reactive metabolite of B(a)P for its mutagenic and
    carcinogenic properties.  The diol-epoxides can be conjugated with
    glutathione or hydrolyse spontaneously to tetrols.  Thus, B(a)P may
    undergo a number of complex simultaneous and sequential
    biotransformations.  The situation is even more complex as the diol-
    epoxides each may exist in 4 optically active isomers (each
    diastereoisomer can be resolved into two enantiomers).  In rat liver
    microsomes the (+)-[7R,8S]-oxide of B(a)P is formed in a 20-fold
    excess relative to the (-)-[7S,8R]-oxide and is stereospecifically
    metabolized by epoxide hydrolase to the (-)-[7R,8R]-dihydrodiol
    (Thakker  et al., 1977), which in turn is further oxidized to (+)-
    B(a)P-7,8-diol-9,10-epoxide-2[7R,8S,9S,10R] (variously called: diol-
    epoxide-2; B(a)PDEI; trans-B(a)PDEI; (+)-anti-B(a)PDE).  B(a)PDEI is
    the predominant diol-epoxide formed from B(a)P-7,8-dihydrodiol in
    almost all tissues examined, and is also the only isomer with high
    tumorigenic activity. It is the predominant isomer found covalently
    bound to DNA (forming the N2-10 -[7, 8alpha, 9alpha-trihydroxy-
    7,8,9,10-tetrahydrol B(a)P]-yl)deoxyguanosine adduct; B(a)PDEI-dGuo)
    in a variety of mammalian cells and organs exposed to B(a)P. 
    Another diol epoxide B(a)PDEI ((-)syn-B(a)PDE; cis-B(a)PDEII;
    (-)diol-epoxide 1) is also formed in significant amounts, while the
    two other diol-epoxides are formed in only very small amounts.  The
    formation of diol-epoxides can also be catalyzed by microsomal
    prostaglandin synthetase, present in a variety of different tissues
    (Pezzuto  et al., 1978; Levin  et al., 1982; Conney, 1982; IARC,
    1983; Cooper  et al., 1983).  In vitro

         An overview of the  in vitro metabolism of B(a)P by various
    human tissues was given by IARC (1983).

         Primary hepatocyte cultures from six human donors metabolized
    B(a)P to a significant extent (24-35 nmol in 24 hours).  The
    predominant extracellular organic solvent-soluble B(a)P metabolites
    were the 9, 10- and 7, 8-dihydrodiols, 9-hydroxy-B(a)P, and a
    mixture of tetrols, but the general ratios of these metabolites
    varied widely among the cells from different donors (Moore & Gould,

         Monolayer cultures of human bronchial epithelial cells
    converted B(a)P to dihydrodiols, phenols, quinone derivatives, and
    polyhydroxylated forms.  Sulfate and glucuronide conjugates of B(a)P
    metabolites were also detected.  Both total metabolism and

    distribution of metabolites showed a 10-fold or greater variation in
    cultures from different specimens.  When the data were divided
    according to smoking status, however, no differences in total
    metabolism, extent of conjugation, or distribution of metabolites
    could be demonstrated between the two groups (Siegfried  et al.,

         The capacity of human hepatocytes to metabolize B(a)P was not
    saturated at up to 100 M of B(a)P, and the predominant metabolites
    produced were a mixture of highly polar B(a)P forms.  The next four
    most prevalent forms of B(a)P metabolites were the 3-hydroxy B(a)P,
    B(a)P-4,5-dihydrodiol, B(a)P-9,10-dihydrodiol, and B(a)P-7,8-
    dihydrodiol.  These metabolites all increased nearly linearly with
    dose.  B(a)P metabolite binding to DNA was associated with the
    amount of unconjugated B(a)P-7,8-dihydrodiol metabolite (Monteith
     et al., 1987).

         In human liver microsomes a four-fold variation in B(a)P
    metabolism was observed.  The levels of expression of cytochromes P-
    450 from five gene subfamilies did not show any correlation with the
    rate of B(a)P metabolism.  The P450IA1 was most effective in
    metabolizing B(a)P, but several other forms of cytochrome P-450 were
    shown to be involved in B(a)P metabolism in humans (Hall  et al.,

         The ability of seven different forms of cytochrome P-450
    purified from rat liver microsomes to metabolize B(a)P was compared. 
    The major 3-methylcholanthrene (MC) inducible cytochrome P-450 (form
    c) exhibits the greatest activity toward B(a)P.  Cytochrome P-450d,
    a minor MC-inducible from, has far lower activity for metabolism of
    B(a)P.  Two phenobarbital (PB)-induced forms (P-450's b and e) had
    low activity.  P-450's a, h, and pregnenolone-16 alpha-carbonitrile
    (PCN) exhibited little activity toward B(a)P (Wilson  et al., 

         3-OH-B(a)P and the 1,6- and 3,6-quinones were the major
    products formed by a reconstituted pulmonary cytochrome P-450MC
    system. The B(a)P-9,10-dihydrodiol was the major dihydrodiol formed
    by pulmonary cytochrome P-450MC.  The addition of purified epoxide
    hydrolase increased the formation of B(a)P-dihydrodiols,
    particularly B(a)P-7,8-dihydrodiol.  Similar results were obtained
    in reconstituted systems of hepatic cytochrome P-450MC (Sagami  et
     al., 1987).

         When incubated in the presence of peroxidising polyunsaturated
    fatty acids such as linoleic acid (C18:2), arachidonic acid (C20:4),
    eicosapentanoic acid (C20:5) or docosahexanoic acid (C22:6) B(a)P
    was converted to oxidized products.  Between 7% and 9% of the B(a)P
    was oxidized in one hour when incubated with arachidonic acid and
    docosahexanoic acid.  1,6-,3,6-, and 6,12-quinone derivatives of

    B(a)P were identified by HPLC.  The products of B(a)P oxidation were
    shown to produce sister chromatid exchange (SCE) in CHV79 cells
    (McNeill & Willis, 1985).

         Homogenates of colonic mucosa from different mouse strains
    metabolized B(a)P to predominantly phenolic derivatives (3-OH- and
    9-OH-B(a)P), and lesser amounts of diols (4,5-, 7,8-, and 9,10-
    B(a)P-diol) and quinones (1,6-, 3,6-, and 6,12-B(a)P-quinone)
    (Anderson  et al., 1982).

         When male C57BL/6 mice were fed a basal semisynthetic diet with
    added various amounts of vitamins and/or types of fibers and/or
    types of fats no differences were observed in the ability of colonal
    mucosal homogenates to metabolize B(a)P. The various diets had no
    effects on beta-naphthoflavone induced metabolism (Anderson  et al.,

         In mouse liver microsomes the 7,8-epoxidation of B(a)P, and the
    9, 10-epoxidation of B(a)P trans-7,8-dihydrodiol coupled with
    covalent binding of the highly reactive diol-epoxide, were shown to
    be mediated by P-450 protein(s) that are responsible for aryl
    hydrocarbon hydroxylase activity and that are coordinately
    controlled by the Ahb allele (Van Canfort  et al., 1985).

         B(a)P metabolites formed by rough and smooth endoplasmic
    reticulum, nuclei, and plasma membrane as well as mitochondrial
    fractions were investigated.  The metabolic profiles produced by the
    two most active fractions, smooth and rough endoplasmic reticulum,
    were very similar to each other but different from those produced by
    the other three preparations.  The metabolite pattern produced by
    incubations containing nuclear fractions differed slightly from that
    produced by the fractions of endoplasmic reticulum, but plasma
    membrane and mitochondria produced markedly different patterns
    (Oesch  et al., 1985).

         When groups of 6-8 male Wistar rats were fed diets containing
    10% of different types of fat, there were significant changes in the
    incorporation of fatty acids into the endoplasmic reticulum of the
    mucosal cells of the small intestine:  the proportions of
    polyunsaturated fatty acids in the endoplasmic reticulum reflected
    the amounts of these fatty acids in the dietary fat.  The rate of
    B(a)P  in vitro oxidation in the intestinal mucosa was dependent on
    the amount and composition of the dietary fat, but the range and
    proportions of the metabolites produced were not effected.  Dietary
    C18:2 (corn oil) was particularly important in elevating the rate of
    B(a)P oxidation, but dietary C20:5 and C22:6 (mackerel oil and cod
    liver oil) also effectively increased the rate of B(a)P oxidation
    (Gower & Wills, 1986).

         Levels of total metabolism of B(a)P by rat and human kidney
    cells were similar, although analysis of specific metabolites of
    B(a)P indicated that species differences existed.  Human kidney
    cells produced the organic-soluble metabolites B(a)P-9,10-diol,
    B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-
    phenol.  Rat kidney cells produced organic-soluble B(a)P-pre-9,10-
    diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone. 
    Both species produced sulfate and glucuronide conjugates of all
    products (Rudo  et al., 1989).

         The metabolism of 1 mM benzo(a)pyrene was studied in isolated
    perfused lung and liver of 5,6-benzoflavone-pretreated rats. 
    Benzo(a)pyrene metabolism by the liver was more rapid than by the
    lung, but total metabolite formation in the lung at the end of a
    120-min perfusion period was comparable to that in the liver.  Lung
    perfusate was characterized by high concentrations of free
    metabolites, diols outweighing phenols; in liver perfusate free
    metabolite concentrations were low, and large quantities of
    metabolites were found as conjugates in the bile at the end of
    perfusion.  The tissue concentrations of free diols and phenols
    including the precursors of the main DNA-binding secondary
    metabolites were higher in the lung than in the liver (Molliere  et
     al., 1987).

         The oesophagi of anaesthetized hamsters were surgically
    catheterized so that radiolabeled material instilled as B(a)P in the
    nose could be collected and analyzed for metabolites.  About 50% of
    the instilled B(a)P was metabolized in the nose and, potentially,
    would have been swallowed in an awake animal.  Using homogenates of
    respiratory and alimentary tissues it was shown that the nose,
    trachea, and lungs, had about equally high activities on a per organ
    basis (5-7 nmol/hour), whereas all other tissues had considerably
    less activity (Dahl  et al., 1985).  In vivo

         When rats were given 14C-B(a)P in oral doses of 10.2, 102, or
    1020 g/rat, 74-79% of the dose was excreted in the faeces in the
    first 48 hours.  Unchanged B(a)P in faeces amounted to 13.0, 7.8,
    and 5.6%, respectively, for the three doses given.  Faecal
    metabolites included 3-OH-B(a)P, 9-OH-B(a)P, B(a)P-3,6-quinone,
    B(a)P-1,6-quinone, and trace amounts of B(a)P-4,5-dihydrodiol, and
    B(a)P-7,8-dihydrodiol.  When rats were fed charcoal-broiled
    hamburger containing 52.7 g B(a)P/kg, 11% (0.06 g/rat) of the
    B(a)P consumed was excreted unchanged in the faeces.  When humans
    ate meals containing charcoal-broiled meat (24.2 g B(a)P/kg) B(a)P
    was not detected in the faeces, despite the fact that each person
    consumed 8.6 g B(a)P (Hecht  et al., 1979).

         B(a)P was administered orally to Wistar rats at doses of 10, 20
    and 50 umol/kg body weight/day 3 consecutive days.  Urine was
    collected for a total of 6 days.  At all dose levels, the urinary
    excretion of 3-OH-benzo[a]pyrene amounted to approximately 0.3% of
    dose.  Three other metabolites were seen in urine, but not
    identified (Jongeneelen  et al., 1984).

