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CARCINOGENICITY OF DIBENZO(a,l)PYRENE

COC/03/S5 - November 2003

Introduction

1. In 1995, at the request of MAFF and the Department of Environment, the COC agreed a hazard-ranking scheme for the carcinogenicity of 25 polyaromatic hydrocarbons (PAHs). 1 It was based on classification into one of 5 categories. The COC in 1995 had accepted the principle that the carcinogenicity of PAHs was additive. Advice on dibenzo(a,l)pyrene (DB(a,l)P) had not been requested in 1995. Since then, air pollution monitoring in the UK had detected the presence of dibenzo(a,l)pyrene in a number of samples.2 An assessment of the relative carcinogenic potency of DB(a,l)P compared to benzo(a)pyrene (claimed to be about 100 times) had been published in an Environmental Health Criteria Document (International Programme on Chemical Safety).3

2. The COC was asked to consider the mutagenicity and carcinogenicity data on dibenzo(a,l)pyrene and to consider to what category of the COC hazard ranking scheme of PAHs benzo(a,l)pyrene could be assigned. Additionally the committee was asked whether the relative carcinogenic potency of DB(a,l)P compared with benzo(a)pyrene could be determined from the available data and in particular whether it was possible to make any statement on the relative potency by the inhalation route of exposure. The Committee considered the available published mutagenicity and carcinogenicity data on DB(a,l)p at its November 2002 meeting.

Introduction to Dibenzo(a,l)pyrene; DB(a,l)P

3. Dibenzo(a,l)pyrene ((DBal)P) CAS Number191-30-0, Chemical Abstracts Name; Dibenzo(def,p)chrysene). There is very little information available on the chemical characteristics of this compound. Its Molecular weight is 302.38 D and its melting point is 162-164^oC. Dibenz(a,l)pyrene occurs in some products of incomplete combustion; it also occurs in fossil fuels.4 It has been identified in mainstream cigarette smoke and products of coal gasification.4 There are few data on levels in the environment. Some measurements have been reported for urban and industrialised areas of Holland and from air quality monitoring in Canada.5,6 It is noted that in some early analyses dibenzo(a,e)fluroanthene has been mistaken for DB(a,l)P.4

Evaluation of DP(a,l)P

4. The following paragraphs give an overview of the mutagenicity and carcinogenicity data on DB(a,l)P. This is followed by a discussion of the studies where the carcinogenicity of, and DNA adducts formed by DB(a,l)P have been compared to equivalent studies with other PAHs. This statement does not present a review of the large number of investigations which have considered the pathways of metabolic activation of DB(a,l)P, although some references to studies with pro-carcinogenic metabolites are made.

Mutagenicity

DB(a,l)P

5. DB(a,l)P was mutagenic in-vitro in Salmonella typhimurium TA 100 and TA 98 in the presence of exogenous metabolic activation (using Aroclor 1254 pre-treated rat liver S-9).7 Positive results have also been reported in an in-vitro cell transformation assay using CH310T1/2 cells, although the significance of these findings for mutagenicity are unclear.8

6. No in-vivo mutagenicity studies were identified in a literature search but DB(a,l)P has been shown to act as an initiator in mouse skin promotion assays.6,10 It is also noted that an increase in mutations of codon 12 and 61 of Ki-ras was documented in pulmonary adenomas from mice treated with single intraperitoneal doses of 0.3-6 mg/kg bw of DB(al)P.11 The distribution of mutations at codon 12 (GGT-TGT, GGT-GTT and GGT-CGT) and of codon 61 (CAA-CTA, CAA-CGA and CAA-CAT) was different from those reported by the same investigators in pulmonary adenomas from control animals. (There are no concurrent in-vitro mutagenicity spectra data for DB (a,l)P.) Finally DB(a,l)P has also been shown to induce DNA adducts in rat mammary tissue following intramammary instillation using ³²P-postlabelling with adduct enrichment via nuclease P1.12

7. Thus the available mutagenicity data in studies where DB(a,l)P has been used in conventional tests is very limited. (This concurs with the conclusion reached in the IPCS Environmental Health Criteria document 202 on PAHS.3)

