111 

## 1. Exposure Data

1.1 Identification of the agent

Chem. Abstr. Services Reg. No .: 50-32-8

Chem. Abstr. Name : Benzo[ a ]pyrene
IUPAC Systematic Name : Benzo[ a ]pyrene
Synonyms : B a P; benzo[ def ]chrysene; 3,4-benzopyrene*; 6,7-benzopyrene*;
benz[ a ]pyrene; 3,4-benz[ a ]pyrene*; 3,4-benzpyrene*; 4,5-benzpyrene* (*alternative numbering conventions)

1

2

3

4

5 6 7 8 9 10 11 12



### C

20 

### H

12 

### Relative molecular mass: 252.31
Description : Yellowish plates, needles from benzene/methanol; crystals may be mono

clinic or orthorhombic Boiling-point : 310–312 °C at 10 mm Hg Melting-point : 179–179.3 °C; 178.1 °C Spectroscopy data : Ultraviolet/visual,
infrared, fluorescence, mass and nuclear magnetic-resonance spectral data have been reported Water solubility : 0.00162 mg/L at 25 °C; 0.0038 mg/L at 25 °C log K

ow



### (octanol–water) : 6.35 Henry’s Law Constant : 0.034 Pa m 3 

### /mol at

20 °C

From IARC (2010 )



## 1.2 Occurrence and exposure

Benzo[ a ]pyrene and other polycyclic aromatic hydrocarbons (PAHs) are widespread environ

mental contaminants formed during incomplete combustion or pyrolysis of organic material.
These substances are found in air, water, soils and sediments, generally at trace levels except near their sources. PAHs are present in some foods and in a few pharmaceutical products based on coal tar that are applied to the skin. Tobacco smoke contains high concentrations of PAHs
( IARC, 2010 ).



## 1.2.1 Exposure of the general population

The general population can be exposed to
benzo[ a ]pyrene through tobacco smoke, ambient air, water, soils, food and pharmaceutical prod

ucts. Concentrations of benzo[ a ]pyrene in 

# BENZO[ a ]PYRENE

Benzo[ a ]pyrene was considered by previous IARC Working Groups in 1972, 1983, and 2005

( IARC, 1973

, 1983

, 2010 ). Since that time new data have become available, which have been incorporated in this Monograph , and taken into consideration in the present evaluation.

### IARC MONOGRAPHS – 100F

sidestream cigarette smoke have been reported to range from 52 to 95 ng/cigarette — more than

three times the concentration in mainstream smoke. Major sources of PAHs in ambient air
(both outdoors and indoors) include residential and commercial heating with wood, coal or other biomasses (oil and gas heating produce much lower quantities of PAH), other indoor sources such as cooking and tobacco smoke, and outdoor sources like motor-vehicle exhaust (especially from diesel engines), industrial emissions and forest fires. Average concentrations of individual PAHs in the ambient air in urban areas typically range from 1 to 30 ng/m 3 

### ; however, concentra

tions up to several tens of nanograms per cubic metre have been reported in road tunnels, or in large cities that make extensive use of coal or other biomass as residential heating fuel. Estimates of PAH intake from food vary widely, ranging from a few nanograms to a few micro

grams per person per day. Sources of PAHs in the diet include barbecued/grilled/broiled and
smoke-cured meats; roasted, baked and fried foods (high-temperature processing); bread, cereals and grains (at least in part from gas/ flame-drying of grains); and vegetables grown
in contaminated soils, or in areas with surface contamination from atmospheric PAH fall-out
( IARC, 2010 ).



## 1.2.2 Occupational exposure

Occupational exposure to PAHs occurs primarily through inhalation and via skin contact. Monitoring by means of ambient air-sampling or personal air-sampling at the workplace, to determine individual PAHs, sets of PAHs or surrogates (e.g. coal-tar pitch vola

tiles) has been used to characterize exposure via inhalation; more recently, biological monitoring methods have been applied to characterize the uptake of certain specific PAHs (e.g. benzo[ a ] pyrene) to be used as biomarkers of total expo

sure ( IARC, 2010 ). Industries where occupational exposure to
benzo[ a ]pyrene has been measured and reported include: coal liquefaction, coal gasification, coke production and coke ovens, coal-tar distillation, roofing and paving (involving coal-tar pitch), wood impregnation/preservation with creosote, aluminium production (including anode manu

facture), carbon-electrode manufacture, chimney sweeping, and power plants. Highest levels of exposure to PAHs are observed in aluminium production (Söderberg process) with values up
to 100 μg/m 3 

### . Mid-range levels are observed in roofing and paving (e.g. 10−20 μg/m 3 

### ) and the lowest concentrations (i.e. at or below 1μg/m 3 

### ) are observed in coal liquefaction, coal-tar distil

lation, wood impregnation, chimney sweeping and power plants ( IARC, 2010 ).



## 2. Cancer in Humans

No epidemiological data on benzo[ a ]pyrene alone were available to the Working Group.



## 3. Cancer in Experimental Animals

Benzo[ a ]pyrene was considered by three previous Working Groups ( IARC, 1973

, 1983

, 2010 ).

In IARC Monograph Volume 3 ( IARC,

1973 ) it was concluded that benzo[ a ]pyrene produced tumours in all species tested (mouse, rat, hamster, guinea-pig, rabbit, duck, newt, monkey) for which data were reported following exposure by many different routes (oral, dermal,
inhalation, intratracheal, intrabronchial, subcu

taneous, intraperitoneal, intravenous). Benzo[ a ] pyrene had both a local and a systemic carcino

genic effect, was an initiator of skin carcinogen

esis in mice, and was carcinogenic in single-dose studies and following prenatal and transplacental

exposures.

112

### Benzo[ a ]pyrene

In IARC Monograph Volume 32 ( IARC, 1983 )

no evaluation was made of studies of carcino

genicity in experimental animals published since
1972, but it was concluded that there is sufficient

evidence for the carcinogenicity of benzo[ a ] pyrene in experimental animals. Carcinogenicity studies with administra

tion of benzo[ a ]pyrene by multiple route of exposure, reported after the initial evaluations, were subsequently reviewed in IARC Monograph
Volume 92 ( IARC, 2010 ) and are summarized
below ( Table 3.1 ). See Table 3.2 for an overview of malignant tumours induced in different animal

species.



## 3.1 Skin application

In several studies in which benzo[ a ]pyrene was applied to the skin of different strains of mice,benign (squamous cell papillomas and keratoacanthomas) and malignant (mainly squamouscell carcinomas) skin tumours were observed
( Van Duuren et al

. , 1973 ; Cavalieri et al

. , 1977

, 1988a ; Levin et al

. , 1977 ; Habs et al

. , 1980

, 1984 ; Warshawsky & Barkley, 1987 ; Albert et al

. , 1991 ; Andrews et al

. , 1991 ; Warshawsky et al

. , 1993 ). No skin-tumour development was seen in AhR −/− 

### mice that lacked the aryl hydrocarbon receptor, whereas the heterozygous and wild-type mice developed squamous-cell carcinomas of the skin
( Shimizu et al

. , 2000 ). In a large number of initiation–promotion
studies in mice, benzo[ a ]pyrene was active as an initiator (mainly of squamous-cell papillomas) when applied to the skin ( IARC, 2010 ).



## 3.2 Subcutaneous injection

In subcutaneous injection studies of benzo[ a ]pyrene, malignant tumours (mainly fibrosarcomas) were observed at the injection site in
mice ( Kouri et al

. , 1980 ; Rippe & Pott, 1989 ) and
rats ( Pott et al

. , 1973a

, b ; Rippe & Pott, 1989 ). No

fibrosarcomas were observed in AhR −/− 

### mice that lacked the aryl hydrocarbon receptor, whereas the heterozygous and wild-type mice did develop
these tumours ( Shimizu et al

. , 2000 ). In another study, male and female newborn Swiss mice that were given benzo[ a ]pyrene subcutaneously showed a significant increase in lung-adenoma incidence and multiplicity
( Balansky et al

. , 2007 ). A single study in 12 strains of hamsters resulted in sarcomas at the site of injection in
both sexes of all 12 strains ( Homburger et al

. , 1972 ).



## 3.3 Oral administration

After administration of benzo[ a ]pyrene by gavage or in the diet to mice of different strains
( Sparnins et al

. , 1986 ; Estensen & Wattenberg,

1993 ; Weyand et al

. , 1995 ; Kroese et al

. , 1997 ; Culp et al

. , 1998 ; Hakura et al

. , 1998 ; Badary

et al

. , 1999 ; Wijnhoven et al

. , 2000 ; Estensen

et al

. , 2004 ), increased tumour responses were observed in lymphoid and haematopoeitic tissues and in several organs, including the lung, forestomach, liver, oesophagus and tongue.
Oral administration of benzo[ a ]pyrene to

XPA

– /– 

### mice resulted in a significantly higher increase of lymphomas than that observed in similarly treated XPA +/– 

### and XPA +/+ 

### mice ( de V ries et al

. , 1997 ). Benzo[ a ]pyrene given by

gavage to XPA

– /– 

### / p53 +/– 

### double-transgenic mice induced tumours (mainly splenic lymphomas
and forestomach tumours) much earlier and at higher incidences than in similarly treated single transgenic and wild-type counterparts. These

cancer-prone XPA –/– 

### or XPA –/– 

### / p53 +/– 

### mice also developed a high incidence of tumours (mainly
of the forestomach) when fed benzo[ a ]pyrene in
the diet ( van O ostrom et al

. , 1999 ; Hoogervorst

et al

. , 2003 ).
Oral administration of benzo[ a ]pyrene by gavage to rats resulted in an increased incidence of mammary gland adenocarcinomas ( el-Bayoumy

et al

. , 1995 ).