         3-OH-B(a)P and mutagenic activity in rat urine were determined
    after oral administration of B(a)P given in three repeated doses of
    10, 20 and 50 mol/kg/bw.  The mutagenic activity of concentrated
    urine samples was assayed with the  Salmonella typhimurium strain
    TA98 in the presence of S9 mix and -glucuronidase.  The urinary
    excretion of 3-0H-B(a)P and mutagens showed a correlation and both
    increased dose-dependently during the sampling period of 6 days
    (Jongeneelen  et al., 1985).

         Female Lewis rats administered 3-OH-B(a)P (50 mg/kg,
    intraperitoneally) excreted metabolites via the bile.  After
    treatment with -glucuronidase and aryl sulphatase a minor, highly
    labile metabolite, tentatively identified as 3,5-dihydroxy-B(a)P,
    was found in addition to 3-OH-B(a)P-7,8-dihydrodiol and 3-OH-B(a)P
    (Ribeiro  et al., 1985).

         When rats (germfree and conventional) were dosed with 14C-
    B(a)P, a large part of the metabolites (9-24% depending on animal
    type and route of excretion) had amphoteric properties, like
    glutathione and cysteine conjugates.  The abundance of conjugates
    sensitive to -glucuronidase and sulphatase was low.  The relative
    amount of acidic conjugates in faeces was much higher in the
    germfree than in the conventional rats, indicating the influence of
    the intestinal flora on the metabolism.  The results support the
    view that the mercapturic acid pathway is a quantitatively important
    route for B(a)P in rats (Egestad  et al., 1987).

         Groups of male Fisher F344 rats were fed cooked, low-fat human
    diets and given 14C-B(a)P (0.6 mol) by gavage.  Potential reactive
    metabolites in the gastrointestinal tract were trapped with magnetic
    polyethyleneimine microcapsules.  Approximately 70% of the dose was
    recovered in the faeces when the diet had a low fibre content, while
    80% was excreted in the faeces when the diet had a high fibre
    content.  One to two % of dose was bound to the microcapsules.  The
    metabolites found bound to microcapsules were B(a)P-3,6-quinone,
    B(a)P-1,6-quinone, an unidentified metabolite, and B(a)P-tetraols.
    Addition of beef protein to the diet increased the amount of
    metabolites bound to the microcapsules (O'Neill  et al., 1990a;

         Approximately one-third of an intravenous dose of 14C-B(a)P
    was excreted within 4 hours in the bile of guinea-pigs fed a normal
    diet.  The extent of excretion was not altered by feeding high-fat

    (17.3% coconut oil) or high-cholesterol (0.1% in 17.2% coconut oil)
    diets.  Hepatic cytochromes P-450 and b5, and B(a)P-hydroxylase
    activity were unaltered by the administration of high-fat and high-
    cholesterol diets.  Pretreatment with low oral doses of B(a)P (6 X 3
    mg/kg body weight) did not induce these parameters in animals given
    any of the diets.  High-fat and high-cholesterol diets altered the
    pattern of B(a)P metabolites in the bile, with significantly
    increased excretion of dihydrodiol glucuronides in both the high-fat
    and high-cholesterol groups.  Hepatic epoxide hydrolase activity and
    glutathione content were unaltered by the high-fat or high-
    cholesterol diets, and therefore cannot explain the alteration in
    the profile of biliary metabolites of B(a)P.  The altered pattern of
    biliary excretion in animals fed high-fat or high-cholesterol diets
    would lead to an increase in the delivery to the colon of
    dihydrodiol metabolites of benzo[a]pyrene (Bowes & Renwick, 1986a).

         Strains of intestinal bacteria from guinea-pigs were capable of
    deconjugating B(a)P metabolites  in vitro.  The hydrolysis
    products, and other  primary oxidative metabolites of B(a)P, were
    stable to further degradation by the strains tested.  B(a)P
    hydroxylase was measurable in the mucosa of the upper intestine, but
    was present in the lower gut only at very low levels in some
    animals.  The activity was inducible, by oral administration of
    B(a)P, in small intestinal mucosa of guinea-pigs fed normal diet but
    not in those fed high-fat and high-cholesterol diets.  Low levels of
    covalent binding of 3H to DNA of liver and gut mucosa were obtained
    in guinea-pigs dosed orally with 3H-B(a)P (app. 100 g).  The
    feeding of high-fat and high-cholesterol diets did not increase this
    binding.  Guinea-pigs fed high-fat and high-cholesterol diets
    excreted a greater proportion of an oral dose of 3H-B(a)P in urine,
    and less in faeces, than animals fed a normal diet (Bowes & Renwick

    2.1.3  Effects on enzymes and other biochemical parameters

         B(a)P and other polycyclic aromatic hydrocarbons (PAHs)
    stimulate their own metabolism by inducing microsomal cytochrome P-
    450 monoxygenases and epoxide hydrolase.  This has been most
    thoroughly studied with 3-methylcholanthrene (3-MC).  The most
    significant isozymes induced belong to the cytochrome P450IA
    subfamily, namely P450IA1 (P4501 in the mouse; P450c in the rat)
    which is the major isozyme induced and P450IA2 (P450d in the rat). 
    The induction is mediated by binding to a cytosolic receptor
    protein, the Ah receptor, and the receptor-inducer complex is
    translocated to the cell nucleus where its binding to a promoter
    sequence in DNA triggers the transcription of genes producing the
    enzymes.  The Ah receptor is genetically controlled by the Ah locus. 
    Strains of mice (e.g. B6) having high affinity receptors are readily
    induced (responsive mice; Ahb/Ahb), while other strains (e.g. C3 and
    D2) having low affinity receptors are much less prone to induction

    (non-responsive mice; Ahd/Ahd).  When the induction is measured  in
     vitro in microsomes using B(a)P as the substrate, the activity is
    termed aryl hydrocarbon hydroxylase (AHH) or B(a)P hydroxylase
    (B(a)PH) activity, and is primarily a determination of the 3-
    hydroxylation of B(a)P.  The AHH activity is inducible not only in
    the liver, but also in most extrahepatic tissues.  Numerous studies
    have shown that AHH induction not only leads to an enhanced turn-
    over of B(a)P, but also lead to enhanced generation of the active
    metabolites (presumably diolepoxides) that bind to cellular
    macromolecules, and induce mutations and cancer (Nebert & Jensen,
    1979; Gelboin, 1980; Conney, 1982; Cooper, 1983).

         In the pregnant Sprague-Dawley rat the  in vivo metabolism of
    3H-B(a)P was markedly increased when the rats had been pretreated
    for three days with 10 mg B(a)P/kg body weight/day or higher doses. 
    This treatment also induced AHH activity in the placenta and the
    fetal liver (Welch  et al., 1972).

         The  in vitro liver microsomal AHH activity was significantly
    increased in rats 24 hours after intraperitoneal doses of 4 mg
    B(a)P/kg body weight and 48 hours after 2 mg B(a)P/kg body weight,
    but not after 1 mg B(a)P/kg body weight.  The  in vivo binding of
    3H-B(a)P to liver DNA showed linear dose-response relationship in
    the dose range 40 g to 1 mg B(a)P/kg body weight, followed by a
    step towards 2 mg/kg, and a shallow linear slope above that value. 
    The binding to liver DNA from an equimolar dose of B(a)P was 35
    times less than the binding found in mouse skin (Lutz  et al.,

         Groups of 6 female Sprague-Dawley rats were fed a fibre-free
    purified diet for 7 days, then they were switched to experimental
    diets for 48 hours.  After another 48 hours, the small intestinal
    mucosa was assayed for B(a)P hydroxylase (B(a)PH) activity. 
    Experimental diets contained 0, 100, 400, 800, or 1200 mg B(a)P/kg
    diet each with and without 10% soft white wheat bran.  Enzyme
    induction with 100 and 400 mg B(a)P/kg diet was partially inhibited
    by bran, but with higher concentrations of B(a)P there was no
    protective effect.  The inhibition in B(a)P-induced B(a)PH activity
    was observed with 10% wheat bran but not with 3.3 or 6.6%. 
    Subsequent studies showed no significant inhibition in B(a)PH
    induction with cellulose or lignin,  whereas all forms of wheat bran
    (hard red, soft white, or finely ground soft white) caused
    significant inhibition.  A diet containing charcoal-broiled beef was
    compared with diets containing raw beef or soybean protein each with
    and without 10% soft white wheat bran.  B(a)PH activity remained low
    with raw beef and soybean protein whether or not fiber was added. 
    However, intestinal B(a)PH activity was raised ninefold by charcoal-
    broiled beef.  The addition of bran reduced B(a)PH activity to 65%
    of that observed with the fibre-free, charcoal-broiled beef diet
    (Clinton & Visek, 1989).

         B(a)P hydroxylase (B(a)PH) activity was measured in homogenates
    of fetal liver (day 18) or of whole embryos of mice on day 9, 10 or
    12 of gestation after oral maternal pretreatment with B(a)P on 3
    consecutive days.  Three oral doses of 17.5 mg B(a)P/kg body weight
    were found to just significantly induce B(a)PH in maternal liver. 
    An induction was observed after pretreatment with 24 mg B(a)P/kg
    body weight in 9-, 10- or 12-day-old whole embryos.  The induction
    was demonstrable in embryos at tissue levels about one order of
    magnitude lower than those required for induction in maternal liver. 
    Treatment with 25 mg B(a)P/kg body weight on 3 consecutive days was
    required to induce B(a)PH in fetal liver on day 18 of gestation. 
    The required B(a)P tissue concentrations were about one half of
    those necessary for induction in maternal liver (Neubert & Tapken,

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The LD50 in the mouse after intraperitoneal injection is about
    250 mg/kg body weight (IARC, 1983).

    2.2.2  Short-term studies

         See special studies.

    2.2.3  Long-term/carcinogenicity studies

         B(a)P is a well documented carcinogen producing mainly local
    tumours in a variety of species after skin application, inhalation
    and/or intratracheal administration, intrabronchial implantation,
    subcutaneous and/or intramuscular administration, and a variety of
    other applications.  Tumours are also induced after intraperitoneal,
    intravenous, and transplacental injection.  Studies showing
    carcinogenicity after oral administration have also been reported. 
    B(a)P appears to be a less potent carcinogen after systemic
    administrations than after local applications (IARC, 1973; 1982;

         The following is a brief summary of carcinogenicity studies
    that have used mainly oral administration of B(a)P.  Mouse


         Groups of 20 female albino mice were treated with single
    intragastric dose of 0.2 mg B(a)P/mouse (corresponding to 6 mg/kg
    body weight).  After 43 weeks a total of 14 tumours in the
    forestomach was observed in 5 of 11 surviving animals.  Single doses

    of 0.05 mg (1.5 mg/kg body weight) and 0.012 mg (0.36 mg/kg body
    weight) produced 0/9 and 2/10 tumours, respectively.  No tumours
    were seen in 9 surviving control animals.  Weekly treatment with
    lime oil enhanced the tumour incidence. No tumours were seen in the
    glandular part of the stomach or other parts of the alimentary canal
    (Pierce, 1961).

         White Swiss mice fed 250 or 1000 ppm of B(a)P in the diet for
    various time periods developed squamous papillomas and carcinomas of
    the forestomach in a dose dependent manner.  All mice fed 1000 ppm
    B(a)P and examined after 86 days had tumours.  In the group fed 250
    ppm B(a)P 25% developed gastric tumours when fed for more than 85
    days. A high incidence of lung adenomas was also observed in these
    mice (Rigdon & Neal, 1966).