Metabolites of DB(a,l)P

8. The pathways leading to metabolic activation of DB(a,l)P are complex and not reviewed in detail in this statement. It has been suggested that stereospecific metabolism may be important with the fjord region syn-and anti-11,12-dihdrodiol 13,,-14-epoxides (DB(a,l)PDE) being of most importance in the activation of BD(a,l)P to DNA reactive metabolites.13

9. Investigations of pathways leading to activation using 3-methylcholanthrene pre-treated liver microsomes from Sprague-Dawley rats and CD-1 mice in Chinese hamster V79 cells showed a preferential steroeselective oxidation of (-) -11R,12R dihydrodiol of DB(a,l)P at the 13,14 position to form the diol-epoxide.14 The authors noted that this finding was consistent with the results of mutagenicity studies in V79 cells using the individual steroeisomers.

Summary: Mutagenicity

10. DB(a,l)P and some of its diol-epoxide metabolites are mutagenic in-vitro. Other PAHs which have been shown to be metabolised to diol-epoxides have also been shown to be mutagenic in-vivo. DB(a,l)P is an initiator in mouse-skin promotion assays, binds DNA in mammary tissue when instilled directly into mammary tissue. There is also limited evidence that DB(a,l)P induces a specific mutational signature in Ki-ras in lung tumours in mice following intraperitoneal administration. Overall the data are consistent with DB(a,l)P being an in-vivo mutagen.

Carcinogenicity of DB(a,l)P

[Dose levels in the following studies have been cited in the units used by the investigators which have included dosed expressed in terms of number of moles or weight of test material dosed. Thus dosages of DB(a,l)P have been reported in terms of nmoles/kg bw in several of the studies reported. 1 nmole of DB(a,l)P is approximately equal to 0.3 µg DB(a,l)P by weight. Additionally 1µmole of DB(a,l)P is approximately equal to 300 µg DB(a,l)P by weight (or 0.3 mg DB(a,l)P by weight.]

Mouse

Dermal administration

11. DB(a,l)P was applied to shaved skin of groups of 22-27 female Swiss mice twice weekly (either 4 or 8 nmol in 100ul of acetone) for 40 weeks. DB(a/l)P was carcinogenic in this study inducing squamous cell carcinomas in 90% of mice at 8 nmol and 70% at 4 nmol.9 Tumours were also recorded at other sites; these included adenoma of the lung, malignant lymphoma of the spleen and malignant lymphoma with multiple organ involvement. A dose level of 8 nmol was the maximum dose that could be applied without erythema occurring. These data are consistent with DB (a,l)P being a potent carcinogen in the mouse.

12. A single dose of 100 nmol DB(a,l)P was applied to the shaved skin of a group of 24, eight week old SENCAR mice. Mice were killed after 27 weeks. It was reported that 7 mice treated with DB(a,l)P each developed a skin tumour (4 papillomas and 3 squamous cell carcinomas).10

Intraperitoneal administration

13. Groups of 70 male A/J male mice (5-6 weeks old) were given a single intraperitoneal administration of DB(a,l)P in tricaprylin at 0.3, 1.5, 3.0, or 6.0 mg/kg bw. Approximately half of the animals were used at various time points up to 28 days for DNA adduct studies. Remaining animals (30-35/group) were subject to necropsy at 8 months post dose and the number of surface lung adenomas counted using a dissecting microscope. A dose level of 0.3 mg/kg bw resulted in 43% Tumour Bearing Animals (TBA), whereas 1.5 mg/kg bw resulted in 97% TBA and doses of 3 mg/kg be and above 100% TBA. The mean number of adenomas per mouse at 6 mg/kg was 16.1 ± 7.26.11 Maximal levels of DNA adducts ocurred between 5-10 days after injection followed by a gradual decrease. The Time Integrated DNA Adduct Levels (TIDAL) were linearly related to dose.