113

### IARC MONOGRAPHS – 100F

114Table 3.1 Carcinogenicity studies of benzo[a]pyrene in experimental animalsSpecies, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsSkin applicationMouse, Swiss ICR/Ha (F)52 wkVan Duurenet al.(1973)0 (untreated), 0 (vehicle control),5 μg/animal, 3 × /wk, 52 wk50/groupSkin T: 0/50, 0/50, 23/50 (46%; 13 P; 10 C)+NR (acetone)Mouse, Swiss (F)38–65 wkCavalieriet al. (1977)0 and 0.396 μmol [0.1 mg] peranimal, twice/wk, 30 wk40/groupSkin T: 0% [0/29], 78.9% [30/38] (7 P, 7 K, 36 C, 1malignant Schwannoma)+99% (acetone)Mouse, C57BL/6J (F)60 wkLevinet al. (1977)Experiment 1 and 2: 0 (DMSO/acetone), 0.02 [5.28 μg], 0.1[26.43 μg], 0.4 [105.75 μg] μmol/animal, once/2 wk, 60 wk (highdose given in two paintings, 30 minapart)Experiment 3: 0 (acetone/NH4OH),0.025 [6.6 μg], 0.05 [13.21 μg], 0.1[26.43 μg] μmol/animal, once/2 wk,60 wk30/groupSkin T (mainly SCC):Experiment 1–0%, 0%, 38% (13 T), 100% (44 T)Experiment 2–0%, 4% (1 T), 50% (15 T), 100% (40T)Experiment 3– 0%, 7% (2 T), 59% (20 T), 91% (24T)+NR (DMSO/acetone (1:3) oracetone/NH4OH (1 000:1))Effective number of animalsnot clearly specifiedAt most, seven animals/groupdied prematurely without askin tumour.Mouse, NMRI (F)63–109 wkHabset al. (1984)0, 2, 4 μg/animal, twice/wk20/groupSkin T: 0/20, 9/20 (45%; 2 P, 7 C), 17/20 (85%; 17C)+> 96% (acetone)Mouse C3H/HeJ (M)99 wkWarshawsky &Barkley (1987)0 (untreated), 0 (vehicle control) or12.5 μg/animal, twice/wk50/groupSkin T: 0/50, 0/50, 48/50 (96%; 47 C, 1 P)+99.5% (acetone)Mouse, Swiss (F)42 wkCavalieriet al. (1988a)0, 0.1 [26.4 μg], 0.4 [105.7 μg] μmol/animal, twice/wk, 20 wk30/groupSkin T incidence: 0/30, 26/29 (90%; SGA, 3 P, 23SCC), 26/30 (90%; 2 P, 26 SCC)+Purified [NR] (acetone)

### Benzo[ a ]pyrene

115Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsMouse, C3H/fCum(M) Experiment 1:15 mo Experiment 2:18 mo Experiment 3:18 moKouriet al. (1980)Experiment 1: 0 (trioctanoincontrol), 0 (DMSO control), 0.9μmol [0.23 mg] in trioctanoin orDMSO,FibroS at injection site:Experiment 1–0/16, 0/20, 15/18 (83%), 12/19 (63%)+Pure (trioctanoin, DMSO)Experiment 2: 0 (trioctanoincontrol), 0 (DMSO control), 0.9μmol [0.23 mg] in trioctanoin or inDMSO,Experiment 2–0/20, 0/18, 14/18 (78%), 7/19 (37%)Experiment 3: 0 (trioctanoin/DMSO, 100:1), 0.9 μmol [0.23 mg]in trioctanoin/DMSO (100:1), 1 ×20 or 40/groupExperiment 3–0/20, 36/40 (90%)Mouse, NR (F)78 wkRippe & Pott (1989)0, 10, 100 μg/animal, 1 × NR/groupS at injection site: 1/30 (3%), 13/30 (43%), 20/30(67%)+NR (tricaprylin)Mouse, Swiss(newborn) (M, F)75–200 dBalanskyet al. (2007)0 and 1.0 mg/animal, 1 ×12–15 M/group, 12–15 F/groupLung A: M – 0/15, 9/12; F – 0/15, 11/12P< 0.001Pure (olive oil)Rat, Wistar (F)~530 dPottet al. (1973a)0, 33, 100, 300, 900, 2 700 μg/animal, 1 ×50/groupT (mainly fibroS) at injection site [incidencederived from dose–response curves]: 2/50 (~4%),4/50 (~8%), 7/50 (~14%), 23/50 (~46%), 35/50(~70%), 38/50 (~76%)+NR (tricaprylin)Rat, NR (F)132 wkRippe & Pott (1989)0 and 1 mg, 1 ×NR/groupS at injection site: 0/24 (0%), 20/24 (83%)+NR (tricaprylin)Rat, NR (F)132 wkRippe & Pott (1989)0 and 15 mg, 1 ×NR/groupS at injection site: 1/24 (4%), 19/24 (79%)+NR (DMSO)Table 3.1 (continued)

### IARC MONOGRAPHS – 100F

116Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syrian, RB(randomly bred),BIO inbred strainsdesignated as: 1.5,4.22, 4.24, 7.88, 12.14,15.16, 45.5, 54.7, 82.73,86.93, 87.20 (M, F)53 wkHomburgeret al.(1972)500 μg/animal, 1 ×25 M/group, 25 F/groupFibroS at injection site:RB–M, 4/25 (16%); F, 6/23 (26%)1.5–M, 5/25 (20%); F, 4/23 (17%)4.22–M, 3/25 (12%); F, 8/25 (32%)4.24–M, not tested; F, 9/25 (36%)7.88–M, 13/25 (52%); F, 5/23 (23%)12.14–M, 3/25 (12%); F, 9/22 (41%)15.16–M, 9/25 (36%); F, 16/25 (64%)45.5–M, 12/25 (48%); F, 7/15 (47%)54.7:–M, 5/25 (20%); F, 5/25 (20%)82.73–M, 4/21 (19%); F, 4/24 (17%)86.93–M, 9/25 (36%); F, 8/25 (32%)87.20–M, 16/25 (64%); F, 11/25 (42%)+NR (tricaprylin)No subcutaneous T observedin historical controlsOral administrationMouse, A/J (F)260 dWeyandet al. (1995)0, 16, 98 ppm (total dose; 0, 11, 67mg) in the diet30/groupLung T: 4/21 (19%; 4 A; 0.19 ± 0.09 A/animal),9/25 (36%*; 7 A, 2 AdC; 0.48 ± 0.14 T/animal),14/27 (52%*; 14 A; 0.59 ± 0.12 A/animal)Forestomach T: (0%) 0/21, (5/25) (20%; 3 P, 2 C;0.24 ± 0.11** T/animal), 27/27 (100%**; 13 P, 14 C;4.22 ± 0.41**)*P< 0.05**P< 0.001NR (gel diet)Mouse B6C3F1 (F)2 yrCulpet al. (1998)0 (acetone control diet), 5 ppm, 25ppm, 100 ppm in the diet48/groupLiver (A): 2/48 (4%), 7/48 (15%), 5/47 (11%), 0/45(0%)Lung (A and/or C): 5/48 (10%), 0/48 (0%), 4/45(9%), 0/48 (0%)Forestomach (P and/or C): 1/48 (2%), 3/47 (6%),36/46 (78% ), 46/47 (98%** **)Oesophagus (P and/or C): 0/48 (0%), 0/48 (0%),2/45 (4%), 27/46 (59%**)Tongue (P and/or C): 0/48 (0%), 0/48 (0%), 2/46(4%), 23/48 (48%***)Larynx (P and/or C): 0/35 (0%), 0/35 (0%), 3/34(9%), 5/38 (13%*)*P< 0.014**P< 0.0014***P< 0.0003****P< 0.0000198.5% (acetone)Mouse, Swiss albino,inbred (F)27 wkBadaryet al. (1999)0 and 1 mg/animal by gavage,twice/wk, 4 wk10/group0, 10/10 (100%) (forestomach P; multiplicity,7.11 ± 1.05)+Highest purity grade (cornoil)Drinking-water contained0.005% ethanolTable 3.1 (continued)

### Benzo[ a ]pyrene

117Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsMouse, CSB–/–or wildtype (CSP±or CSB+/+)(M, F)52 wkWijnhovenet al.(2000)0 and 13 mg/kg bw by gavage, 3 × /wk, 13 wk6–18 M/group, 6–13 F/groupWild-type: 5/27 (14 M, 13 F; 19%; 4 bronchioloalveolar A, 2 lymphoma), 17/29* (18 M, 11 F; 59%;6 bronchiolo-alveolar A, 10 forestomach P, 2forestomach SCC, 2 histiocytic S, 2 hepatocellularA, 1 intestinal AdC, 1 skin P)CSB–/–: 0/13 (6 M, 7 F), 7/12** (6 M, 6 F; 58%; 2bronchiolo-alveolar A, 2 uterine S, 1 forestomachSCC, 1 intestinal AdC, 1 skin histiocytic S)*P= 0.0023**P= 0.0017NR (soya oil)Rat, Crl:CD(SD)BR (F)49 wkel-Bayoumyet al.(1995)0 and 50 μmol/animal, once/wk,8 wk by gavage30/groupMammary T incidence: 11/30 [37%] [incidencenot clearly specified] (8 desmoplastic A, 2 A, 1AdC), 29/30 (96.7%; 8 fibroA**, 17 desmoplasticA*, 7 A, 22 AdC**)Numbers of mammary T: controls,14 desmoplastic A, 2 A, 1 AdC; treated animals,14 fibroA*, 35 desmoplastic A, 11 A, 56 AdC***P< 0.05**P< 0.0199% (trioctanoin)Intraperitoneal injectionMouse, B6C3F1; C3A/JF1 (M, F)90 wk or lifetimeVesselinovitchet al.(1975a,b)0, 75, 150 μg in 10 μL/g bw, 1 × at 1,15, 42 d of age30–63/group, 96–100 controls/groupB6C3F1 mice (all ages combined):Liver T (A and hepatocellular C)–M, 1/98 (1%), 69/162 (43%), 81/165 (49%); F, 0/96(0%), 7/147 (5%), 10/126 (8%)Lung T (A and AdC)–M, 7/98 (7%), 57/162 (35%), 73/165 (44%); F, 2/90(2%), 53/147 (36%), 50/126 (40%)Forestomach T (P and SCC)–M, 0/98 (0%), 39/162 (24%), 64/165 (39%); F, 0/96(0%), 22/147 (15%), 40/126 (32%)Lymphoreticular T (mainly reticulum-cell S)–M, 2/98 (2%), 104/314 (33%) (high- and low-dosegroups combined); F, 2/96 (2%), 148/281 (53%)(high-and low-dose groups combined)+NR (trioctanoin)Table 3.1 (continued)

### IARC MONOGRAPHS – 100F

118Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsVesselinovitchet al.(1975a,b)Contd.C3A/JF1 mice (all ages combined):Liver T (A and hepatocellular C)–M, 3/97 (3%), 30/148 (20%), 33/137 (24%); F, 0/100(0%), 1.3% 2/126 (1.3%), 2/153 1.3%)Lung T (A and AdC)–M, 49/97 (49%), 1 438/148 (93%), 125/137 (91%); F,26/100 (26%), 115/126 (91%), 141/153 (92%)Forestomach T (P and SCC)–M, 0/97 (0%), 18/148 (12%), 42/137 (31%); F, 0/100(0%), 18/126 (14%), 31/153 (20%)Lymphoreticular T (mainly reticulum-cell S)–M, 0/97 (0%), 26/285 (9%); F, 2/100 (2%), 50/278(18%) (high- and low-dose groups combined)+Mouse, CD-1 (M, F)1 yrWislockiet al. (1986)0 and 560 nmol [148 μg] (total dose;given as 1/7, 2/7, 4/7 on PND 0, 8,15)37 M/group, 27 F/groupLiver T: M, 2/28 (7%; 2 A), 18/37* (49%; 11 A, 7C*); F, no liver T foundLung T: M, 1/28 (4%; 1 A), 13/37** (35%; 13 A); F,0/31, 13/27** (48%) (13 A)Malignant lymphoma: M, 1/28 (4%), 2/37 (5%); F,1/31 (3%), 4/27 (15%)*P< 0.005**P< 0.05> 99% (DMSO)Mouse, CD-1 (M, F)52 wkLavoieet al. (1987)0 and 1.1 μmol [290 μg] (total dose;given as 1/6, 2/6, 4/6 on PND 1, 8,15)17 M/group, 14–18 F/groupLiver T: M, 1/17 (6%; 1 H), 13/17* (76%; 9 hepaticA, 4 H); F, 0/18, 0/14Lung A: M, 0/17, 14/17* (82%); F, 0/18, 9/14**(64%)*P< 0.005**[P< 0.0005]> 99% (DMSO)Mouse, Swiss-WebsterBLU: Ha(ICR) (M, F)26 wkBusbyet al. (1989)0 and 59.5 μg (total dose; given as8.5, 17, 34 μg on PND 1, 8, 15)NR/groupLung T: M, 12/91 (13%; 12 A, 1 AdC; 0.15 ± 0.04 T/mouse), 13/28 (46%; 13 A; 0.71 ± 0.19 A/mouse); F,7/101 (7%; 7 A; 0.08 ± 0.03 A/mouse), 18/27 (67%;18 A, 1 AdC; 1.19 ± 0.21 T/mouse)+> 99% (DMSO)statistics NRMouse, NR, newborn(M, F)30 wkRippe & Pott (1989)0, 10, 100 μg/animal, 1 ×NR/groupLung T: 13% [5/38] (0.13 T/animal), 16% [5/31](0.23 T/animal), 64% [21/33] (2.52 T/animal)+NR (saline solution + 1%gelatine + 0.4% Tween 20)Type of lung tumour NR;statistics NRMouse, A/J (F)260 dWeyandet al. (1995)0 (untreated), 0 (vehicle control),1.79 mg/animal, 1 ×29–30/groupLung T: 7/30 (23%; 7 A; 0.27 ± 0.12 A/animal),11/30 (37%; 11 A; 0.43 ± 0.11 A/animal), 29/29*(100%; 27 A, 2 AdC; 15.8 ± 1.28** T/animal);forestomach T: 0/30 (0%), 0/30 (0%), 24/29** (83%;15 P, 9 C; 1.83 ± 0.25** T/animal)*P< 0.05**P< 0.001NR (tricaprylin)Table 3.1 (continued)