         No increases in stomach tumours were found in mice after 110
    days of treatment with diets containing up to 30 ppm B(a)P, while
    40-45 ppm for 110 days induced stomach tumours in about 10% of the
    mice, and more than 70% of mice fed 50-250 ppm for 122-197 days had
    stomach tumours.  A diet containing 250 ppm B(a)P fed for different
    time periods produced the following incidences of stomach tumours;
    one day, 0%; 2-4 days, 10%; 5-7 days, 30-40%; 30 days, 100% (Neal &
    Rigdon, 1967).

         Groups of 19-20 female Ha/ICR mice were fed 300 ppm or 100 ppm
    B(a)P in their diets for 17 and 31 weeks, respectively.  The
    percentages of mice with forestomach tumours were 68% and 55%,
    respectively.  In 12 female A/HeJ mice fed 1000 ppm B(a)P in their
    diet for 12 weeks all mice developed tumours of the forestomach
    (Wattenberg, 1972).

         Groups of 20 female Ha/ICR mice (9 weeks old) were fed a diet
    containing 300 ppm B(a)P for 6 weeks and thereafter maintained on
    control diet for 14 weeks.  Forty percent of the mice developed
    forestomach tumours.  The tumours were inhibited by concurrent
    feeding with disulfiram (Wattenberg, 1974).

         In a similar experiment where 17 female Ha/ICR mice 9 weeks of
    age were fed 300 ppm B(a)P for 6 weeks followed by 14 weeks on
    control diet 41% of the mice developed forestomach papillomas.  The
    tumours were completely inhibited by benzyl isothiocyanate added to
    the diet (Wattenberg, 1977).

         Groups of 9 female Ha/ICR mice (9 weeks old) were fed diets
    containing 200 or 300 ppm B(a)P for 12 weeks.  In the 200 ppm group
    66%, and in the 300 ppm group 100% of the mice had forestomach
    papillomas.  AHH activity was elevated in the forestomach, glandular
    stomach and lung, but not liver of these mice (Triolo  et al.,

         Eight doses of 1.5 mg B(a)P were administered twice a week for
    four weeks to 20-24 ICR mice (5 weeks old).  After 25 weeks the
    average number of forestomach papillomas per mouse was approximately
    5.  Nitrite (0.05% in drinking-water) and soy sauce (20% in a
    refined diet) together significantly reduced the number of neoplasms
    per animal (Benjamin  et al., 1988).

         Eight organosulfur compounds from garlic and onions were
    studied for their inhibitory effects on B(a)P-induced neoplasia of
    forestomach and lung of female A/J mice when administered 96 and 48
    hours prior to carcinogen challenge.  B(a)P (2 mg/mouse) was given
    perorally 3 times with two week intervals to groups of 15 mice.  The
    study was terminated 26 weeks after the first B(a)P dose. 
    Approximately 3 papillomas per mouse were seen in the forestomach
    and approximately 15 adenomas per mouse in the lung after B(a)P. 
    Allylic compounds inhibited B(a)P-induced neoplasia of the
    forestomach while the saturated analogs were almost without
    inhibitory activity.  All the allylic compounds induced increased
    glutathione S-transferase (GST) activity in the forestomach, but
    varied in their capacity to induce GST in lung, liver and small
    bowel.  Their saturated analogs produced little or no induction
    (Sparnins  et al., 1988).

         Nomilin, a limonoid found in edible citrus fruits and an active
    inducer of glutathione S-transferase activity in the liver and small
    intestinal mucosa of female ICR/Ha mice was found to inhibit
    perorally B(a)P-induced (1 mg per animal twice a week for 4 weeks)
    neoplasia in the forestomach.  The number of mice with tumours after
    18 weeks was reduced from 100 to 72%, and the number of tumours per
    mouse was significantly decreased as a result of nomilin treatment
    (Lam & Hasegawa, 1989).

         Lung tumours

         The induction of lung adenomas and leukaemia in mice after 140
    days on a diet containing 250 ppm B(a)P has been reported (Rigdon &
    Neal, 1969).

         The induction of lung adenomas after peroral administration of
    B(a)P was confirmed in groups of 15 female A/HeJ mice.  B(a)P was
    given by intubation at a dose of 6 mg B(a)P (2x3 mg B(a)P), and
    repeated after 3 weeks.  After 19 weeks an average of 16 pulmonary
    adenomas per mouse was found compared to 0 in the controls.  The
    induction of lung tumours was almost abolished by treatment of the
    mice with beta-naphthoflavone (an inhibitor of cytochrome P4501)
    for 3 weeks prior to B(a)P (Wattenberg & Leong, 1970).

         All 24 female A/HeJ mice given two doses of 3 mg B(a)P/mouse by
    gavage at 14 day interval developed pulmonary adenomas after 20
    weeks (Wattenberg, 1973).

         All 12 female A/HeJ mice (10 weeks old) given two peroral
    intubations with 3 mg B(a)P/mouse with two week interval and
    maintained on control diet for another 21 weeks developed lung
    adenomas (100%; 7.8 tumours/mouse).  Disulfiram did not inhibit
    tumour induction (Wattenburg, 1974).

         A single intraperitoneal dose of 100 mg/kg body weight of BaP
    to groups of 20 male A/J mice produced pulmonary adenomas in all
    mice within 6 months.  The average number of tumours per mouse was
    10.2.  Three phenolic compounds (ferulic, chlorogenic and ellagic
    acids) inhibited markedly the number of tumours per mouse but not
    the number of mice with tumours (Lesca, 1983).


         Oral B(a)P doses estimated to be between 6 and 12 mg/kg body
    weight/day induced leukaemia in non-responsive (Ahd/Ahd) mice after
    100 or more days, but not in responsive mice (Ahb/Ahd).  It was
    suggested that a higher dose is obtained in the bone marrow of non-
    responsive mice than in responsive mice (Nebert & Jensen, 1978).

         When given to 42-day old mice a single intraperitoneal
    injection of 75-100 g B(a)P/kg body weight produced a high
    frequency of lymphoreticular tumours after 90 weeks.  When B(a)P was
    given to either 1-day or 8-day old mice the tumour incidences were
    lower (Vesselinovitch  et al., 1975).

         Intracolonical administration

         Groups of 50 male and 50 female Swiss mice (6 weeks old)
    received one or 10 intracolonical instillations of 200 g B(a)P/g
    body weight.  The treatment had no effect on survival of the mice
    compared to controls.  The single administration of B(a)P induced
    malignant lymphomas (42% in females, 12% in males compared to 14%
    and 0% in controls) and forestomach tumours (10% in females, 4% in
    males compared to 2% and 0% in controls).  In the mice given 10
    doses of B(a)P neoplasia of oesophagus (10% in females, 0% in males;
    2% and 0% in controls), anus (12% in females, 10% in males; 0% and
    0% in controls), and skin (22% in females, 26% in males; 0% and 2%
    in controls) were seen in addition to malignant lymphomas (36% in
    females; 14 in males) and forestomach tumours (22% in females; 20%
    in males) (Toth, 1980).

         Groups of 45 female Ha/ICR mice and 38 female C57B1/6 mice were
    given 1 mg B(a)P/mouse intrarectally once weekly for 14 weeks, and
    thereafter maintained on control diet until 18 months.  In the
    Ha/ICR mice 27/37 (73%) developed primary lung tumours (25% in
    control mice) and 16/17 (94%) mice had forestomach tumours (20% in
    controls).  Mammary tumours occurred in 23% of the treated mice
    compared to 9% in the control mice.  In the C57B1/6 mice 94%

    developed forestomach tumours (21% in controls), 28% lymphomas (2%
    in controls), and 16% peritoneal sarcomas (0 in controls), 28%
    lymphomas (2% in controls), and 16% peritoneal sarcomas (0 in
    controls).  No colonic neoplasms were found in any of the mice after
    18 months (Anderson  et al., 1983).

         Newborn mouse

         Subcutaneous or intraperitoneal injection of 20-40 g
    B(a)P/mouse during the first day of life have produced lung adenomas
    and/or hepatomas in different strains of mice after 50-60 weeks. 
    Malignant lymphoma and mammary adenocarcinoma have also been
    reported (Peitra  et al., 1961; Roe & Waters, 1967; IARC, 1973).

         Intraperitoneal administration of a total dose of 1400 nmol
    B(a)P-7,8-dihydrodiol/mouse to newborn Swiss-Webster mice on days 1,
    8, and 15 of life produced more malignant lymphomas and pulmonary
    adenomas than the equimolar dose of B(a)P.  B(a)PDEI ((+)-anti-
    B(a)PDE) produced the same incidence of pulmonary adenomas at a much
    lower dose (28 nmol/mouse) (Kapitulnik  et al., 1977; 1978).  Of
    the four optical enantiomers of the diastereoisomeric B(a)P-7,8-
    diol-9,10-epoxides only B(a)PDEI had an exceptional pulmonary
    tumorigenicity in newborn Swiss-Webster mice when given in total
    doses of 7 (71% of mice with tumours) or 14 nmol (100% of mice with
    tumours) (Buening  et al., 1978).

         Transplacental route

         Subcutaneous injection of 2-4 mg B(a)P to pregnant ICR/Ha mice
    on days 11, 13, and 15 of the pregnancy resulted in an increased
    incidence of lung adenomas (62%) in the offspring when they were 28
    weeks of age.  The treatment also increased skin carcinogenesis of
    B(a)P in the offspring (Bulay & Wattenberg, 1970).

         Direct injection of B(a)P, B(a)P-4,5-oxide (4-20 nmol/foetus),
    and B(a)PDE (racemic mixture) (0.4-4 nmol/foetus) to fetal Swiss
    mice on day 15 of intrauterine growth produced pulmonary adenomas at
    12-15 weeks of age (Rossi  et al., 1983).  Rat

         Nine female Sprague-Dawley rats (50-65 days of age) were given
    a single oral dose of 100 mg B(a)P.  Within 60 days, 8/9 rats
    developed tumours of the mammary gland (papillary adenocarcinomas)
    (Huggins & Yang, 1962).

         In a study with Sprague-Dawley rats of both sexes (3 1/2 months
    of age) daily doses of 2.5 mg B(a)P/rat for 8-12 months induced
    papillomas of the oesophagus and forestomach in 3 out of 40 animals
    (Gibel, 1964).

         B(a)P was administered for 87-131 weeks to groups of 32 male
    and 32 female Sprague-Dawley rats either as an admixture to the diet
    or by gavage in an  aqueous 1.5% caffeine solution.  B(a)P in
    solution was given as doses of 0.15 mg/kg bw either: 1) 5 days per
    week (annual dose: 39 mg/kg bw ), 2) every 3rd day (18 mg/kg
    bw/year), 3) or every 9th day (6 mg/kg bw).  When mixed in the diet
    the doses were: 4) 0.15 mg/kg body 5 days a week (39 mg/kg bw/year)
    or 5) 0.15 mg/kg bw every 9th day (6 mg/kg bw/year).  Similar groups
    given either caffeine solution or control diet served as controls. 
    A significant increased number of rats with forestomach papillomas
    was observed in group 1 to 4 (14, 25, 11, and 9 rats with tumours,
    respectively) compared to 1 in group 5, and 2 and 3 tumours in the
    control groups.  No other tumours were found significantly different
    from control levels (Brune  et al., 1981).