### Benzo[ a ]pyrene

119Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsMouse, B6C3F1,infant(M, F)26 wk, 39 wk, 52 wkRodriguezet al. (1997)0 (untreated), 0 (vehicle controls),125, 250, 375 μg/7 g bw, 1 ×> 30 M/group, > 30 F/groupLiver T (M):At wk 26: 0/41, 0/58, 0/29, 0/25, 3/34 (9%;multiplicity, 1.0); at wk 39: 0/34, 0/59, 6/26 (23%;multiplicity, 1.0), 13/34 (38%; multiplicity, 1.9),15/23 (65%; multiplicity, 1.9); at wk 52: 4/64 (6%;multiplicity, 1.0), 3/63 (5%; multiplicity, 1.0), 13/29(45%; multiplicity, 1.8), 14/27 (52%; multiplicity,2.2), 19/24 (79%; multiplicity, 2.5)No liver T in F+NR (corn oil)No forestomach tumoursMouse, CD-1 (M)12 moVon Tungelnet al.(1999)0, 100, 400 nmol [26, 111 μg]/animal (total dose; given as 1/7, 2/7,4/7 on PND 1, 8, 15)24/groupLiver T: 3/20 (15%; 1 A, 2 C; 1.7 T/liver section),5/21 (24%; 4 A, 1 C; 1.5 T/liver section), 9/20 (45%;7 A*, 2 C; > 2.3 T/liver section)Lung T: 4/20 (20%; 4 A; 1.0 T/lung section), 1/21(5%; 1 A; 1.0 T/lung section), 9/20 (45%; 7 A, 2 C;1.9 T/lung section)*P= 0.0234> 99% (DMSO)Rat, Wistar (F)~112 wkRolleret al. (1992)0 and 5 mg/animal, 1 ×NR/groupAbdominal mesothelioma and S: 3/41 (7.3%),33/37 (89.2%)+NR (3:1 mixture oftricaprylin/ beeswax)Limited reportingRat, Wistar (F)~116 wkRolleret al. (1992)5 mg/animal, 1 ×NR/groupAbdominal mesothelioma and S: 19/38 (50%);historical controls, 11/369 (3%)+NR (saline solution)No control; limited reportingof tumour dataInhalationHamster, Syriangolden (M)LifetimeThyssenet al. (1981)0, 2.2, 9.5, 46.5 mg/m3, 4.5 h/d, 7 d/wk, 10 wk; thereafter 3 h/d, 7 d/wk(total average doses: 0, 29, 127, 383mg/animal)24/group (+ animals added duringthe study)Respiratory tract T:(polyps, P, SCC)–0/27, 0/27, 34.6% [9/26; 3 nasal, 8 laryngeal,1 tracheal], 52% [13/25; 1 nasal, 13 laryngeal, 3tracheal; no bronchogenic T]Upper digestive tract T:(polyps, P, SCC)–0/27, 0/27, 26.9% [6/26; 6 pharyngeal,1 forestomach], 56% [14/25; 14 pharyngeal,2 oesophageal, 1 forestomach]+NR (0.1% saline solution);particle size, > 99% diameter0.2–0.5 μm, > 80% diameter0.2–0.3 μmSurvival decreased for highdose-exposed animals (59wk)vsother groups (96 wk).Table 3.1 (continued)

### IARC MONOGRAPHS – 100F

120Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsIntrapulmonaryinjectionRat, OM (F)64 (high-dosegroup)–133 wk(untreated controls)Deutsch-Wenzelet al.(1983)0 (untreated), 0 (vehicle control),0.1, 0.3, 1.0 mg/animal, 1 ×35/groupLung T: [0/35] (0%), [0/35] (0%), [10/35] (28.6%) (4epidermoid C; 6 pleomorphic S), [23/35] (65.7%)(21 epidermoid C; 2 pleomorphic S), [33/35](94.3%) (33 epidermoid C)+99.1% (1:1 mixture of beeswaxand trioctanoin)Rats, F344/NSlc (M)104 wkIwagawaet al. (1989)0, 0.03, 0.1, 0.3, 1.0 mg/animal, 1 ×NR/groupLung T: 0/40, 1/29 (3%; 1 undifferentiated T), 7/30(23%; 6 SCC, 1 undifferentiated T), 22/29 (76%; 20SCC, 2 undifferentiated T), 9/13 (69%; 9 SCC)+NR (1:1 mixture of beeswax/tricaprylin)Rat, Osborne-Mendel(F)134 wk (low-dosegroup)–140 wk (vehiclecontrols)Wenzel-Hartunget al.(1990)0 (untreated), 0 (vehicle control),30, 100, 300 μg/animal, 1 ×35/groupLung T: [0/35] (0%), [0/35] (0%), [3/35] (8.6%; 3SCC), [11/35] (31.4%; 11 SCC), [27/35] (77.1%; 27SCC).+99.6% (beeswax/trioctanoinmixture of varyingcomposition)SCC predominantlykeratinizedRat, F344/DuCrj (M)100 wkHorikawaet al. (1991)0, 50, 100, 200 μg/animal, 1 ×9–10/groupLung T: 0/19, 0/10, 3/10 (30%; 2 SCC, 1 AdSC), 4/9(44.4%; 3 SCC, 1 undifferentiated T)+NR (1:1 mixture of beeswax/tricaprylin)IntratrachealadministrationRat, Wistar-WU/Kisslegg (F)124–126 wkPottet al. (1987)0 and 1 mg/animal, once/wk, 20 wkNR/groupLung T: 0/40, 7/36 (19%; 1 A, 5 SCC, 1 mixedAdC/SCC)+NR (0.9% saline solution)Rat, Sprague-Dawley(M, F)Controls, 131 wk;treated animals,112 wkSteinhoffet al. (1991)0 and 0 (physiological saline),7 mg/kg bw/instillation(physiological saline with Tween60), once/2 wk, 44 wk20 or 50/groupM: 0/50, 0/50, 19/20 (95%; 19 malignant lung T)F: 0/50, 0/50, 19/20 (95%; 18 malignant, 1 benignlung T)+NR (physiological salinesolution with or withoutTween 60)Limited histologyTable 3.1 (continued)

### Benzo[ a ]pyrene

121Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (M, F)78 wkFeron (1972)0 (only for M), 1 mg/animal, once/wk, 36 wk35/groupRespiratory tract T/adenomatoid lesions:M–6/27 (22%; 1 tracheal P, 5 pulmonaryadenomatoid lesion), 19/29 (66%; 1 tracheal P,17 SCC, 26 pulmonary adenomatoid lesion, 5 A,1 AdC, 1 SCC)F–22/27 (81%; 1 laryngeal SCC, 16 tracheal SCC,2 bronchial A, 1 AdC, 21 pulmonary adenomatoidlesion, 8 A, 1 AdC)+> 99% (0.9% saline solution)No female controls; StatisticsNRHamster, Syriangolden (M)78 wkFeronet al. (1973)0, 0.0625, 0.125, 0.25, 0.5, 1.0 mg/animal, once/wk, 52 wk30/groupRespiratory tract T:0/29, 3/30 (10%; 3 tracheal P, 1 pulmonary A),4/30 (13%; 1 tracheal P, 4 pulmonary A), 9/30(30%; 5 tracheal P, 7 pulmonary A), 25/29 (86%;2 tracheal polyp, 9 P, 5 SCC, 1 AdSC, 1 fibroS,2 bronchial polyp, 1 P, 2 SCC, 1 AdSC), 26/28(93%; 6 tracheal P, 11 SCC, 1 AdSC, 1 bronchialpolyp, 2 P, 4 SCC, 2 AdSC, 4 AdC, 1 anaplasticC, 16 pulmonary A, 4 SCC, 3 AdSC, 1 AdC, 2anaplastic C)+NR (0.9% saline solution)Hamster, Syriangolden (M, F)M, 67–88 wk; F, 60–88wkHenryet al. (1973)0, 13.3–15.5 mg/animal, once/wk,8 wk50/group, 25 controls/groupRespiratory tract T:Controls–1 tracheal polyp, 6 pulmonary bronchiolaradenomatoid lesions/47 animalsTreated animals–26/65 (40%; 1 nasal polyp; 6 laryngeal polyps,1 P, 1 A, 1 AdC, 7 tracheal polyps, 1 AdC, 1SCC, 1 fibroS, 2 bronchial AdC, 13 pulmonarybronchiolar adenomatoid lesion, 3 A, 5 AdC,1 SCC, 2 anaplastic C, 1 mixed C, 1 myelogenousleukaemia, 1 neurofibroS)T at other sites:Controls–1 renal ATreated animals–3 blast-cell leukaemia, 2 adrenocortical A, 1 renalAdC, 1 oesophageal fibroS, 1 haemangioma+NR (0.5% gelatine in 0.9%saline solution)Tumour data for M and Fcombined; statistics NRTable 3.1 (continued)

### IARC MONOGRAPHS – 100F

122Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (M, F)60 wkKobayashi (1975)0 and 1 mg/animal, once/wk, 30 wk20–32 M/group, 20–28 F/groupRespiratory tract T:M–0/20, 11/26 (42.3%; 1 laryngeal polyp, 1tracheal polyp, 1 P, 1 bronchial SCC, 9 lung A, 7AdC, 3 SCC, 1 anaplastic C, 2 AdSC)F–0/20, 14/26 (53.8%; 1 laryngeal P, 2 trachealpolyps, 1 bronchial SCC, 10 lung A, 3 AdC, 1SCC)+NR (0.9% saline)Hamster, Syriangolden (M, F)78 wkKruysse & Feron(1976)0 (untreated), 0 (vehicle controls),1 mg/animal, once/2 wk, 52 wk17 or 40/groupRespiratory tract T:M–0/40, 0/40, 13/14 (93%; 2 laryngeal P, 1 SCC, 4tracheal P, 3 SCC, 1 anaplastic C, 1 S, 1 bronchialSCC, 1 AdC, 5 pulmonary A, 1 AdC)F–0/40, 0/40, 7/12 (58%; 2 tracheal P, 3 SCC,1 bronchial P, 5 pulmonary A)+> 99% (saline solution)Hamster, Syriangolden (M)100 wkSellakumaret al.(1976)0 (untreated), 3 mg/animal, once/wk, 10 wk48/groupRespiratory tract T:0/48, 7/48 (15%; 2 laryngeal P, 4 tracheal P, 1 lungA)T at other sites:6/48 (13%; 3 forestomach P, 2 lymphoma, 1anaplastic C), 26/48 (54%; 21 forestomachP, 1 skin melanoma, 1 liver haemangioma,1 adrenocorticoA, 3 adrenocorticoC)+> 99% (0.9% saline solution)Hamster, Syriangolden (M, F)Experiment 1: up to89 wk for M and 70 wkfor FExperiment 2: up to83 wk for M and 68 wkfor FKetkaret al. (1977)Experiment 1:0, 4, 8, 16 mg in 0.9% salinesolution/animal, 1 ×30/groupRespiratory tract T:Experiment 1–M 0/24, 3/30 (10%; 1 laryngeal P, 1 tracheal P,1 lung S), 5/28 (18%; 1 laryngeal SCC, 1 tracheal P,4 lung S), 4/27 (15%; 3 tracheal P, 1 lung A, 1 S)F 0/28, 3/29 (10%; 1 tracheal P, 2 lung A), 1/30(3%; 1 lung A), 3/28 (13%; 1 laryngeal P, 2 lung A)+97% (0.9% saline solution orTris buffer)Experiment 2:0, 4, 8, 16 mg in Tris buffer/animal, 1 ×30/groupExperiment 2–M 0/27, 5/24 (21%; 1 tracheal P, 5 lung A), 13/25(52%; 1 laryngeal P, 7 tracheal P, 4 lung A, 3 AdC),8/27 (30%; 2 laryngeal P, 1 SCC, 3 tracheal P, 3lung A)F 0/27, 3/27 (11%; 2 tracheal P, 1 lung AdC), 2/29(7%; 2 tracheal P), 8/29 (28%; 1 laryngeal P, 4tracheal P, 5 lung A)+Table 3.1 (continued)

### Benzo[ a ]pyrene

123Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (M, F)81 wkFeron & Kruysse(1978)0 (untreated), 0 (vehicle controls),0.35, 0.7 mg/animal, once/wk, 52wk15 or 30/groupRespiratory tract T:M–0/30 (untreated and vehicle controlscombined), 4/29 (14%; 2 tracheal P, 1 bronchialP, 2 pulmonary A), 19/30 (63%; 1 laryngeal P, 5tracheal P, 1 SCC, 1 anaplastic C, 1 S, 2 bronchialP, 1 AdC, 11 pulmonary A, 2 AdC, 1 SCC, 1anaplastic C)F–0/28 (untreated and vehicle controlscombined), 3/27 (11%; 1 laryngeal P, 1 bronchial P,1 pulmonary A), 7/24 (29%; 1 tracheal P, 2 SCC, 1bronchial AdC, 5 pulmonary A)+> 99% (0.9% saline solution)Statistics NRHamster, Syriangolden (M, F)Average survival upto 41 wk for M and 35wk for FKetkaret al. (1978)0, 0.1, 0.33, 1.0 mg/animal, once/wk30/groupRespiratory tract T:M–0/29, 5/26 (19%; 5 bronchiogenic A), 7/29(24%; 5 tracheal P, 2 bronchiogenic A), 6/27 (22%;5 tracheal P, 2 bronchiogenic A)F–0/30, 12/30 (40%; 1 tracheal P, 1 SCC, 10bronchiogenic A), 10/28 (36%; 7 tracheal P, 5bronchiogenic A, 1 SCC), 6/30 (20%; 3 tracheal P,3 bronchiogenic A, 3 SCC)+97% (10% bovine serumalbumin)Average survival time muchlower in the high-dose groupthan in the other groupsHamster, Syriangolden (M, F)Lifetime, up to 90 wkStenbäck & Rowland(1978)0, 3 mg large particles, 3 mg smallparticles/animal, once/wk, 18 wk48 (M + F)/groupRespiratory tract T (M + F combined): 0/46,31/47 (66%; 5 laryngeal P, 12 tracheal P, 20 SCC,2 unspecified T, 2 bronchial P, 9 SCC, 3 A, 2anaplastic C), 5/46 (11%; 1 laryngeal P, 1 SCC,4 tracheal P)+99.4% (0.9% saline solution);particle size by weight:large-98% < 30 μm, 90% < 20μm, 36% < 10 μm, 10% < 5μm; small-98% < 10 μm, 79%< 5 μm, 5% < 1 μmHamster, Syriangolden (M)Average survival up to88 wkKetkaret al. (1979)0 (untreated), 0 (vehicle controls),0.125, 0.25, 0.5, 1.0 mg/animal,once/wk30/groupRespiratory tract T:0/29, 0/28, 9/29 (31%; 2 laryngeal polyps/P,1 tracheal P, 1 SCC, 2 lung A, 2 SCC, 5 AdC),24/29 (83%; 1 nasal SCC, 2 laryngeal polyps/P, 4tracheal P, 9 SCC, 5 lung A, 5 SCC, 11 AdC), 19/29(66%; 1 laryngeal P, 2 SCC, 5 tracheal P, 11 SCC,7 lung SCC, 2 AdC), 9/29 (31%; 1 laryngeal P, 1SCC, 1 tracheal P, 5 SCC, 1 lung A, 4 SCC)P< 0.001, alltreated groups97% (Tris buffer + 0.9%saline solution); particlesize: majority < 10 μm butparticles up to 80 μm alsopresentAverage survival in twohighest-dose groups muchlower than that in the othergroups due to many earlydeaths from pulmonarylesions other than tumoursTable 3.1 (continued)

### IARC MONOGRAPHS – 100F

124Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (M, F)105 wkFeronet al. (1980)0 (untreated), 0 (gelatine in 0.9%saline), 0.5, 1.0 mg fine particles,0.5, 1.0 mg coarse particles, 1.0 mgwide-range particles/animal, once/wk, 52 wk30–35/groupRespiratory tract T:M–0/29, 2/34 (6%; 2 laryngeal P), 7/34 (21%; 1laryngeal P, 6 tracheal P, 1 lung A), 6/31 (19%;2 laryngeal P, 1 tracheal P, 1 S,1 pulmonaryA), 13/31 (42%; 2 laryngeal P, 3 tracheal P, 9pulmonary A), 25/34 (74%; 2 laryngeal P, 9tracheal P, 4 SCC, 2 S, 1 pulmonary A, 1 AdC),23/34 (68%; 2 laryngeal P, 1 SCC, 6 tracheal P, 2SCC, 1 bronchial P, 1 SCC, 13 pulmonary A, 2AdC, 2 anaplastic C)F–0/28, 2/33 (6%; 1 tracheal P, 1 pulmonaryA), 2/34 (6%; 1 bronchial P, 1 A), 5/32 (16%;1 laryngeal P, 2 tracheal P, 3 pulmonary A), 9/32(28%; 2 laryngeal P, 5 tracheal P, 6 pulmonary A),19/32 (31%; 4 tracheal P, 1 SCC, 1 S, 1 bronchial P,7 pulmonary A, 1 AdC), 11/34 (34%; 1 laryngealP, 3 tracheal P, 2 bronchial P, 7 pulmonary A,1 AdC)+NR; particles size by weight:fine, 77% < 5.2 μm, 60%< 3.9 μm; coarse, 77% < 42μm, 3% < 16 μm; wide-range,72% < 30 μm, 19% < 10 μm(gelatine in 0.9% salinesolution)Statistics NRHamster, Syriangolden (M)129 wkGodleskiet al. (1984)0 and 5 mg/animal, once/wk, 15 wk80/groupMalignant T: 4/80 (5%; 1 multicentricundifferentiated lung C, 3 lymphoma), 25/80*(31%; 9 SCC, 2 undifferentiated C of therespiratory tract, 5 lymphoma, 1 SCC, 2 AdCof the gastrointestinal tract, 2 soft-tissue T, 1hepatoma, 2 mouth SCC, 1 skin C)*P <0.001> 99% (0.5% gelatine in 0.9%saline solution)Intratracheal administration of combinations of benzo[**a**]pyrene and ‘particles/fibres’Rat, Sprague-Dawley(M, F)Up to 130 wkSteinhoffet al. (1991)0, (untreated), 0 (physiologicalsaline), 10–40 mg/kg bw Bayferrox130 (96.2% cubic α-Fe2O3),10–40 mg/kg bw Bayferrox 920(86.1% fibrous α-FeOOH), 7 mg/kgbw, 7 mg/kg bw + 10–40 mg/kg bwBayferrox 130, 7 mg/kg bw +10–40 mg/kg bw Bayferrox 920,~once/2 wk, 44–~130 wk20 or 50/groupLung T:M–0/50, 0/50, 0/50, 0/50, 19 malignant T in 20animals, 21 malignant and 1 benign T in 20animals, 17 malignant and 1 benign T in 20animalsF–0/50, 0/50, 0/50, 1 malignant and 1 benignT in 50 animals, 18 malignant and 1 benign Tin 20 animals, 16 malignant T in 20 animals,17 malignant and 2 benign T in 20 animals+NR (physiological salinesolution with or withoutTween 60);Bayferrox 130,Bayferrox 920Limited histologyTable 3.1 (continued)

### Benzo[ a ]pyrene

125Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (M, F)Lifetime (up to 140wk)Saffiottiet al. (1972)Experiment 10, 50 mg ferric oxide, 5 mg +45 mg ferric oxide, 12.5 mg +37.5 mg ferric oxide/ animal,1 × Experiment 2(2 groups/dose level)5 mg + 5 mg ferric oxide,10 mg + 10 mg ferric oxide, 15 mg +15 mg ferric oxide, once/wk, 15 wk23–110 M/group, 18–107 F/groupExperiment 1:Respiratory tract T–M 0/45, 0/101, 3/92 (3%; 1 tracheal polyp, 1P, 1 bronchial A), 3/27 (11%; 1 bronchial A, 1bronchogenic SCC, 1 anaplastic C)F 0/44, 0/89, 4/97 (4%; 1 tracheal polyp, 1 P, 1bronchiolar A, 1 AdC), 6/33 (18%; 1 bronchialP, 1 A, 2 bronchogenic SCC, 1 anaplastic C,2 bronchiolar A)Forestomach P–M 5/45 (11%; 6 T), 5/101 (5%; 5 T), 15/92 (16%; 35T), 8/27 (30%; 16 T)F 2/44 (5%; 2 T), 2/89 (2%; 3 T), 5/97 (5%; 5 T),4/33 (12%; 6 T)+NR (0.9% saline solution);ferric oxideExperiment 2:Respiratory tract T (M + F combined)–7/50 (14%; 2 tracheal P, 1 SCC, 1 bronchial P, 1 A,2 SCC, 1 anaplastic C, 1 pulmonary SCC),8/58 (14%; 2 tracheal polyps, 1 bronchial polyp,2 SCC, 2 AdC, 2 pulmonary A, 2 AdC), 17/61(28%; 2 tracheal polyps, 2 P, 5 SCC, 5 bronchialSCC, 1 pulmonary A, 1 SCC, 1 AdC, 1 anaplasticC), 25/60 (42%; 4 tracheal polyps, 3 P, 3 SCC,4 anaplastic C, 1 bronchial P, 1 SCC, 2 anaplasticC, 4 AdC, 1 A, 2 pulmonary SCC, 2 anaplasticC, 6 A), 25/39 (64%; 1 tracheal P, 10 SCC, 1anaplastic C, 3 bronchial P, 7 SCC, 11 anaplasticC, 2 AdC, 2 pulmonary SCC, 2 A), 35/55 (64%;2 laryngeal SCC, 11 tracheal P, 1 polyp, 12SCC, 1 carcinoS, 2 fibroS, 16 bronchial SCC, 10anaplastic C, 6 AdC, 3 A, 2 pulmonary A)+Forestomach T–M 8/22 (36%; 13 P, 1 SCC),6/28 (21%; 9 P), 11/34 (32%; 28 P, 1 SCC), 11/30(37%; 18 P), 5/22 (23%; 10 P), 1/28 (4%; 1 P)F 9/28 (32%; 14 P), 6/30 (20%; 9 P), 5/27 (19%; 20P), 8/30 (27%; 10 P, 1 SCC), 5/17 (29%; 11 P), 3/27(11%; 3 P)Table 3.1 (continued)