         Intraperitoneal administration of B(a)P (50 mg/kg bw) 18 hours
    after partial hepatectomy induced enzyme-altered foci in rats
    subsequently promoted with 2-acetylaminofluorene/CC14. 
    Pretreatment of rats with 3-MC 66 hours before partial hepatectomy
    and 84 hours before B(a)P administration, increased the number of
    enzyme-altered foci (Dock  et al., 1988).  Hamster

         Bi-weekly administration of 2-5 mg B(a)P by gavage produced 5
    papillomas of the stomach in 67 hamsters treated for one to five
    months, seven papillomas and two carcinomas in 18 hamsters treated
    for six to nine months and five papillomas in eight hamsters treated
    for 10-11 months (Dontenwill & Mohr, 1962).  In another experiment a
    diet containing 500 ppm B(a)P was given to 13 hamsters four days per
    week for up to 14 months.  A total of 12 tumours (two in oesophagus,
    eight in the forestomach and two in the intestine) were seen in
    eight hamsters (Chu & Malmgren, 1965).

         A low incidence of large bowel neoplasms was induced in a group
    of 30 male Syrian hamsters of the inbred strain BIO15.16 by
    intrarectal instillation of B(a)P.  The hamsters were given 0.8 mg
    B(a)P once weekly for 30 weeks.  The experiment was terminated after
    1 year.  The incidence of colon neoplasms was 6% with B(a)P exposure
    (2/30 adenocarcinomas versus 0/15 in control hamsters) (Wang  et
     al., 1985).  Reproduction studies

         Oral B(a)P (120 mg/kg/day) given to pregnant Ahd/Ahd mice (non-
    responsive mice) between gestational days 2 and 10 produced more
    intrauterine toxicity and malformations in Ahd/Ahd (non-responsive)
    than in Ahb/Ahd (responsive) embryos.  Pharmacokinetic studies with
    3H-B(a)P in the diet showed that in the pregnant Ahd/Ahd mouse
    little induction of B(a)P metabolism occurred in the intestine and

    liver, leading to much larger amounts of B(a)P reaching the embryos. 
    In the pregnant Ahb/Ahd mouse B(a)P metabolism was greatly enhanced
    in the intestine and liver; this led to less B(a)P reaching the
    embryos, and much less intrauterine toxicity and malformations. 
    More toxic metabolites (especially B(a)P 1,6- and 3,6-quinones) were
    shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos
    (Legraverend  et al., 1984).

         B(a)P (50 mg/kg bw) was given subcutaneously to pregnant rats
    at different stages of gestation.  B(a)P affected the reproductive
    performance of pregnant rats by significantly increasing the number
    of resorptions and fetal wastage, and by decreasing the fetal weight
    (Bui  et al., 1986).

         The teratogenicity of B(a)P, B(a)P-4,5-oxide, and a racemic
    mixture of B(a)PDEI was investigated after direct (intrauterine)
    injection into embryonal Swiss mice.  The compounds were injected at
    doses ranging from 0.4 to 16.0 nmol/embryo on days 10, 12, and 14 of
    development.  The transplacental effects of B(a)P given at the same
    gestational days and a comparable dose level (47.5 mol/kg bw) were
    also evaluated.  The fetuses were examined when they were 18 days
    old.  On the basis of gross external and internal malformations,
    B(a)PDEI appeared to be the most potent embryotoxic and teratogenic
    compound tested, causing 85% embryolethality and 100% malformed
    fetuses in the group treated on day 10 (0.4 nmol/embryo) of
    intrauterine development.  There were 61 and 27% of fetuses
    malformed following B(a)PDEI treatment on days 12 and 14 of
    gestation, respectively (2 and 4 nmol/embryo).  The effects of this
    B(a)P metabolite were very specific and malformations such as
    exencephaly, thoraco- and gastroschisis, phocomelia, and oedema were
    found.  The administration of B(a)P (both transplacental and direct
    intraembryonal injection) and B(a)P-4,5-oxide caused no significant
    increase of malformed foetuses in any of the developmental stages
    considered (Barbieri  et al., 1986).

         Mouse endometrial cell microsomes showed inducible cytochrome
    P-450 mediated oxidation of B(a)P(-)-trans 7,8-dihydrodiol to B(a)P-
    7,8-dihydrodiol 9,10-epoxides.  Embryos obtained from pregnant mice
    on day 3 post-coitum were incubated with endometrial cell microsomes
    and B(a)P(-)-trans-7,8-dihydrodiol at various concentrations from 0
    to 1.0 M.  Following the incubation, the embryos were transferred
    to pseudopregnant surrogate mothers which were sacrificed 7 days
    later.  The number of surrogate mothers remaining pregnant following
    transfer was reduced significantly at the highest concentration of
    B(a)P(-)-trans-7,8-dihydrodiol.  Blastocyst implantation and
    decidual swelling volume was reduced in a concentration dependent
    manner (Iannaccone  et al., 1984).

    2.2.5  Special studies on genotoxicity

         B(a)P has been extensively studied for mutagenic activity and
    is used as a positive control in a variety of short-term tests.  It
    was active in assays for bacterial DNA repair, bacteriophage
    induction and bacterial mutation; mutation in  Drosophila
     melanogaster; DNA binding, DNA repair; sister chromatid exchange,
    chromosomal aberration, point mutation and transformation in
    mammalian cells in culture; and tests in animals  in vivo, 
    including DNA binding, sister chromatid exchange, chromosomal
    aberration, sperm abnormality and the somatic specific locus (spot)
    test (IARC, 1983).

          In vitro

         Isolated monkey hepatocytes were more efficient than human
    hepatocytes in their capacity to activate B(a)P into products
    mutagenic towards  Salmonella typhimurium TA 1538.  It was shown
    that monkey liver preparations seem to possess a higher
    monooxygenase activity towards B(a)P than human liver preparations
    (Neis  et al., 1986).

         B(a)P was assayed for mutagenicity in the Ames test, in the
    presence of hepatic post-mitochondrial preparations isolated from
    the mouse, rat, hamster, pig and man.  B(a)P gave a positive
    mutagenic response only in the presence of activation systems
    derived from the hamster (Phillipson & Ioannides, 1989).

         No mutagenic metabolites of B(a)P were detected in the
    perfusate from an isolated perfused rat-liver system using either
    the Ames test or a bioluminescence test for genotoxic agents.  The
    bile showed strong genotoxic activity especially in the presence of
    the deconjugation enzymes -glucuronidase and arylsulfatase (Ben-
    Itzhak  et al., 1985).

         Liver microsomal enzymes from male Sprague-Dawley rats
    pretreated with Aroclor 1254 were more effective in inducing B(a)P-
    mediated mutagenesis in the  Salmonella/mammalian microsome
    mutagenicity test than microsomes from DDT treated rats.  DDT-
    induced microsomes yielded a greater proportion of B(a)P-4,5-oxide
    and its metabolic product B(a)P-4,5-dihydrodiol than did Aroclor-
    induced microsomes (Bonin  et al., 1985).

         Rat small-intestinal microsomes were compared with liver
    preparations for their ability to activate B(a)P using the
     Salmonella mutagenicity assay.  At lower doses (less than 1 g
    B(a)P/plate) comparatively high levels of activation were obtained
    with intestinal microsomes.  This could be due to preferential
    formation of the mutagenic 4,5-oxide with the intestinal microsomes,
    as opposed to the putative major active metabolite, the 7,8-diol-
    9,10-epoxide (Walters & Combes, 1986).

         Liver microsomes from rats with vitamin A deficiency enhanced
    the mutagenicity of B(a)P in  Salmonella typhimurium TA 98 (Alzieu
     et al., 1987).

         Selenium inhibited the S9 dependent mutagenicity of B(a)P and a
    number of its metabolites to  Salmonella typhimurium strain TA100. 
    The results suggested that selenium modified the metabolism and
    hence the mutagenicity of B(a)P to TA100 by affecting mixed-function
    oxidase and/or epoxide hydratase activity in both the rat and
    hamster liver S9 activation systems.  Differences were reflected in
    decreased amounts of the strongly mutagenic B(a)P-7,8-dihydrodiol
    and increased amounts of 4,5- and 9,10-dihydrodiols that were weakly
    mutagenic (Teel, 1984).

         Selenium (sodium selenite) decreased the mutagenicity of B(a)P
    in  Salmonella typhimurium strains TA98 and TA1000 (Prasanna  et
     al., 1987).

         Small amounts of seminal fluid strongly enhanced the
    mutagenicity of B(a)P in the  Salmonella/microsome test.  The
    effect was found only in the presence of S9 mix for metabolic
    activation (Rivrud, 1988).

         Using different scavengers of active oxygen species (superoxide
    dismutase, catalase, mannitol and dimethylfuran) in the Ames
     Salmonella assay to determine the role of the reactive oxygen
    species in the B(a)P mutagenesis process, it was suggested that
    singlet oxygen (inhibited by dimethylfuran) may play an important
    role in promoting B(a)P mutagenicity (Wei  et al., 1989).

         The mutation frequency (resistance to ouabain) induced by B(a)P
    was substantially inhibited dose dependently by haemin in Chinese
    hamster V79 cells co-cultivated with X-irradiated hamster embryo
    cells.  The mutagenicity of B(a)P (1 microgram/ml) on V79 cells was
    reduced 6.5% by haemin, 52% by biliverdin, 73% by protoporphyrin and
    85% by chlorophyllin (Katoh  et al., 1983).

         Studies on the cytotoxicity and mutagenicity to 6-thioguanine
    resistance by S9-activated B(a)P in asynchronized and synchronized
    Chinese hamster V79 cells suggested the presence of a specific hot
    spot in the cell cycle for mutagenesis by the B(a)P in cultured
    hamster cells (Ochi  et al., 1985).

         Irradiated second passage Wistar rat embryo (WRE) cells were
    used as activator cells for B(a)P; V79 Chinese hamster lung cells
    were used as the target cells and exposed to 3H-B(a)P for 5, 24 and
    48 hours under the conditions of a cell-mediated mutation assay.  A
    correlation was found between mutation induction and the amount of
    B(a)PDEI-deoxyguanosine (dGuo) adduct in the V79 cells (Sebti  et
     al., 1985).

         The triol 3-OH-B(a)P-7,8-diol was not mutagenic in  Salmonella
     typhimurium strains TA 97, TA 98, TA 100 or TA 1537 or in V79
    Chinese hamster cells (6-thioguanine) when no exogenous metabolizing
    system was added. In the presence of S9 mix, the triol was 5-18
    times more mutagenic than 3-OH-B(a)P in strains TA 97, TA 100 and TA
    1537, but both compounds showed similar mutagenic potencies with
    strain TA 98.  B(a)P-7,8-diol, like the triol, showed mutagenic
    effects only in the presence of S9 mix.  In V79 cells, the diol was
    a potent mutagen, while the triol showed only very weak mutagenic
    effects (Glatt  et al., 1987).

         B(a)P did not effect development and induction of sister
    chromatid exchanges (SCEs) in cultured mouse blastocysts when added
    at a concentration of 1 uM (Spindle & Wu, 1985).

         Induction of SCE in the mouse hepatoma cell line TAOc1B(a)Prc1
    by B(a)P was due to the production of B(a)P-7,8-dihydrodiol.  This
    metabolite did not appear to be produced by another mouse hepatoma
    cell line B(a)Prc1.  TAOc1B(a)Prc1 required only 40 nM B(a)P to
    induce a 2-fold increase in SCE frequency (Schaefer & Selkirk,

         B(a)P-dependent mutagenesis was strongly inhibited in a
    concentration-dependent manner by 7,8-benzoflavone in a murine
    keratinocyte cell-mediated mutagenesis assay (Reiners, 1985).