### IARC MONOGRAPHS – 100F

126Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (F)Presumably lifetimePottet al. (1973b)340 μg in tricaprylin,340 μg in Tween 60/saline solution,340 μg in Tween 60/saline solution+ 850 μg atmospheric dust/animal,45 × within a period of 6.5 mo(total dose, ~15 mg; dust, 38 mg)48/groupRespiratory tract T (benign and malignant T ofthe larynx, trachea or bronchi):2/48 (4%), 14/48 (29%), 16/48 (33%)+NR (tricaprylin, Tween 60/saline solution); atmosphericdust from Bochum, Germany(particle size < 5 μm)Hamster, Syrian (M, F)100 wkSellakumaret al.(1973)0 (untreated), 3 mg + 3 mg ferricoxide, 3 mg + 6 mg ferric oxide,3 mg + 9 mg ferric oxide, once/2wk, 20 wk36/group, 193 controls/groupRespiratory tract T (M + F combined):0/193, 26/67 (39%; 3 laryngeal polyp, 3 P, 3 SCC,7 tracheal polyp, 6 P, 2 SCC, 2 bronchial polyp,5 SCC, 9 AdC, 1 anaplasic C, 7 lung A, 1 AdC),28/64 (44%; 1 laryngeal polyp, 3 P, 6 SCC, 3tracheal polyp, 9 P, 3 SCC, 3 bronchial polyp, 1 P,4 SCC, 3 AdC, 1 anaplastic C, 7 lung A, 4 AdC),26/66 (39%; 3 laryngeal polyp, 6 SCC, 6 trachealpolyp, 11 P, 1 SCC, 1 bronchial polyp, 1 P, 4 SCC,4 AdC, 2 anaplastic C, 6 lung A, 6 AdC)Forestomach T:M–0/193 (M + F), 17/32 (53%; 37 P), 10/31 (32%;16 P, 1 SCC), 6/35 (17%; 15 P)F–0/193 (M + F), 10/35 (29%; 30 P), 12/33 (36%;25 P), 15/31 (48%; 33 P)+NR (0.9% saline solution);ferric oxideHamster, Syriangolden (M, F)Lifetime (up to 120wk)Stenbäcket al. (1975)0 (untreated),2 mg + 1 mg magnesium oxide/animal, once/wk, 20 wk, 3 mg + 3mg ferric oxide/animal, once/wk,15 wk48 or 90/groupRespiratory tract tumours (M + F combined):0/89, 32/45 [71%] (11 laryngeal P, 3 SCC, 1tracheal polyp, 20 P, 5 SCC, 1 AdC, 1 bronchialP, 3 A, 8 AdC, 9 SCC, 1 AdSC), 31/44 (70%;10 laryngeal P, 4 SCC, 8 tracheal P, 12 SCC, 2anaplastic C, 2 bronchial P, 4 A, 2 AdC, 17 SCC, 3anaplastic C)+NR (0.2% saline solution);ferric oxide, magnesiumoxideTable 3.1 (continued)

### Benzo[ a ]pyrene

127Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsHamster, Syriangolden (M, F)Lifetime (up to 100wk)Stenbäck & Rowland(1979)0 (untreated), 0 (saline), 0 (gelatinein saline), 3 mg silicon dioxide insaline, 1.5 mg manganese dioxidein saline, 3 mg in saline, 3 mg ingelatine/saline, 3 mg + 3 mg silicondioxide in saline, 1.5 mg + 1.5 mgmanganese dioxide in saline/animal, once/wk, 20 wk50/groupAll T (M + F combined): 2/100 (2%; 2lymphoma), 1/48 (2%; 2 forestomach P), 2/45 (4%;2 lymphoma), 0/48 (0%), 2/48 (4%; 1 forestomachP, 1 lymphoma), 18/46 (39%; 1 laryngeal P, 1SCC, 4 tracheal P, 15 forestomach P), 11/47 (23%;2 tracheal P, 1 SCC, 3 bronchial SCC, 1 splenichaemangioma, 1 adrenal cortical A, 1 lymphoma,2 forestomach SCC), 25/48 (52%; 1 laryngeal SCC,8 tracheal P, 2 SCC, 3 bronchial SCC, 6 lung A,3 AdC, 10 forestomach P, 1 thyroid A, 1 uterinefibroma, 1 A, 1 lymphoma), 20/48 (42%; 1laryngeal P, 3 tracheal P, 1 SCC, 1 bronchial SCC,24 forestomach P, 1 ovarian fibroma, 1 thyroid A,2 forestomach SCC, 1 squamous-cell fibroma)+> 99% (saline, 0.5% gelatinein saline); manganesedioxide, silicon dioxideHamster, Syriangolden (M, F)82 wkReynderset al. (1985)0 and 8 mg + 6 mg ferric oxide/animal, once/wk, 6 wk35/groupRespiratory tract T:M–0/32, 12/24 (50%; 15 T: 3 laryngeal P, 1tracheal P, 1 SCC, 2 bronchial polyp, 2 SCC,1AdC, 3 pulmonary SCC, 1 AdSC, 1 AdC)F–0/35, 9/26 (35%; 12 T: 1 laryngeal P, 5 trachealP, 2 bronchial polyp, 2 pulmonary SCC, 1 AdSC,1 AdC)+NR (0.9% saline solution);ferric oxideBuccal pouchHamster, Syriangolden (M)Up to 40–44 wk withinterim kills after 5,20, and 24–32 wkSoltet al. (1987)Painting of both buccal pouch with0, 20 mM solution/animal, twice/wk, 20 wk28/group, 20 controls/groupForestomach P: 0/6, 8/10* (after 40–44 wk)Buccal pouch SCC: 0/6, 1/10 (after 40–44 wk)*[P< 0.01]NR (paraffin oil)Intramammary or intramamillary administrationRat, Sprague-Dawley(F)20 wkCavalieriet al. (1988a)0 and 0 (untreated contralateralmammary gland), 4 [1 mg],16 μmol [4.2 mg] (5thrightmammary gland), 1 ×20/groupMammary gland T: [0/20] (0%), [0/20] (0%),[10/20] (50%; 6 AdC, 4 fibroS), [16/20] (80%;8 AdC, 2 fibroA, 10 fibroS)+> 99% (no vehicle)Table 3.1 (continued)

### IARC MONOGRAPHS – 100F

128Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsRat, Sprague-Dawley(F)45 wkCavalieriet al. (1988a,b)0 and 4 μmol [1 mg]/mammarygland (2nd, 3rd, 4th and 5thmammary gland on both sidesinjected), 1 ×20/groupEpithelial mammary T: [3/20] (15%; 3 fibroA),[14/20] (70%; 13 AdC, 3 fibroA); multiplicity:controls, 3/3 [1]; treated rats: AdC, 18/13 [1.4];fibroA, 4/3 [1.3]Mesenchymal (mammary) T: [0/20] (0%), [11/20](55%; 11 fibroS; multiplicity, 20/11 [1.8])Skin T: [0/20] (0%), [9/20] (45%; 9 SCC;multiplicity, 11/9 [1.2])+> 99% (trioctanoin)Rat, Sprague-Dawley(F)24 wkCavalieriet al. (1991)0, 0.25 [66 μg], 1 μmol [264 μg]/mammary gland (the 2nd, 3rd, 4thand 5th on both sides), 1 ×20/groupEpithelial mammary gland T: 1/18 (6%; 1 fibroA),1/20 (5%; 1 AdC), 0/20 (0%)Mesenchymal (mammary) T: 0/18 (0%), 6/20(30%; 6 fibroS; multiplicity, 7/6), 8/20 (40%; 8fibroS; multiplicity, 10/8)Skin T: 0/18 (0%), 0/20 (0%), 1/20 (5%; 1 SCC)+> 99% (trioctanoin)Statistics NRIntracolonic instillationMouse, Swiss albino(M, F)120 wkToth (1980)0, 200, 2000 μg/g bw (total doses);control and high-dose group, 10 × /wk instillations of 0 and 200 μg,respectively; low-dose group,1 instillation50/group/sexMalignant lymphoma:M–0/50, 6/50* (12%; 1 histiocytic, 4 lymphocytic,1 mixed), 7/50** (14%; 2 histiocytic, 3lymphocytic, 2 mixed)F–11/49 (22%; 5 histiocytic, 6 lymphocytic),21/50*** (42%; 5 histiocytic, 16 lymphocytic),18/49 (36%; 6 histiocytic, 8 lymphocytic, 4 mixed)Oesophagus T:M–no tumourF–0/49, 0/50, 5/49 (10%)Forestomach T:M–0/50, 2/50 (4%; 2 P), 10/50  (20%; 9 P,1 SCC)F–1/49 (2%; 1 SCC), 5/10 (20%; 3 P, 2 SCC),11/49** ** (22%; 9 P, 2 SCC)*P< 0.04**P< 0.02***P< 0.053 P< 0.006** ***P< 0.000198% (olive oil)Anal and skin tumoursprobably due to release ofbenzo[a]pyrene through theanal orificeTable 3.1 (continued)

### Benzo[ a ]pyrene

129Species, strain (sex)DurationReferenceDosing regimenAnimals/group at startIncidence of tumoursResult orsignificancePurity (vehicle)CommentsToth (1980)Contd.Anal T :M–0/50, 0/50, 7/50** (14%; 4 P, 3 SCC)F–0/49, 1/50 (2%; 1 P), 6/49* (12%; 1 P, 4 SCC, 1K)Skin T :M–1/50 (2%; 1 K), 0/50, 13/50***** (26%; 5 P,7 SCC, 1 K)F–0/49, 2/50 (4%; 2 SCC), 11/49**** (22%; 4 P, 5SCC, 2 K)Mouse, C57Bl/6 (F)18 moAndersonet al. (1983)0 (untreated, olive oil orβ-naphthoflavone in olive oil),1 mg/animal (in olive oil), once/wk, 14 wk45–60/groupForestomach P: 7/34 (21%; multiplicity, 1.1 ± 0.4),17/18* (94%; multiplicity, 3.2 ± 2.3*)Peritoneal S: 0/40, 5/32*Lymphoma: 1/40 (2.5%), 9/32* (28%)*P< 0.0599% (olive oil, enzymeinducer β-naphthoflavone)No colon tumours foundIntravaginal applicationMouse, C57B1 (F)5 moNäslundet al. (1987)Cotton swab soaked in acetone(controls) or 1% solution ofbenzo[a]pyrene in acetone, twice/wk10 or 76/group0/10, 17/76 (22%; invasive cervical C)+NR (acetone)Intrafetal injectionMouse, Swiss (M, F)12 wkRossiet al. (1983)0, 0.4, 4.0, 9.9, 19.8 nmol[0, 0.1, 1, 2.6, 5.2 μg]/animal, 1 ×43–56/groupLung A (M + F combined):0/37, 1/39 (3%), 10/42 (25%), 10/38 (26%), 12/31(39%)+> 99%(trioctanoin-acetone mixture(1:1))A, adenoma; AdC, adenocarcinoma; AdSC, adenosquamous carcinoma; bw, body weight; C, carcinoma; d, day or days; DMSO, dimethyl sulfoxide; F, female; H, hepatoma; K,keratocanthoma; M, male; min, minute or minutes; mo, month or months; NH4OH, ammonium hydroxide; NR, not reported; P, papilloma; PND, postnatal day; S, sarcoma; SCC,squamous-cell carcinoma; SGA, sebaceous gland adenoma; T, tumour;vs, versus; wk, week or weeks; yr, year or yearsTable 3.1 (continued)

### IARC MONOGRAPHS – 100F



## 3.4 Intraperitoneal injection

In a series of studies in newborn and adult mice, intraperitoneal injection of benzo[ a ]pyrene
increased the incidence of liver (adenomas and carcinomas) and lung (adenomas and adenocar

cinomas) tumours and, occasionally, forestomach (squamous cell papillomas and carcinomas) and lymphoreticular tumours ( Vesselinovitch et al

. , 1975a

, b ; Wislocki et al

. , 1986 ; Lavoie et al

. , 1987 ; Busby et al

. , 1989 ; Rippe & Pott, 1989 ; Mass

et al

. , 1993 ; Nesnow et al

. , 1995 ; Ross et al

. , 1995 ; Weyand et al

. , 1995 ; Rodriguez et al

. , 1997 ; Von Tungeln et al

. , 1999 ). In one study in rats with a single intraperito

neal injection of benzo[ a ]pyrene, a high incidence

of abdominal mesotheliomas and sarcomas was
observed ( Roller et al

. , 1992 ).



## 3.5 Inhalation

In a lifetime inhalation study ( Thyssen et al

. , 1981 ) in male hamsters, benzo[ a ]pyrene induced dose-related increases in the incidence of papil

lomas and squamous-cell carcinomas in both the upper respiratory tract (nose, larynx and trachea) and the upper digestive tract (pharynx, oesophagus and forestomach). 

## 3.6 Intrapulmonary injection

Dose-related increases in the incidence of malignant lung tumours (mainly epidermoid and squamous-cell carcinomas and a few pleo

morphic sarcomas) were found after injection of
benzo[ a ]pyrene into the lung of rats ( DeutschWenzel et al

. , 1983 ; Iwagawa et al

. , 1989 ; WenzelHartung et al

. , 1990 ; Horikawa et al

. , 1991 ).



## 3.7 Intratracheal administration

Intratracheal administration of benzo[ a ] pyrene alone or mixed with particulates and suspended in saline with or without suspendents resulted in benign and malignant respiratory
tumours in mice ( Heinrich et al

. , 1986a ), rats
( Pott et al

. , 1987 ; Steinhoff et al

. , 1991 ) and in
numerous studies in hamsters ( IARC, 2010 ). This

treatment also induced forestomach tumours in
hamsters ( Saffiotti et al

. , 1972 ; Sellakumar et al

. , 1973 ; Smith et al

. , 1975a

, b

, Stenbäck & Rowland,

1979 ). Larger benzo[ a ]pyrene particles were generally more effective than smaller ones. Mice that lack the nucleotide excision-repair

gene XPA ( XPA –/– 

### mice) showed a stronger lungtumour response after intratracheal instillation
of benzo[ a ]pyrene than did their similarly treated

XPA +/+ 

### and XPA +/– 

### counterparts ( Ide et al

. , 2000 ).

130 

### Table 3.2 Summary of reports of malignant tumours clearly induced in experimental animals by

benzo[ a ]pyrene

Organ

site/
species Lung Trachea Larynx Forestomach Liver Lymphoid

tissue (lymphoma) Sarcoma (injection
site) Skin Mammary gland

Mouse x x x x x x

Rat x x x

Hamster x x x x x

### Benzo[ a ]pyrene



## 3.8 Buccal pouch application

Repeated application of benzo[ a ]pyrene to the buccal pouch mucosa of male hamsters resulted in a high incidence of forestomach papillomas
( Solt et al

. , 1987 ).



## 3.9 Subcutaneous tracheal grafts transplantation

In one study conducted in rats transplanted with subcutaneous rat tracheal grafts exposed to beeswax pellets containing various amounts of
benzo[ a ]pyrene, a high incidence of squamouscell carcinomas was reported ( Nettesheim et al

. , 1977 ).



## 3.10 Intramammilary administration

In three studies in rats, benign and malig

nant mammary gland tumours developed after intrammilary injection of benzo[ a ]pyrene
( Cavalieri et al

. , 1988a

, b

, 1991 ).



## 3.11 Intracolonic instillation

In three experiments in mice, intraco

lonic instillation of benzo[ a ]pyrene induced lymphomas and a variety of benign and malig

nant tumours in various organs including the
forestomach ( Toth, 1980 ; Anderson et al

. , 1983 ).



## 3.12 Intravaginal application

Intravaginal application of benzo[ a ]pyrene in mice produced invasive cervical carcinoma;
no such tumours were seen in controls ( Näslund

et al

. , 1987 ). 

## 3.13 Intrafetal injection

In one study in male and female Swiss mice, intrafetal injection of benzo[ a ]pyrene produced lung adenomas ( Rossi et al

. , 1983 ).



## 4. Other Relevant Data

Benzo[ a ]pyrene is a carcinogen that induces tumours in many animal species. Some of the examples relevant for this review are: lung
tumours in mice, rats, and hamsters; skin tumours
in mice; liver tumours in mice; forestomach tumours in mice and hamsters; and mammary gland tumours in rats ( Osborne & Crosby, 1987 ; IARC, 2010 ). In humans, occupational exposures
to benzo[ a ]pyrene-containing mixtures have

been associated with a series of cancers: coke production: lung; coal gasification: lung, bladder; paving and roofing: lung; coal tar distillation: skin; soots: lung, oesophagus, haematolymphatic system, skin; aluminum smelting: lung, bladder; tobacco smoking: lung, lip, oral cavity, pharynx, oesophagus, larynx, bladder ( IARC, 1984

, 1985

, 1986

, 2010 ).

Studies on the mechanisms of action of
benzo[ a ]pyrene have been reviewed ( Xue & Warshawsky, 2005 ; IARC, 2010 ).



## 4.1 Metabolism

Benzo[ a ]pyrene is metabolized by both phase-I and phase-II enzymes to form a series of arene oxides, dihydrodiols, phenols, and quinones and their polar conjugates with glutathione, sulfate, and glucuronide ( Osborne & Crosby, 1987 ).
Benzo[ a ]pyrene-7,8-diol is a key metabolite that is formed by the action of epoxide hydrolase on
benzo[ a ]pyrene-7,8-epoxide. This dihydrodiol can be further metabolized by cytochrome P450s
(CYPs) to a series of benzo[ a ]pyrene-7,8-diol9,10-epoxides, which form one class of ultimate carcinogenic metabolites of benzo[ a ]pyrene.

131

### IARC MONOGRAPHS – 100F

Both CYPs and peroxidases (e.g. prostaglandin-H synthase) can oxidize benzo[ a ]pyrene. The major cytochrome P450s involved in the formation of diols and diolepoxides are CYP1A1, CYP1A2
and CYP1B1 ( Eling et al

. , 1986 ; Shimada, 2006 ). Cytochrome P450s are inducible by benzo[ a ] pyrene and other PAHs through binding to the aryl hydrocarbon-receptor (AhR) nuclear complex, leading to changes in gene transcrip

tion of CYPs and phase-II enzymes. Mice lacking the AhR receptor are refractory to benzo[ a ] pyrene-induced tumorigenesis ( Shimizu et al

. , 2000 ). Both CYPs and peroxidases can form radical cations by one-electron oxidation. These cations comprise another class of ultimate carci

nogenic metabolites ( Cavalieri & Rogan, 1995 ). Some polymorphisms in human CYPs and phase-II enzymes (glutathione S -transferases,

uridine 5 ′ -diphosphate glucuronosyltransferases and sulfotransferases modulate susceptibility
to cancer ( Shimada, 2006 ). In another meta

bolic pathway, benzo[ a ]pyrene-7,8-dihydrodiol
is oxidized to benzo[ a ]pyrene-7,8-quinone by enzymes of the aldo-keto reductase (AKR1) family. Among these, gene polymorphisms that influence susceptibility have been iden

tified. NAD(P)H: quinone oxidoreductase-1 (NQO1) catalyses the reduction of benzo[ a ] pyrene quinones to hydroquinones, which may be re-oxidized and generate reactive oxygen species. Polymorphisms in this gene have also
been described ( Penning & Drury, 2007 ; IARC,

2010 ). The current understanding of mechanisms underlying benzo[ a ]pyrene-induced carcino

genesis in experimental animals is almost solely based on two complementary pathways: those of the diolepoxides and the radical cations. Each provides a different explanation for the effects observed in experimental animals in specific

tissues. 