         B(a)P was mutagenic and induced chromosomal aberrations in the
    L5178Y/TK+/- mouse lymphoma assay and induced sister chromatid
    exchanges  in vivo using the mouse peripheral blood lymphocyte
    culture system (Klingerman  et al., 1986).

         A recombinant plasmid containing the thymidine kinase (TK) gene
    (pAGO; 6.36 kilobases) was reacted  in vitro with B(a)PDEI.  Upon
    transection of mouse LTK-cells with modified plasmid or modified TK
    gene, none or only a few TK-positive cells were obtained, in
    contrast to the formation of many colonies after transection with
    the unmodified plasmid (gene), B(a)P itself, and phenanthrene oxide,
    a weakly reactive but noncarcinogenic chemical (Schaefer-Ridder  et
     al., 1984).  The genomic level of DNA cytosine methylation was
    significantly diminished in dividing BALB/3T3 CL1-13 cells treated
    with B(a)P.  The decrease in DNA 5-methylcytosine levels was
    concentration dependent over the range of 0.1 to 1.0 g/ml when
    determined at the end of the 16 hour treatment period.  (+/-)-SYN-
    B(a)PDE was implicated as the active metabolite causing the effect
    (Wilson & Jones, 1984).

         Suspensions of rat colon epithelial cells metabolized B(a)P
    into products mutagenic for human P3 teratoma cells.  Mutagenesis in
    the P3 cells was indicated by an acquired resistance to 6-
    thioguanine (Oravec  et al., 1986).

         B(a)P was cytotoxic and enhanced viral transformation in the
    Syrian hamster embryo/simian adenovirus SA7 (SHE/SA7) viral
    enhancement assay (Lubet  et al., 1986).

         The effects of glucuronide conjugation on B(a)P-induced
    cytotoxicity and mutagenicity were studied using the CHO/HGPRT assay
    with a rat liver homogenate preparation containing Mixed Function
    Oxidase (MFO) system cofactors (S9 mix) and uridine diphosphate a-D-
    glucuronic acid (UDPGA).  A reduction of B(a)P and B(a)P 6-OH-
    induced cytotoxicity of glucuronide conjugation was probably due to
    the elimination of cytotoxic phenols and quinones.  Since B(a)P 7,8-
    diol is a poor substrate for UDP-glucuronyltransferase enzymes, no
    effects on B(a)P-induced mutagenicity or B(a)P 7,8-diol-induced
    cytotoxicity and mutagenicity were observed (Recio & Hsie, 1984).

         Arachidonic acid (AA), a prostaglandin precursor, significantly
    potentiated sister-chromatid exchange (SCE) induction  in vitro by
    B(a)P in the aryl hydrocarbon hydroxylase (AHH)-inducible human
    hepatoma C-HC-4 cells, and to a lesser extent in the non-inducible
    rat tumour AH66-B and R1 and Chinese hamster Don-6 cells, all of
    which were less sensitive than C-HC-4 cells.  Indomethacin
    completely eliminated the potentiating effect of AA on SCE induction
    by B(a)P (Abe, 1986).

         A continuous B(a)P dose as low as 0.02 M for 20 days produced
    a significant increase in mutant fraction at the 6TG-resistance
    (HGPRT) locus in metabolically competent human lymphoblastoid cells. 
    The long-term, low-dose protocol (0-1 M for up to 20 days) was
    significantly more efficient at inducing mutations than a short-
    term, high-dose protocol (0-10 M for 1 day) (Danheiser  et al.,

         The active metabolite of B(a)P, B(a)PDEI caused cytotoxicity
    and induced mutations in normally repairing or nucleotide excision
    repair-deficient diploid human fibroblasts.  Mutations induced in a
    defined gene sequence, supF, when plasmid containing adducts formed
    by the compound replicated in human 293 cells were mainly base
    substitution mutations, predominantly G:C to T:A transversions
    (Maher  et al., 1987).

         In a cell-mediated mutagenesis assay, treatment of human
    mammary epithelial cells with B(a)P resulted in significant rates of
    mutagenesis in cocultured V-79 cells.  No such effect was found with
    rat cells under identical conditions.  The most significant
    qualitative difference in B(a)P metabolism between the two species
    was the ability of the rat, but not the human, mammary epithelial
    cells to conjugate significant amounts of B(a)P to glucuronic acid. 
    Other aspects of carcinogen metabolism, including production of the
    precursors to known active metabolites of B(a)P were similar (Moore
     et al., 1986).

          In vivo

         B(a)P and two of its major metabolites, B(a)P-4,5-oxide, and
    B(a)P-7,8-diol were investigated for mutagenicity in  Salmonella
     typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-
    mediated assay.  B(a)P and B(a)P-4,5-oxide were not mutagenic under
    any experimental conditions.  B(a)P-7,8-diol was inactive with the
    strain TA1538 but was mutagenic with the strains TA98 and TA100. 
    The effect was potentiated by pretreatment of the host mice with the
    cytochrome P-450 inducer 5,6-benzoflavone (Glatt  et al., 1985).

         B(a)P induced dose-related nuclear damage (micronuclei,
    pyknotic nuclei and karyorrhectic bodies) in colonic epithelial
    cells of female C57BL/6J mice within 24 hours when administered
    intrarectally in single doses of 0-200 mg/kg body weight.  This
    damage was reduced when mice ingested the plant phenols, caffeic,
    ferulic and ellagic acids, and quercetin at levels of 4% or BHA at
    2% in the diet for 1 week prior to the B(a)P challenge (100 mg/kg
    bw) (Wargovich  et al., 1985).

         Several organosulfur compounds in high concentrations, most
    notably allyl mercaptan, benzyl mercaptan, and phenylethyl mercaptan
    were active in inhibiting peroral B(a)P (100 mg/kg body weight)
    nucleotoxicity to the colon of C57BL/6J mice (Wargovich & Eng,

         B(a)P was tested in a micronucleus test using peroral
    administration (62.5, 125, 250, and 500 mg/kg) to males of the MS/Ae
    and CD-1 mouse strains.  Initially, an acute toxicity study lasting
    3 days was done to estimate LD50s.  The LD50 was larger than 1600
    mg/kg in the 2 strains.  The full-scale micronucleus tests indicated
    that B(a)P induced micronuclei dose-dependently in each strain
    (Awogi & Sato, 1989).

         Male B6C3F1 mice were injected intraperitoneally with B(a)P
    (25, 75, 150, 300 mg/kg body weight).  Twenty-four hours later
    lymphocytes were cultured for analysis of SCE in B lymphocytes. 
    B(a)P induced significant dose-related increases in SCE frequency. 
    At the highest concentration B(a)P induced a 3.1-fold increase in
    SCE frequency compared to concurrent controls (Kligerman  et al.,

         Male C57BL/6 mice and male Sprague-Dawley rats were injected
    intraperitoneally with doses of B(a)P ranging from 10 to 100 mg/kg. 
    After 24 hours the peripheral blood lymphocytes (PBLs) were analyzed
    for both DNA adduct formation by 32p-postlabeling and SCE induction
    following lymphocyte culture.  B(a)P induced similar, but not
    identical, SCE induction following lymphocyte culture.  B(a)P
    induced similar, but not identical, SCE dose-response curves for
    each species.  After B(a)P administration, the major DNA adduct,

    B(a)PDEI-dGuo, was approximately 10-fold more prevalent in the PBLs
    of the mouse (300-1200 attomol/g DNA) than those of the rat (20-130
    attomol/g DNA).  Thus, for equivalent amounts of B(a)PDEI-dGuo, a
    greater number of SCEs are induced in the rat than the mouse
    (Kligerman  et al., 1989).

         B(a)P at 62.5 mg/kg body weight given by gavage to female Brown
    Norway rats failed to induce unscheduled DNA synthesis in
    parenchymal liver cells isolated 5 or 18 hours after the
    administration.  In contrast, alkaline elution showed that at 5
    hours after administration of B(a)P a considerable number of alkali-
    labile sites was present in the DNA of both intestinal cells and
    parenchymal liver cells, but not in that of non-parenchymal liver
    cells (Mullaart  et al., 1989).

    2.2.6  Special studies on macromolecular binding

         The implication of covalent binding of B(a)P metabolites to DNA
    for B(a)P carcinogenicity in skin and lung of experimental animals
    has been intensively studied in cell culture systems and in vivo. 
    Several reviews have been published (Gelboin, 1980: Levin et al.,
    1982; Pelkonen & Nebert, 1982; Conney, 1982; IARC, 1983; Cooper et
    al., 1983; Grover, 1986).  The metabolites responsible for binding
    to DNA were the B(a)P-7,8-diol-9,10-oxide (B(a)PDEI) and the 9-OH-
    B(a)P-4,5-oxide.  Mutagenicity and carcinogenicity studies on a
    variety of metabolites seem to indicate that B(a)PDEI is the
    putative carcinogenic metabolite of B(a)P, although the correlation
    between binding of this metabolite to DNA and tumour initiating
    ability has not always proven perfect.

          In vitro

         B(a)P induced genotoxic effects and DNA adduct levels were
    determined in several short-term bioassay systems: cytotoxicity,
    gene mutation, and sister chromatid exchange in Chinese hamster V79
    cells; cytotoxicity, gene mutation, and chromosome aberrations in
    mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus
    transformation in Syrian hamster embryo cells; and cytotoxicity and
    morphological transformation in C3H10T1/2CL8 mouse embryo
    fibroblasts.  Both total B(a)P-DNA binding and specific B(a)P-DNA
    adducts were measured.  B(a)PDE I-dGuo was one of the major adducts
    identified in all bioassay systems.  DNA binding and genotoxic
    responses varied significantly between bioassays.  Each genetic end
    point was induced with a differing efficiency on a per adduct basis. 
    However, the relationships between frequency of genetic effect or
    morphological transformation and B(a)P-DNA binding or B(a)PDE I-dGuo
    were linear within a given assay (Arce  et al., 1987).

         The cytochrome P-450-dependent metabolism of 3H-B(a)P by
    cultured primary keratinocytes prepared from BALB/C mouse epidermis

    was found to be largely inhibited by the dietary plant phenol,
    ellagic acid.  The intracellular enzyme-mediated binding of B(a)P to
    mouse keratinocyte DNA was also largely inhibited in a dose-
    dependent fashion (Mukhtar  et al., 1984).

          In vitro studies demonstrated that mouse serum sequesters
    B(a)PDEI and protects it from hydrolysis.  Four hours after B(a)P
    administration to mice (i.p.), mouse serum produced two adduct spots
    when incubated with salmon sperm DNA.  The major adduct co-
    chromatographed with a B(a)PDEI adduct standard.  B(a)PDEI-DNA
    adducts in tissues were highest in liver, lung and spleen, with
    kidney and stomach levels significantly lower (Ginsberg & Atherholt,

         The formation of 3H-B(a)P adducts with calf thymus DNA was
    studied  in vitro in the presence of microsomes prepared from the
    isolated labyrinth zone of the rat placenta, the haematopoietic
    erythroblast cells of the fetal liver, the fetal liver, as well as
    the maternal liver.  Pregnant rats were induced with -
    naphthoflavone on day 17 of gestation and the microsomes prepared
    one day later.  The levels of covalent binding (pmol/mg DNA/mg
    microsomal protein) for maternal liver, fetal liver, placenta and
    erythroblast cells were: 28.4, 2.4, 0.31, and 3.9, respectively. 
    Major adducts were identified as the 9-OH-4,5-oxide adduct and the
    B(a)PDEI adduct (Salhab  et al., 1987).