## 4.2 Diolepoxide mechanism

The diolepoxide mechanism for benzo[ a ] pyrene features a sequence of metabolic trans

formations: benzo[ a ]pyrene → benzo[ a ]pyrene7,8-oxide (by CYP1A1 and CYP1B1) → benzo[ a ] pyrene-7,8-diol (by epoxide hydrolase) → benzo[ a ] pyrene-7,8-diol-9,10-epoxides (by CYP1A1 and
CYP1B1) ( Xue & Warshawsky, 2005 ). Each class

of metabolic intermediate has been shown to be genotoxic and carcinogenic ( Osborne & Crosby,

1987 ). The stereochemistry of the metabolic
transformation of benzo[ a ]pyrene to diols and diolepoxides is an important component of this

mechanism of action. Due to the creation of chiral carbons during the metabolic conversions, many of the metabolic intermediates of benzo[ a ] pyrene have multiple streochemical forms (enan

tiomeric and diastereomeric). As the metabolism proceeds the complexity of the stereo-chemical forms increases, eventually leading to four
benzo[ a ]pyrene-7,8-diol-9,10-epoxides [(+)- and
(-)- anti , (+)- and (-)- syn ]. Diolepoxides react with DNA, mainly with the purines, deoxy

guanosine and deoxyadenosine, and each diolepoxide can form both cis and trans adducts thus giving a total of 16 possible benzo[ a ]pyrene-7,8- diol-9,10-epoxide DNA adducts. However, in most cases far fewer DNA adducts are actually observed. The most ubiquitous benzo[ a ]pyrene

adduct detected in isolated mammalian DNA after metabolic conversion in metabolically competent mammalian cells in culture, or in
mammals, is the N 2 

### -deoxyguanosine adduct,
(+)- N 2 

### -10S-(7 R ,8 S ,9 R -trihydroxy-7,8,9,10- tetrahydrobenzo[ a ]pyrene)-yl)-2’-deoxyguanosine (BPDE-deoxyguanosine), derived

from 7 R ,8 S -dihydroxy-9 R ,10 R -epoxy-7,8,9,10- tetrahydrobenzo[ a ]pyrene ( anti -benzo[ a ]pyrene7,8-diol-9,10-epoxide, or BPDE). This adduct was first fully identified after isolation from benzo[ a ] pyrene-treated human and bovine bronchial explants ( Jeffrey et al

. , 1977 ). This diolepoxide

is considered to be an ultimate, DNA-reactive,

132

### Benzo[ a ]pyrene

metabolite of benzo[ a ]pyrene ( Osborne & Crosby, 1987 ). The anti -benzo[ a ]pyrene-7,8- diol-9,10-epoxide can form both stable and unstable (so-called ‘depurinating’) adducts with DNA, mediated by electrophilic carbonium
ions ( Chakravarti et al

. , 2008 ). In vivo

, anti

benzo[ a ]pyrene-7,8-diol-9,10-oxide produces stable adducts that were formed primarily with guanines in many species and organs ( IARC,

2010 ).
Mice treated with benzo[ a ]pyrene had

anti -benzo[ a ]pyrene-7,8-diol-9,10-epoxideN

2 

### -deoxyguanosine adducts in their lung tissue, while the lung tumours induced by benzo[ a ] pyrene had G→T and G→A mutations in the

K

i 

### - Ras gene at codon 12 ( Mass et al

. , 1993 ). In
mice treated with benzo[ a ]pyrene the major stable DNA adduct in the epidermis was the

anti -benzo[ a ]pyrene-7,8-diol-9,10-oxide-deoxy

guanosine adduct ( Melendez-Colon et al

. , 1999 ).
Skin tumours from benzo[ a ]pyrene-treated mice or in preneoplastic skin from benzo[ a ]pyrenetreated mice had G→T mutations in codon 13 and

A→T mutations in codon 61 of the Ha- Ras gene ( Chakravarti et al

. , 2008 ).
Benzo[ a ]pyrene-induced skin tumours

harboured G→T transversion mutations in the Tp53 tumour-suppressor gene ( Ruggeri et al

. , 1993 ). The anti -benzo[ a ]pyrene-7,8-diol-9,10- oxide-DNA adducts occurred at guanine posi

tions in codons 157, 248, and 273 of the TP53 gene

in anti -benzo[ a ]pyrene-7,8-diol-9,10-epoxidetreated human HeLa cells. The same positions are the major mutational hotspots found in human lung cancers ( Denissenko et al

. , 1996 ).



## 4.3 Radical-cation mechanism

The radical-cation mechanism for benzo[ a ] pyrene has been studied exclusively in connec

tion with mouse-skin tumorigenesis ( Cavalieri &
Rogan, 1995 ). One-electron oxidation of benzo[ a ] pyrene by CYPs or peroxidases creates a radical cation localized on carbon 6, as a consequence of the ionization potential and geometric configu

ration. In mouse skin, this radical cation gives

rise to the formation of covalent adducts with guanine (at the C8 carbon and N7 nitrogen) and adenine (at the N7 nitrogen). These adducts are unstable and are thought to generate apurinic sites in mouse skin. However, only low levels of apurinic sites were measured in the epidermis
of mice treated with benzo[ a ]pyrene ( MelendezColon et al

. , 1999 ) and no studies to date have shown an increase in the number of apurinic sites in lung tissues treated with benzo[ a ]pyrene. In

two in vivo studies, rats treated intraperitoneally
with benzo[ a ]pyrene were shown to excrete
7-(benzo[ a ]pyrene-6-yl)- N7 -guanine in faeces
and urine, while the same adduct was detected in lung tissue of mice treated intraperitoneally with
benzo[ a ]pyrene ( Rogan et al

. , 1990 ; Banasiewicz

et al

. , 2004 ). Skin papillomas obtained from mice treated topically with benzo[ a ]pyrene showed mutations (at guanine and/or adenine) at codons
12, 13 and 61 in the Ha- Ras oncogene ( Wei et al

. , 1999 ). Similar studies in preneoplastic skin from
benzo[ a ]pyrene-treated mice showed Ha- Ras mutations at codons 13 and 61 ( Chakravarti

et al

. , 2008 ). The anti -benzo[ a ]pyrene-7,8-diol9,10-epoxide can also form depurinating DNA adducts at guanine and adenine (at the N7 nitrogen). The distribution and chemical natureof the depurinating adducts (from both radicalcation and diolepoxide intermediates) in mouse

skin and the distribution and chemical nature of the specific benzo[ a ]pyrene-induced mutations in mouse-skin papillomas have been reported
( Chakravarti et al

. , 2008 ).



## 4.4 Other activation mechanisms of
benzo[ a ]pyrene

4.4.1 Meso-region mechanism

The mechanism of meso-region biomethylation and benzylic oxidation features biomethylation of benzo[ a ]pyrene to 6-methylbenzo[ a ]

133

### IARC MONOGRAPHS – 100F

pyrene, with S -adenosylmethione as the carbon
donor ( Flesher et al

. , 1982 ). This process has

been shown to occur in vitro , and in vivo in rat liver ( Stansbury et al

. , 1994 ). 6-Methylbenzo[ a ] pyrene is further metabolized by CYPs to 6-hydroxymethylbenzo[ a ]pyrene ( Flesher

et al

. , 1997 ) and then conjugated to sulfate by 3 ′ -phosphoadenosine-5 ′ -phosphosulfate sulfotransferase to 6-[(sulfooxy)methyl]-
benzo[ a ]pyrene. This reactive sulfate ester forms

DNA adducts in vivo ( Stansbury et al

. , 1994 ).
These benzo[ a ]pyrene-DNA adducts have only
been measured in rat liver ( Surh et al

. , 1989 ), which is not a target for benzo[ a ]pyrene-induced carcinogenesis. There is no evidence to date that this mechanism operates in lung.



## 4.4.2 Mechanism via formation of orthoquinone/ reactive oxygen species

This mechanism features enzymatic oxidation
of benzo[ a ]pyrene-7,8-diol to the ortho -quinone, benzo[ a ]pyrene-7,8-quinone, by aldo-keto
reductases ( Mangal et al

. , 2009 ). Benzo[ a ]pyrene7,8-quinone can react with DNA to yield both stable and depurinating DNA adducts in vitro ( McCoull et al

. , 1999 ; Balu et al

. , 2006 ) and can also undergo repetitive redox cycling which generates reactive oxygen species that damage
DNA ( Penning et al

. , 1999 ). In human A549 lung-tumour cells benzo[ a ]pyrene-7,8-quinone increased the formation of 8-oxo-deoxyguano

sine and DNA strand-breaks ( Park et al

. , 2008 ; Mangal et al

. , 2009 ). In a yeast reporter-assay,
benzo[ a ]pyrene-7,8-quinone (in the presence of redox cycling) induced 8-oxo-deoxyguanosine

formation and G→T transversions in the Tp53 tumour-suppressor gene. The mutational spectra induced in the yeast reporter-assay closely matched those seen in DNA from human lung
tumours ( Shen et al

. , 2006 ). Benzo[ a ]pyrene-7,8- quinone inhibited the activity of protein kinase C in MCF-7 cell lysates suggesting an ability to alter cell signalling ( Yu et al

. , 2002 ). Rats treated with benzo[ a ]pyrene showed increased urinary concentrations of 8-oxo-deoxyguanosine, but lower levels in liver and lung tissues. This suggested that reactive oxygen species are gener

ated during the CYP-dependent metabolism of
benzo[ a ]pyrene, but induction of DNA-repair mechanisms may reduce these levels in target
tissues ( Briedé et al

. , 2004 ). To date this mecha

nism has been studied only in in-vitro systems. It is noted that formation of reactive oxygen species is not limited to the redox cycling of

the ortho -quinone of benzo[ a ]pyrene (benzo[ a ] pyrene-7,8-quinone). There are several other
sources of benzo[ a ]pyrene-induced reactive
oxygen species. In vivo , both mice and rats
metabolize benzo[ a ]pyrene to benzo[ a ]pyrene1,6-quinone, benzo[ a ]pyrene-3,6-quinone
and benzo[ a ]pyrene-6,12-quinone and these quinones enter into redox cycling and induce
mutations ( Osborne & Crosby, 1987 ; Joseph &
Jaiswal, 1998 ). Many of the reactive intermedi

ates of benzo[ a ]pyrene (oxides, diol-epoxides, radical cations) and quinone-generated reactive oxygen species can disrupt the balance of cellularoxidants and anti-oxidants by reducing the antioxidant levels thus leading to an imbalance and an excess of reactive oxygen species.



## 4.4.3 Aryl hydrocarbon-receptor mechanism

The AhR regulates the transcription of a series of genes including Cyp1A1

, Cyp1A2,
Nqo1

, Aldh3a1 (encoding aldehyde dehydro

genase 3A1), UGT1a6 (uridine 5 ′ -diphosphateglucuronosyl transferase), and Gsta1 (glutathione

S -transferase A1). All these genes are activated by AhR-ligands, including benzo[ a ]pyrene, via the AhR-mediated aromatic hydrocarbon response element. The AhR plays a role in the response to oxidative stress in cell-cycle regulation and apop

tosis. In addition, the CYP1A1/1A2-mediated metabolism generates oxidative stress ( Nebert

et al

. , 2000 ). Mitochondrial hydrogen-peroxide production was induced by an AhR-ligand in

134

### Benzo[ a ]pyrene

wild-type mice but not in AhR −/− 

### knockout mice
( Senft et al

. , 2002 ). These mice were shown to be refractory to benzo[ a ]pyrene-induced carci

nogenicity ( Shimizu et al

. , 2000 ). Benzo[ a ] pyrene induced oxidative stress in mouse lung
( Rajendran et al

. , 2008 ).



## 4.4.4 Immunosuppression mechanism

Benzo[ a ]pyrene induces immunosupres

sion in adult mice by altering the cell-mediated responses ( Wojdani & Alfred, 1984 ). Immune development in offspring is also altered following in utero exposure to benzo[ a ]pyrene
( Urso & Gengozian, 1984 ). It is postulated that PAHs, including benzo[ a ]pyrene, act princi

pally through their AhR-mediated CYP-derived metabolites (diolepoxides, quinones) to activate oxidative and electrophilic signalling pathways in lymphoid and nonlymphoid cells, including myeloid cells, epithelial cells, and other cell types. Furthermore, there is evidence that PAHs suppress immunity by p53-dependent path

ways, by modulating signalling pathways in lymphocytes via non-genotoxic mechanisms, and by oxidative stress ( Burchiel & Luster, 2001 ).