         Primary cultures of epithelial cell aggregates and fibroblasts
    derived from mammary tissue from female Wistar rats able to
    metabolize B(a)P and at least 7 DNA-adducts were isolated and
    analyzed.  None of the adducts showed chromatographic properties
    characteristic of adducts formed by B(a)PDEI or other known
    electrophilic metabolites of B(a)P.  Similar profiles of adducts
    were obtained from mammary DNA of rats that had been treated with
    B(a)P by injection into their mammary fat pads.  In contrast, when
    B(a)P was administered by intraperitoneal injection to female Wistar
    rats, B(a)PDEI-DNA adducts were detected in each of seven tissues,
    including mammary gland, that were examined (Phillips  et al.,

         The covalent binding of B(a)P to calf thymus DNA brain
    microsomes isolated from control and 3-methylcholanthrene (3-MC)
    treated rats was investigated.  Treatment of rats with 3-MC resulted
    in a 1.5-fold increase in the brain microsomal mediated covalent
    binding of 3H-B(a)P to DNA (Das  et al., 1985b).

         Using rat liver nuclei or hepatocytes incubated with B(a)P and
    B(a)PDEI it was found that B(a)P binds more readily to DNA of active
    chromatin and nuclear matrix than to bulk chromatin.  Selectivity
    was not due to the subnuclear location of enzymes which activate to
    B(a)PDEI (Obi  et al., 1986).

         Primary cultures of epithelial and fibroblast cells derived
    from human oral mucosa were studied for the ability to activate
    B(a)P.  The cells were exposed to B(a)P for 18 hours.  B(a)P tetrols
    and diols were the major metabolites formed by primary cultures of
    epithelial and fibroblast cells derived from human oral mucosa.  The
    epithelial cells had a much higher rate of biotransformation of
    B(a)P as measured by binding to cellular DNA.  The major B(a)P-DNA
    adduct was formed by the reaction of B(a)PDEI with the exocyclic 2-
    amino group in guanine.  In contrast to human cells, B(a)P phenols
    and B(a)P 9,10-diol were the major metabolites produced by primary
    epithelial and fibroblast cells derived from rat buccal mucosa.  The
    DNA binding levels of B(a)P in the two rat cell types were
    identical, and the binding level was several-fold lower than in the
    human epithelial cells (Autrup  et al., 1985).

          In vitro activation of B(a)P to protein-binding forms in high
    yield was obtained with human and rat blood cells.  A simple
    combination of unsaturated fatty acid, i.e., linoleic or arachidonic
    acid, and haematin or haemin resulted in activation of B(a)P
    (Nemota, 1986).

         Normal human mammary epithelial cell cultures and the human
    mammary carcinoma T47D cell line were exposed to 3H-B(a)P for 24
    hours, and the levels of binding were 81 and 182 pmol B(a)P/mg DNA
    in normal and T47D cultures, respectively.  Analysis of B(a)P-
    deoxyribonucleoside adducts demonstrated the presence of three
    adducts in both cells:  (+)-anti-B(a)PDE-dGuo (B(a)PDEI-dGuo), (-)-
    anti-B(a)PDE-dGuo, and syn-B(a)PDE-dGuo.  Thus evidence was provided
    that (-)-anti-B(a)PDE is formed in cell systems and reacts with DNA
    in cells to form (-)-anti-B(a)PDE-dGuo (Pruess-Schwartz  et al.,

         The major B(a)P adduct formed in human mammary epithelial cells
    was identified as B(a)PDEI-Guo.  This adduct was only formed at very
    low levels in rat mammary epithelial cells.  The rat cells contained
    a large proportion of syn-B(a)PDE adducts, and other unidentified
    B(a)P-DNA adducts (Moore  et al., 1987).

         Studies on the metabolism of B(a)P in randomly proliferating
    and confluent cultures of human skin fibroblast cells suggested that
    factors other than random modification of DNA by B(a)P might have a
    significant role in the expression of a transformed phenotype and
    that metabolism and transformation are not directly related. 
    Furthermore, confluent dense cultures with a heightened capability
    for metabolism of B(a)P were more active in the detoxification of
    B(a)P than in the production of the metabolites associated with
    cellular transformation (Cunningham  et al., 1989).

         The formation of adducts of B(a)P metabolites on DNA was
    investigated in endometrial tissue from humans, hamsters, mice, and

    rats. B(a)PDEI was the predominant adduct identified in all the
    species studied.  The amount of B(a)PDEI bound to DNA from human
    endometrium was approximately 3 times higher than to DNA from
    hamster tissue.  Among the three animal species examined, the level
    of this adduct was highest in hamsters and lowest in rats (Kulkarni
     et al., 1986).  

         Human colon and bronchus tissue explants were incubated with
    3H-B(a)P.  The total percentage of metabolism of B(a)P was 8-59% in
    bronchus and 11-23% in colon.  B(a)P was found to bind covalently to
    the DNA of human bronchi from 15 cases at a mean of 42 pmol/10 mg
    DNA, and to the DNA of human colon from 6 cases at a mean of 66.5
    pmol/10 mg DNA.  The range among individuals was within one order of
    magnitude.  Human bronchus explant DNA contained one adduct:  (+/-)-
    B(a)PDEI-dGuo.  DNA obtained from the lung or liver of rats given
    2.0 mg/kg doses of 3H-B(a)P by intraperitoneal injection contained
    3 DNA adducts in liver and two were observed in lung DNA
    hydrolysates.  One adduct from each organ cochromatographed with
    (+/-)-B(a)PDEI-dGuo; however, the major adduct in each case eluted
    earlier (Garner  et al., 1985).

          In vivo

         The administration of B(a)P topically to pregnant C3H mice
    during days 13-17 of gestation resulted in adduct formation in the
    haemoglobin of the mother and progeny.  Exposure to a total maternal
    body burden of 500 g B(a)P during the last 5 days of delivery
    resulted in an average level of 6.35 pg of anti-diolepoxide
    metabolite covalently attached per mg of haemoglobin analyzed in the
    mother and 1.40 pg in the newborn animals.  Concomitant adduct
    formation in the DNA of the skin with B(a)P in the progeny was not
    observed (Shugart & Matsunami, 1985).

         Occurrence and persistence of DNA damage in the hepatic and
    pulmonary tissues of fetal (days 12, 15 and 18 of pregnancy),
    newborn (days 1 and 7) and adult (days 82-85) CD1 mice exposed to
    selected doses of B(a)P (10 mg/kg body weight) were studied by
    utilizing the alkaline elution technique.  This approach indicated
    that 15-day-old fetuses were the most sensitive to B(a)P
    genotoxicity.  B(a)P at dose levels of 0, 2 and 10 mg/kg body weight
    was injected intraperitoneally into pregnant females or directly
    into single fetuses and the fetal livers and lungs recovered 2, 4,
    24 and 48 hours later.  The results showed that the maximum DNA
    damage is present at 4 hours following B(a)P treatment and it almost
    disappeared at 48 hours irrespective of the route of B(a)P
    administration.  The effects where markedly magnified by Aroclor
    pretreatment (Bolognesi  et al., 1985).

         In female ICR mice pregnancy lowered the binding of B(a)PDEI to
    liver and lung DNA by 29-41%, but not the binding of other
    metabolites (Lu  et al., 1986a).

         Using the 32P-postlabeling method the binding of B(a)P (200
    mol/kg body weight) to the DNA of various maternal and fetal
    tissues was determined.  B(a)P was administered to pregnant ICR mice
    on day 18 of gestation.  B(a)P was bound to the DNA of maternal and
    fetal liver, lung, kidney, heart, brain, intestine, skin, maternal
    uterus, and placenta, with organ-specific quantitative and
    qualitative differences.  B(a)P exhibited no obvious tissue
    preference in either maternal or fetal organs.  The fetal adduct
    levels were generally lower than the corresponding maternal adduct
    levels (Lu  et al., 1986b).

         The diolepoxide metabolite of B(a)P could be detected bound
    covalently to the haemoglobin of erythrocytes isolated from mice
    previously exposed to 400 g B(a)P given intraperitoneally (Shugart,

         Following a single oral administration of 80 umol/kg of B(a)P
    to male BALB/c mice, a 32P-postlabeling assay showed that after 24
    hours the highest levels of total DNA adducts were found in the
    skin, followed by lung, liver and kidney.  The main adduct
    identified was B(a)PDEI-deoxy-guanosine 3',5'-bisphosphate (Schurdak
    & Randerath, 1989).  

         The distribution and macromolecular binding of 3H-B(a)P was
    examined in the skin, liver, lung, and stomach of SENCAR and BALB/c
    mice following topical or oral administration of B(a)P (50 mg/kg
    body weight) at time periods ranging from 0.5 to 48 hours.  Levels
    of labeled material in skin were higher, and the binding of B(a)P to
    epidermal DNA was greater following topical administration than
    following oral administration for mice of both strains.  Following
    oral administration of 3H-B(a)P greater levels of radioactivity
    were found in  liver, lung, and stomach tissue than after topical
    administration (Morse & Carlson, 1985).

         It has been reported that B(a)P is able to produce papillomas
    of the skin in male SENCAR mice after a single oral administration
    of 10 or 30 mg/kg body weight.  When 3H-B(a)P was administered as
    single doses orally or topically to male SENCAR mice, high
    concentrations were found in the skin following topical application,
    but very little  material reached this target organ following oral
    administration.  In contrast, the internal organs generally
    contained more material after oral administration.  The binding of
    labelled compound to DNA, RNA, and protein generally reflected the
    distribution data, thus more compound was bound in the stomach,

    liver, and lung after oral administration compared to topical
    application, whereas the opposite was true for the skin (Carlson  et
     al., 1986).

         The  in vivo formation of B(a)P metabolite-DNA adducts has
    been characterized in a variety of target and nontarget tissues of
    A/HeJ mice and rabbits.  Tissues included were lung, liver,
    forestomach, colon, kidney, muscle, and brain.  The major adduct
    identified in each tissue was the (+)-(anti)-B(a)PDEI-dGuo adduct. 
    A (+/-)-syn-B(a)PDEII-dGuo adduct, a (-)-(anti)-B(a)PDEI-dGuo
    adduct, and an unidentified adduct were also observed.   The adduct
    levels were unexpectedly similar in all the tissues examined from
    the same B(a)P-treated animal.  In mice given perorally 11.9 g/kg
    body weight the range was 12-28 fmol/g DNA whereas in mice given
    1190 g/kg body weight the range was 2.7-6.1 pmol/g DNA.  For
    example, the B(a)PDEI-DNA adduct levels in muscle and brain of mice
    were approximately 50% of those in lung and liver at each oral B(a)P
    dose used.  Adduct levels formed in vivo in several cell types of
    lung and liver were also examined.  Macrophages, type II cells, and
    Clara cells from lung and hepatocytes and non-parenchymal cells from
    liver were isolated from B(a)P-treated rabbits.  B(a)PDEI-
    deoxyguanosine adduct was observed in each cell type and, moreover,
    the levels were similar in various cell types.  (Stowers & Anderson,

         B(a)P-DNA adducts were analyzed in hepatic and pulmonary cells
    isolated from rabbits 24 hours after intravenous administration of
    3H-B(a)P (1 mg/kg).  The major adduct in each of the cell types
    analyzed was (+)-anti-B(a)PDEI-dGuo, but (+/-)-syn-B(a)PDEII-dGuo
    and very low levels of (-)-anti-B(a)PDEI-dGuo and an unidentified
    adduct were also observed.  The level of the major adduct was
    similar in each of the isolated cell types and was at least as high
    in cells with very low cytochrome P-450-dependent monooxygenase
    activity (hepatic nonparenchymal cells and alveolar macrophages) as
    in those with higher activity (hepatocytes, alveolar type II cells,
    and Clara cells) (Horton  et al., 1985).

         The 7-B(a)PDE-Gua adduct was identified in urine from partially
    hepatectomized male Wistar rats treated intraperitoneally with 0,
    10, 50 or 100 g 3H-B(a)P in the urine.  Less than 0.6% to 0.15% of
    the doses were excreted as 7-B(a)PDE-dGua.   In vitro studies using
    human PLC/5 cells showed that the 7-BPDE-dGua adduct is very labile
    and is released to the medium with a half life of 3 hours leaving
    apurinic sites in the DNA (Autrup & Seremet, 1986).

         The administration of the 3H-B(a)PDEI-DNA adduct, whether by
    intraperitoneal or intravenous injection, to male Wistar rats
    resulted in the majority of the radioactivity being recovered in the
    faeces.  Excretion was rapid: within 24 hours post-injection, 45% of

    the applied dose was recovered in the faeces.  HPLC analysis of
    radioactive material extracted from the faeces by methanol showed
    that it contained a single component which co-chromatographed with
    3H-B(a)PDEI-dGuo (Tierney  et al., 1987).

         Animals dosed for 7 days with retinyl acetate (80 mg/kg body
    weight/day), 13-cis-retinoic acid (13cisRA) (120 mg/kg body
    weight/day), and N-(4-hydroxyphenyl)-retinamide (4HPR) (600 mg/kg
    body weight/day), and showed a 38, 27, and 40% reduction in binding
    of 3H-B(a)P (2 mg/kg body weight given intraperitoneally) to liver
    DNA and a 29, 32, and 21% reduction in binding to stomach DNA,
    respectively, when B(a)P was administered on the eighth day, and the
    tissues were harvested 24 hours later.  Binding to lung DNA was
    reduced by 23 and 11%, respectively, in the 13cisRA- and 4HPR-dosed
    rats.  No differences were observed in binding to kidney (McCarthy
     et al., 1987).

         Following a single intraperitoneal injection of 3H-B(a)P, more
    B(a)P was bound to liver DNA recovered from rats fed a diet
    containing 20% menhaden fish oil (rich in omega-3 fatty acids) for
    11 days at all time intervals tested (16, 24, 48, and 192 hours)
    than was found from rats fed 0.5% menhaden oil.  The increased
    binding of 3H-B(a)P to liver DNA of rats fed the high level of
    menhaden oil may be due, in part, to increases in the MFO
    responsible for B(a)P activation (as suggested by increased
    cytochrome P-450 level and total B(a)P hydroxylase activity)
    (Dharwadkar & Wade, 1987).

         Groups of 10 weanling male Wistar rats were subjected to
    different levels of food restriction (0, 20, 40, and 60%
    restriction).  After 20 weeks on the diets there was significant
    increase in the binding of 3H-B(a)P to hepatic DNA in 40 and 60%
    food restricted animals (in vivo experimention), although this was
    not observed under in vitro conditions. There was a decrease in
    binding to pulmonary DNA and no change for renal DNA (Jagadeesan &
    Krishnaswamy, 1989).

         Blocking of  in vivo arachidonic acid dependent prostaglandin
    endoperoxide synthetase with acetylsalicylic acid (200 mg/kg body
    weight) did not affect the  in vivo activation of B(a)P to
    metabolites capable of interacting irreversibly with cellular
    macromolecules in guinea pig liver, lung, kidney, spleen, small
    intestine, colon, and brain (Garattini  et al., 1984).

    2.2.7  Special studies on immunotoxicity

         Young (3-6 months), middle-aged (16-18 months) and aged (23-26
    months) mice were exposed  in vitro and  in vivo to B(a)P.  The
    generation of cells producing antibody to the T-dependent antigens
    of sheep erythrocytes was observed to be suppressed in all age

    groups.  Significantly, aged mice were shown to exhibit a greater
    percent suppression of antibody responses than young or middle-aged
    mice both  in vitro and  in vivo (Lyte & Bick, 1985).

         When B(a)P was incorporated into a T-dependent antibody (TDAb)-
    producing spleen cell culture system, dose- and time-dependent
    inhibition of plaque-forming cell responses was observed.  Addition
    of B(a)P at concentrations as low as 0.002 g/ml resulted in
    suppression of the TDAb plaque-forming cell response.   In vitro
    incorporation of B(a)P into polyclonal antibody-generating cultures
    also resulted in a dose-related inhibition.  Fourteen-day exposure
    of mice to B(a)P (40 mg/kg body weight/day) resulted in 98%
    suppression of the TDAb response.  These studies suggest that the
    suppressive effects of B(a)P are multicellular in origin, occur
    apart from the carcinogenic effects, and cannot be attributed merely
    to cellular toxicity (Blanton  et al., 1986).

         Progeny from B(a)P exposed (150 g/g body weight) primiparous
    mothers, injected during the second trimester of pregnancy, were
    severely compromised immunologically.  After 12-18 months the
    progeny developed high incidences of hepatomas, lung adenomas and
    adenocarcinomas, reproductive tumours, and lymphoreticular tumours
    (Urso & Gengozian, 1980).  When B(a)P was administered postnatally
    (after 1 week) both immune suppression and tumour incidence was
    substantially lower (Urso & Gengozian, 1982).

         Pregnant C3H/Anf mice were exposed to 150 g B(a)P/gram body
    weight during fetogenesis (day 11-17 of gestation) and the progeny
    were assayed for humoral and cell mediated immune responses at
    different time intervals after birth.  Immature offspring (1-4 wk)
    were severely suppressed in their ability to produce antibody-
    (plaque-) forming cells (PFC) against sheep red blood cells (SRBC)
    and in the ability of their lymphocytes to undergo a mixed
    lymphocyte response (MLR).  A severe and sustained suppression in
    the MLR and the PFC response occurred from the fifth month up to 18
    months.  Tumour incidence in the B(a)P-exposed progeny was 8- to 10-
    fold higher than in those encountering corn oil alone from 18 to 24
    months of age (Urso & Gengozian, 1984).  Immunodeficiency
    (abnormalities in the T cell-mediated responses caused by disruption
    of T cell differentiation) occur early after birth (1 week) and
    persists for 18 months (Urso & Johnson, 1987).

         Pregnant C3H/Anfcum mice injected i.p. with 150 g B(a)P/g body
    weight at day 12.  Within 5 days after injection, a 2- to 4-fold
    reduction in leukocytes was observed when compared to controls which
    persisted into the 10th postpartum day.  The erythrocytes were also
    significantly reduced but not to the same degree (1.2- to 1.5-fold). 
    Depression in white blood cells was attributed to lymphocyte
    depletion (Urso  et al., 1988).  In the thymus, there was an

    exacerbated depression in the amounts of thymocytes during pregnancy
    relative to the controls, which was sustained postpartum.  In the
    spleen changes in the differentiation potential of T precursor cells
    were indicated (Urso & Johnson, 1988).

         The role of B(a)P metabolism in the suppression of the  in
     vitro humoral immune response was determined as the inhibition of
    antibody-forming cells (AFC) of splenocyte cultures.  Addition of
    B(a)P or various B(a)P-diols in combination with addition of the
    cytochrome P-450 inhibitor, alpha-naphthoflavone suggested that the
    B(a)P-induced suppression of the  in vitro AFC response is mediated
    by B(a)P metabolites generated by cytochrome P-450 present within
    splenocytes (Kawabata & White, 1987).

         B(a)P administration (200 mg/kg body weight, i.p.) to female
    B6C3F1 mice resulted in suppression of polyclonal responses and
    substantial DNA adduct formation in mouse splenic keukocytes (SPL). 
    SPL adduct levels were similar to those in liver, lung, kidney, and
    stomach.   In vitro studies showed that SPL exhibited low AHH
    activity and ability to form DNA-adducting metabolites, and that
    rapid and dose related DNA adduct formation in SPL required the
    addition of liver S9 (Ginsberg  et al., 1989).

    2.2.8  Special studies on bone marrow toxicity

         Severe reduction in peripheral blood leucocytes was seen after
    daily oral administration of B(a)P (120 mg/kg body weight/day) for
    10-50 days in non-responsive mice (DBA/2), while only a mild effect
    was seen in responsive mice (C57B1xDBA/2 F1).  The responsive mice
    were protected from bone marrow toxicity by marked induction of
    B(a)P metabolism in the gastro-intestinal tract and liver (Nebert
     et al., 1980).

         Severe bone marrow depression with almost complete destruction
    of pluripotent haematopoietic stem cells was seen in non-responsive
    female DBA/2 mice after oral B(a)P (125 mg/kg body weight/day) for
    13 days.  Extreme resistance to bone marrow toxicity was observed in
    responsive BDF1 mice fed for 19 days (Anselstetter & Heimpel, 1986).

         The inducibility of P-4501 in the inbred mouse strains C57BL/6N
    in the liver and intestine afforded protection against the
    myelotoxic effect on bone marrow induced by B(a)P.  In the DBA/2N
    mouse strain having a poor-affinity Ah receptor, 120 mg/kg body
    weight/day of B(a)P led to death within 3 weeks due to bone marrow
    toxicity.  B(a)P in the growth medium, in direct contact with
    cultured myeloid cells, was more toxic to C57BL/6N than DBA/2N
    cultured cells.  Oral B(a)P induced P-4501 (measured by B(a)P-7,8-
    dihydrodiol formation) in C57BL/6N but not DBA/2N intestine and
    liver.  In the bone marrow of oral B(a)P-treated C57BL/6N and DBA/2N
    mice, the magnitude of P-4501 induction was about the same.  Mice

    having the high-affinity receptor, and therefore the P-4501
    induction process in the intestine and liver, were protected from
    oral B(a)P-induced myelotoxicity (Legraverand  et al., 1983).

    2.2.9  Special studies on atherosclerosis

         Treatment of chickens for up to 20 weeks with weekly injections
    of B(a)P (0.1, 1.0, and 10 mg/kg body weight/week) resulted in
    significant increases in incidence and size of atherosclerotic
    lesions of the abdominal aorta at the two higher doses used. 
    Administration of a single dose of B(a)P followed by the tumour
    promoter TPA for 20 weeks did not have any effect (Bond  et al.,

         Weekly B(a)P injections of 0.1, 10 or 100 mg/kg body weight
    were given to White Carneau Pigeons for 6 months.  Only the high
    dose treatment induced atherosclerosis in the aorta of the pigeons
    (Revis  et al., 1984).

         The incidence of atherosclerotic lesions of aorta was increased
    in mice given 0.15 mg 3-methylcholanthrene/kg body weight and
    thereafter maintained on an atherogenic diet for 14 weeks.  The
    increase was significantly greater in responsive mice than in non-
    responsive mice (Paigen  et al., 1986).