## 4.4.5 Epigenetic mechanisms

Benzo[ a ]pyrene and/or its metabolites have been shown to increase cell proliferation in several human cell lines, including terminally differenti

ated human bronchial squamous epithelial cells and in lung-cancer cells where increased expres

sion of the Cdc25B gene (cell-division cycle 25B) was observed, along with reduced phos

phorylation of Cdk1 (cyclin-dependent kinase
1) ( Oguri et al

. , 2003 ). Treatment with benzo[ a ] pyrene increased the number of human embryo lung-fibroblasts in the G1–S transition via the activation of c-Jun, through the p53-dependent PI-3K/Akt/ERK (phosphatidylinositol-3-kinase/ protein kinase β/extracellular signal-regulated kinase) pathway ( Jiao et al

. , 2008 ). Benzo[ a ]pyrene and/or its metabolites also affect apoptosis. Benzo[ a ]pyrene induced apop

tosis in human MRC-5 lung fibroblasts via the
JNK1/FasL (c-Jun N-terminal kinase 1/Fas Ligand) and JNK1/p53 signalling pathways ( Chen

et al

. , 2005 ). Apoptosis induced by anti -benzo[ a ] pyrene-7,8-diol-9,10-epoxide in H460 human lung-cancer cells was associated with induction of Bak (BCL2-antagonist/killer) and with activa

tion of caspase, but it was independent of Bcl-2
( Xiao et al

. , 2007 ). Altered DNA methylation has been reported to be associated with exposure to benzo[ a ] pyrene and/or its metabolites. After treatment of immortalized bronchial epithelial cells with

anti -benzo[ a ]pyrene-7,8-diol-9,10-epoxide, the concentration of cytosine-DNA methyltrans

ferase-1 was increased and was associated with hypermethylation of the promoters of 5–10 genes, including members of the cadherin gene-family
( Damiani et al

. , 2008 ).



## 4.5 Human exposure to PAH-rich

mixtures

4.5.1 Biomarkers of exposure and effect

Molecular-epidemiological studies of cancer associated with occupational and environmental exposures to PAH have provided biomarkers that may be used to estimate internal exposure as well

as to inform about molecular mechanisms that may be relevant to cancer causation, particularly in defining the early events in the carcinogen

esis process. Biomarkers can be detected in the
target organ, in surrogate tissues, or in tumours. These can be categorized into biomarkers of

exposure , which are generally specific to the PAH of concern (e.g. DNA or protein adducts), biomarkers of effect (e.g. genotoxic and cytoge

netic effects, 8-oxo-deoxyguanosine, sister chromatid exchange (SCE), micronuclei, chro

mosomal aberrations, mutations in oncogenes, tumour-suppressor genes, or indicator genes),

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### IARC MONOGRAPHS – 100F

and biomarkers of susceptibility (DNA-repair
enzymes, e.g. XPA, XPC – xeroderma pigmen

tosum complementation groups A and C), bioac

tivation enzymes (e.g. CYPs), detoxification enzymes (e.g. GSTs), and mutagenic metabolites
in urine ( Kalina et al

. , 1998 ; Pilger et al

. , 2000 ; Simioli et al

. , 2004 ; Raimondi et al

. , 2005 ; Vineis & Husgafvel-Pursiainen, 2005 ; Matullo et al

. , 2006 ; Farmer & Singh, 2008 ; Gyorffy et al

. , 2008 ). Although biomarkers of effect and suscepti

bility are generally not unique to any specific PAH exposure, several these biomarkers may provide insight into the mechanism of carcino

genesis induced in humans by PAHs or PAH-rich

exposures.



## 4.5.2 Exposure to benzo[a]pyrene and relationship with specific biomarkers

Biomarkers of exposure to complex mixtures
that contain benzo[ a ]pyrene have been studied in populations exposed in industrial settings: coke production, coal-tar distillation, the aluminium industry, roofing and paving with coal-tar pitch, coal gasification, chimney sweeping, and iron and steel founding. Most if not all of these biomarkers are genotoxic markers. Populations of patients who undergo coal-tar therapy and groups exposed to combustion emissions, and

tobacco smokers have also been evaluated. Studies on biomarkers of exposure are dominated by those focusing on the anti -benzo[ a ]pyrene7,8-diol-9,10-oxide-DNA adduct, the most commonly studied PAH-DNA adduct because of the availability of specific analytical methods
and standards ( Gyorffy et al

. , 2008 ). In one studythe depurinating adducts resulting from radicalcation formation, viz . 7-(benzo[ a ]pyrene-6-yl) guanine and 7-(benzo[ a ]pyrene-6-yl)adenine were found in the urine of women exposed to
coal smoke ( Casale et al

. , 2001 ). Concomitantly,

several biomarkers of effect have also been evalu

ated in these studies: chromosomal aberrations, sister chromatid exchange ( Kalina et al

. , 1998 ), DNA damage (measured by the comet assay) and 8-oxo-deoxyguanosine formation ( Marczynski

et al

. , 2002 ). It is important to note that these genotoxic effects observed in humans in rela

tion to exposure to benzo[ a ]pyrene-containing mixtures have also been observed in experi

mental studies where benzo[ a ]pyrene or anti

benzo[ a ]pyrene-7,8-diol-9,10-epoxide has been shown to induce sister chromatid exchange
( Pal et al

. , 1980 ; Brauze et al

. , 1997 ), chromo

somal aberrations, micronuclei ( Kliesch et al

. , 1982 ), DNA damage ( Nesnow et al

. , 2002 ), and 8-oxo-deoxyguanosine ( Thaiparambil et al

. , 2007 ). Tobacco smoke, dietary habits and indoor ambient air are also important sources of expo

sure to benzo[ a ]pyrene, which has been impli

cated as one of the components of tobacco smoke related to the induction of lung cancer in smokers
( Watanabe et al

. , 2009 ). In a large study of 585 smokers and nonsmokers, smoking and diet were highly correlated with anti -benzo[ a ]pyrene-7,8-
diol-9,10-oxide-DNA adduct levels ( Pavanello

et al

. , 2006 ). Several studies have demonstratedmoderately increased levels of 8-oxo-deoxyguanosine from lungs, sperm, and leukocytes ofsmokers. Increased urinary excretion of 8-oxodeoxyguanosine has also been reported ( Hecht,

1999 ). In rats exposed to benzo[ a ]pyrene via oral,
intratracheal and dermal routes, anti -benzo[ a ] pyrene-7,8-diol-9,10-oxide-DNA adducts were formed in white blood cells independently of the exposure route and their numbers correlated with those found in lung DNA, suggesting that

anti -benzo[ a ]pyrene-7,8-diol-9,10-oxide-DNAadduct levels in white blood cells may be used as a surrogate for pulmonary anti -benzo[ a ]pyrene7,8-diol-9,10-oxide-DNA adducts ( Godschalk

et al

. , 2000 ).

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### Benzo[ a ]pyrene



## 4.5.3 Relationship of biomarkers to human

cancer

Mutations in TP53 are common in lung cancers

from smokers and less common in nonsmokers.

These mutations are G→T transversions with hotspots in codons 157, 248 and 273 ( Hainaut
& Pfeifer, 2001 ; Pfeifer et al

. , 2002 ) and they are

associated with anti -benzo[ a ]pyrene-7,8-diol9,10-oxide-DNA adducts. The active metabolite

anti -benzo[ a ]pyrene-7,8-diol-9,10-oxide causes a unique spectrum of TP53 mutations distinct

from those found in cancers that are not associ

ated with smoking ( Campling & el-Deiry, 2003 ). Similar G→T mutations have been reported in lung tumours from nonsmoking Chinese women whose tumours were associated with exposure to PAHs from smoke generated by burning smoky

coal in unventilated homes. The mutations were clustered at the CpG rich codons 153–158 of the

TP53 gene, and at codons 249 and 273. The muta

tion spectrum was fully consistent with exposure
to PAHs ( DeMarini et al

. , 2001 ).



## 4.6 Synthesis

Benzo[ a ]pyrene is metabolically activated to a series of reactive intermediates by CYP450and related enzymes under control of the arylhydrocarbon receptor. There is strong evidence
that the benzo[ a ]pyrene diolepoxide mecha

nism operates in mouse-lung tumorigenesis, while there is also strong evidence that both the radical-cation and the diolepoxide mechanisms are involved in mouse-skin carcinogenesis. The meso-region mechanism has been studied only
in rat liver, while the mechanism that involves

the formation of ortho -quinone/reactive oxygen species has only been studied in vitro , although reactive oxygen species can be formed in vivo by
other benzo[ a ]pyrene-mediated mechanisms. All these pathways reflect genotoxic mechanisms, as they involve alterations to DNA. Benzo[ a ]pyrene is pleotropic and has the ability to affect many cell- and organ-based systems. Therefore, there are probably many modes of carcinogenic action operating to different extents in vivo . These

include mechanisms that involve AhR, oxidative stress, immunotoxicity and epigenetic events.
Based on the best available, consistent and strong experimental and human mechanistic
evidence it is concluded that benzo[ a ]pyrene contributes to the genotoxic and carcinogenic effects resulting from occupational exposure to complex PAH mixtures that contain benzo[ a ] pyrene. The most commonly encountered – and most widely studied – mechanistically relevant

DNA lesion is the anti -benzo[ a ]pyrene-7,8-diol9,10-oxide-DNA adduct. The formation of this

adduct is consistent with anti -benzo[ a ]pyrene7,8-diol-9,10-epoxide-associated genotoxic effects in surrogate tissues and with the muta

tion pattern in the TP53 gene in lung tumours from humans exposed to PAH mixtures that
contain benzo[ a ]pyrene. The fact that those
PAH mixtures and benzo[ a ]pyrene itself induce genotoxic effects like sister chromatid exchange,
chromosomal aberrations, micronuclei, DNA damage (comet assay) and 8-oxo-deoxyguano

sine, supports the notion that benzo[ a ]pyrene

contributes to human cancer.



## 5. Evaluation

There is sufficient evidence for the carci

nogenicity of benzo[ a ]pyrene in experimental

animals. [No epidemiological data on benzo[ a ]pyrene alone were available to the Working Group.] The genotoxic mechanism of action of
benzo[ a ]pyrene involves metabolism to highly reactive species that form covalent adducts
to DNA. These anti-benzo[ a ]pyrene-7,8-diol9,10-oxide-DNA adducts induce mutations in

the K- RAS oncogene and the TP53 tumoursuppressor gene in human lung tumours, and

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### IARC MONOGRAPHS – 100F

in corresponding genes in mouse-lung tumours. Exposure to benzo[ a ]pyrene and benzo[ a ]pyrenecontaining complex mixtures also induce other genotoxic effects, including sister chromatidexchange, micronuclei, DNA damage and 8-oxodeoxyguanosine, all of which can contribute to the carcinogenic effects of benzo[ a ]pyrene and
benzo[ a ]pyrene-containing complex mixtures in exposed humans.
Benzo[ a ]pyrene is carcinogenic to humans (Group 1)

. In making the overall evaluation, the Working Group took the following into consideration: The strong and extensive experimental evidence for the carcinogenicity of benzo[ a ] pyrene in many animal species, supported by the

consistent and coherent mechanistic evidence from experimental and human studies provide biological plausibility to support the overall
classification of benzo[ a ]pyrene as a human carcinogen (Group 1).



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