    2.3  Observations in humans

         Polyclonal and monoclonal antibodies against B(a)PDEI-DNA
    adducts have been developed and used in radioimmunoassays or
    competitive ELISA assays in order to monitor human exposure to B(a)P
    (Perera  et al., 1982; Santella  et al., 1984; 1985).  The
    monoclonal antibodies developed are not specific for B(a)PDEI-
    adducts but also measure adducts from other PAHs (Santella, 1988).
    The human monitoring studies have primarily been conducted on
    occupationally exposed individuals and smokers.

         B(a)P-DNA adducts were detected at low levels (0.08-0.16
    fmol/g DNA) in lung DNA from five of 27 patients with lung cancer
    (Perera  et al., 1982).

         B(a)PDE-DNA adducts were detected in white blood cells from 7
    of 28 roofers and 7 of 20 foundry workers (range 0.04 to 2.4 fmol/g
    DNA), and in two of 9 samples from non-occupationally exposed
    volunteers (Shamsuddin  et al., 1985).

         Among the DNA samples from peripheral blood lymphocytes from 30
    aluminium plant workers only one sample was found to contain a peak
    similar to B(a)PDEI-DNA when measured by synchronous fluorescence
    spectrophotometry (Vahakangas  et al., 1985).

         In coke oven workers exposed to high levels of PAHs,  B(a)PDEI-
    DNA adducts were detected in peripheral blood lymphoctyes from 18 of
    27 individuals.  In 9 controls no adducts were detected (Harris  et
     al., 1985).

         A mean level of 1.7 fmol B(a)PDEI-DNA adduct/g DNA measured by
    radioimmunoassay was seen in 13 of 38 lymphocyte DNA samples from
    coke oven workers exposed to high levels of PAHs.  Four of the
    samples were positive for B(a)PDEI-DNA in a synchronous fluorescence
    spectrophotometry assay.  These were also the samples having the
    highest levels of DNA adducts (1.0 to >13.7 fmol/g DNA) as
    measured by radioimmunoassay (Haugen  et al., 1986).

         Blood monocytes from 45 selected patients with lung cancer and
    30 healthy controls were incubated with 3H-B(a)P for 30 hours.  The
    DNA adducts were significantly elevated in 22 patients (non-smokers)
    with early age cancer (4.34 fmol/micrograms of DNA).  In 12 familial
    cases (at least one first degree relative with lung cancer), a
    slight elevation (2.77 fmol/micrograms of DNA) was not statistically
    significant in comparison to healthy controls.  B(a)P-DNA adduct
    levels did not differ significantly between smokers and nonsmokers
    (Rudiger  et al., 1985).

         DNA from placental specimens of smokers showed a small but not
    statistically significant increase in B(a)PDEI-DNA adducts compared
    to controls.  The mean adduct levels were 1.65 and 0.96 fmol/g DNA,
    respectively.  Using the 32P-postlabelling assay, an adduct not
    derived from B(a)P was detected in 16 of 17 smokers compared to 3 of
    14 nonsmokers (Everson  et al., 1986).

         In lymphocyte DNA of smokers and nonsmokers the number of
    samples with detectable B(a)PDE-DNA adducts was much lower than in
    placental DNA, but also did not differ between groups (Perera  et
     al., 1987).

         Specimens of human lung, uterine cervix, ovary, and placenta
    were studied for the presence of B(a)PDEI-DNA adducts by using
    rabbit anti-B(a)PDE-DNA antibody and light microscopic
    immunocytochemistry. B(a)PDEI-DNA antigenicity was detected in the
    bronchial epithelial cells, cervical epithelium, oocytes, luteal
    cells, corpora albicans, and hyalinized media of arteries within the
    ovaries and trophoblastic cells of the placental villi (Shamsuddin &
    Gan, 1988).

         Breast epithelial cells from 10 donors were screened for the
    existence of DNA adducts using the 32P-postlabeling assay.   In
     vitro studies had shown that the major B(a)P-DNA adduct formed by
    human mammary epithelial cells  in vitro was B(a)PDEI-dGuo.  This
    adduct did not appear to be formed by rat mammary cells exposed to
    B(a)P in vitro.  However, 32P-postlabeling analysis of mammary

    epithelial cell DNA from rats exposed to B(a)P in vivo indicated
    that B(a)PDEI-dGuo was a major B(a)P-DNA adduct under these exposure
    conditions.  When the mammary epithelial cells from 10 human donors
    were screened for DNA adducts formed  in situ, cells from three
    donors exhibited distinct adduct patterns.  None of these adducts
    appeared to be B(a)PDEI-dGuo (Seidman  et al., 1988).

         In 40 of 81 lung cancer cases and 36 of 67 non cancer controls,
    PAHs (B(a)PDEI)-DNA adducts in leukocytes were not significantly
    related to age, sex, ethnicity, amount of cigarette smoking, dietary
    charcoal, or caffeine consumption.  The mean adduct level was about
    0.4 fmol/g DNA.  When subjects were stratified by smoking status
    (current, former, and nonsmoker), lung cancer cases who were current
    smokers had significantly higher levels of adducts than current
    smoker controls.  A seasonal variation was observed, with peak in
    adduct levels during July-October.  The study indicates that
    B(a)PDEI-DNA adducts reflect a pervasive and variable "background"
    (Perera  et al., 1989).  

         B(a)PDEI-dGuo and other apparent PAHs-DNA adducts were detected
    in human peripheral lung tissue samples from 9 of 17 individuals
    using HPLC-linked synchronous fluorescence spectrophotometry and
    radioimmunoassay.  No correlation between occupational or smoking
    history was seen.  A number of other (non-PAHs) adducts were also
    detected (Wilson  et al., 1989).


         Although benzo [a]pyrene was the substance on the agenda, the
    Committee recognized that this was only one member of a class of
    more than 100 compounds belonging to the family of polycyclic
    aromatic hydrocarbons found in food and that they should be
    considered as a class.  

         The Committee reviewed and discussed data from studies on the
    toxicity of benzo [a]pyrene, with particular emphasis on its
    toxicity after ingestion.  The results demonstrated many different
    toxic effects of this compound.

         In mice, orally administered benzo [a]pyrene consistently
    produced tumours of the forestomach and lung, and the few available
    studies in rats showed tumours of the oesophagus, forestomach and
    mammary gland.  Tumours at other sites, such as lymphoreticular
    tumours in mice, were also reported.  The Committee noted that fetal
    and newborn mice are especially vulnerable to the pulmonary and
    lymphatic tumorogenicity of benzo [a]pyrene administered either by
    direct injection or transplacentally.

         The genotoxicity of benzo [a]pyrene both  in vitro and  in
     vivo, is well documented, and this compound is consequently used
    as a positive control substance in these types of studies.  The
    Committee noted that IARC had found inadequate evidence for
    carcinogenicity of benzo [a]pyrene in humans, but sufficient
    evidence for carcinogenicity to animals and for activity in short-
    term genotoxicity tests.

         The Committee also noted that in mice oral doses of 120 mg of
    benzo [a]pyrene per kg of body weight and above caused intrauterine
    toxic effects and fetal malformations when administered during

         The immunotoxicity and bone-marrow toxicity of benzo [a]pyrene
    in mice were also considered.  In the immunotoxicity studies, in
    which pregnant mice received a single dose of benzo [a]pyrene at
    150 mg per kg of body weight intrapreitoneally, the resultant
    offspring were severley immunosuppressed.  The Committee noted that
    this effect may have led to the subsequent widespread development of
    tumours in these animals.   

         Many studies have implicated benzo [a]pyrene-ne-7,8-diol-9,10-
    oxide (BPDEI) as the proximate carcinogenic metabolite of
    benzo [a]pyrene.  This metabolite binds covalently to DNA, induces
    mutations and transformations in short-term tests, and is a highly
    potent carcinogen in mouse skin.  However,  the Committee noted that
    studies in which benzo [a]pyrene was administered perorally to mice

    and rabbits showed that the level of binding of BPDEI to DNA was
    similar in all tissues examined (target tissues as well as non-
    target tissues).

         The Committee also considered studies in which the levels of
    BPDEI-DNA adducts were measured in human tissues, although none of
    these studies were aimed at monitoring benzo [a]pyrene exposure
    from food.  The Committee noted that the levels of DNA adducts were
    elevated in only a few individuals, who were believed to have been
    exposed to high levels of benzo [a]pyrene.  The levels of BPDEI-DNA
    adducts in humans were unrelated to factors such as age, sex,
    ethnicity, number of cigarettes smoked, or caffeine consumption.

         It was concluded that, for the purpose of evaluation, the most
    significant toxicological effect of benzo [a]pyrene was its
    carcinogenic activity.

         The Committee had before it data from studies on
    benzo [a]pyrene levels in various food and estimated dietary
    intakes.  These data demonstrated the wide-ranging levels of
    benzo [a]pyrene in food and that these levels were dependent on
    factors such as where the food was grown (i.e., industrialized or
    non-industrialized area), how it was processed (e.g., smoking or
    drying), and how it was cooked (e.g., charcoal grilling).  In turn,
    dietary intakes varied considerably, some consumers being exposed to
    high levels of the substance.

         The Committee noted that the estimated average daily intake of
    benzo [a]pyrene by humans was about four orders of magnitude lower
    than the level reported to be without effect on the incidence of
    tumours in an experiment in rats in which benzo [a]pyrene was
    incorporated in the diet.  However, the Committee was unable to
    establish a tolerable intake for benzo [a]pyrene, based on the
    available data.

         Nevertheless, the large difference between estimated human
    intakes of  benzo [a]pyrene and the doses producing tumours in
    animals suggests that any effects on human health are likely to be
    small.  Despite this, the considerable uncertainties in risk
    estimation require that efforts should be made to minimize human
    exposure to benzo [a]pyrene as far as is practicable.

         The Committee was informed that a long-term carcinogenicity
    study in rats in which benzo [a]pyrene is being administered by
    gavage has been initiated to investigate the dose-response
    relationship for the tumorogenicity of this compound.


         The Committee acknowledged the complexities of the problem of
    reducing exposure to B(a)P and other PAHs.  Furthermore, it noted
    that B(a)P exposure constitutes only a fraction of consumers'
    exposure to PAHs and that some other members of this class of
    compounds, not evaluated at this meeting, also exhibit a similar
    toxicology profile to that of B(a)P and may thus contribute to the
    overall carcinogenic risk.  In this regard, strategies to minimize
    B(a)P exposure would also be effective in reducing overall exposure
    to PAHs.  These include practices that consumers can effect, such as
    cleaning fruits and vegetables thoroughly to remove any surface
    contamination and, prior to barbecuing meats, trimming excess fat to
    minimize "flare-ups" and cooking in a fashion that prevents contact
    of the food with any flames.  Measures that can be taken by the food
    industry include conversion to indirect heating for drying foods,
    switching to non-coal-fired roasters (e.g., for roasting coffee
    beans), using protective coverings (e.g., cellulose casing) when
    smoking foods conventionally, and ensuring compliance with limits
    for PAHs in food additives specificed by national and or
    international bodies.  The Committee urged the application of these
    measures to minimize contamination of food with polycyclic aromatic
    hydrocarbons, including benzo [a]pyrene.


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    See Also:
       Toxicological Abbreviations
       BENZO[a]PYRENE (JECFA Evaluation)
       Benzo[a]pyrene (IARC Summary & Evaluation, Volume 32, 1983)