Rat

Intramammary instillation

14. Groups of 20 Sprague-Dawley rats (8 weeks old) were given intramammary doses of 0.25 or 1µmol/gland in 50µl of trioctanoin. Administration was to the nipple region of glands 2, 3, 4, 5 on the right and left sides. The development of tumours was monitored for the following 24 weeks. Animals with tumours >2cm were killed. Full necropsies were undertaken in all animals and mammary glands and any other abnormal tissues examined microscopically. All animals given DB(a,l)P developed mammary tumours (predominantly mammary epithelial adenocarcinmas with a smaller number of mesenchymal fibrosarcomas and squamous cell carcinomas of the skin). The number of tumours/TBA animal was 10.8/animal at 1umol and 6.6/animal at 0.25 µmol.10 (Lung tumours were not specifically assessed in this study).

Initiation-Promotion models

15. Groups of 24 female SENCAR mice (eight weeks of age) received a single dermal dose to shaved dorsal skin of 33.3, 100 or 300 nmols DB(a,l)P in 100µl acetone. One week later promotion treatments using 12-O-tetradecanoylphorbol-13-acetate (TPA, 3.24 nmol/100µl acetone, twice weekly) were begun. All mice treated with DB(a,l)P developed erythemas after the first application of TPA. Severity was proportional to DB(a,l)P dose. Promotion treatments were therefore stopped until the fourth week of the study. Promotion treatment was re-started and continued for 11 weeks. Animals killed after 16 experimental weeks.10

16. In a second experiment, the same investigators, undertook a further initiation-promotion study using dose levels of 4, 20, 100 nmols DB(a,l)P. In concurrent studies, initiation-promotion investigations were undertaken using DB(a,l)P 11,12 dihydrodiol and DB(a,l)P8,9-dihydrodiol. Erythema was noted in the 100 nmol DB(a,l)P group after 10 days. Promotion was delayed till the third experimental week. Promotion treatments (as above) were undertaken for a further 24 weeks.10

17. In the first experiment it was noted that tumours were already present in some animals at 100 and 300 nmol DB(a,l)P before promotion treatment was resumed in the 4th experimental week. An increase in the percent TBAs was reported compared to acetone control (No tumours seen in controls) of 96%, 92% and 100% at 33.3, 100 and 300 nmol respectively. The number of tumours/animal was increased in a dose-related fashion, 3.29, 5.29, 6.26 respectively. An increase in percent tumour bearing animals and numbers of tumours/animal was recorded in the second experiment at all dose levels of DB(a,l)P but the dose-response was noted to show an inversion. The authors considered this was most likely due to a more severe toxicity of DB(a,l)P in the second experiment. However it is noted that 92% of surviving animals at 4 nmol had tumours (6.96/animal). It was also documented that substantial carcinogenic response was recorded in initiation-promotion studies with DB(a,l)P11,12- dihydrodiol but not with DB(a,l)P 8,9-dihydrodiol using the same treatment regime as with DB(a,l)P.10

18. In a subsequent study, the same research group investigated initiation-promotion of DB(a,l)P and DB(a,l) 11,12-dihydrodiol in female SNECAR mice using dermal application of 0.25 or 1nmol in 100µl acetone. Promotion, twice weekly for 27 weeks, was undertaken using a lower dose of TPA (2.16 nmol in 100µl acetone) in order to reduce sensitivity to erythema induced by these chemicals. DB(a,l)P induced 2.6 tumours/mouse and 0.79 tumours/mouse at 1 nmol and 0.25 nmol respectively. DB(a,l)P 11,12-dihydrodiol induced 0.17 tumours/mouse at 1 nmol but was reported to be virtually inactive at the lower dose level. Tumours were seen following treatment with 1 nmol DB(a,l)Pin mice after 5 weeks of TPA promotion.9

19. Initiation-promotion assays in mice using dermal application of enantiomerically pure 11,12-dihydrodiols of DB(a,l)P revealed that (-) -11R,12R trans-dihydrodiol of DB(a,l)P induced skin tumours in 93% of animals (4-5/animal) after a single induction dose of 10 nmole whereas the (+) -11S,12S trans- dihydrodiol of DB(a,l)P induced no tumours at 10 nmole and only 13% of animals had tumours following application of 20 nmoles. 15 The results of this study were consistent with the in-vitro mutagenicity studies in V-79 cells using these enantiomers.13

Summary: Carcinogenicity

20. DB(a,l)P is carcinogenic in mice following dermal or intraperitoneal application. DB(a,l)P is also carcinogenic in rats following intramammary instillation. It acts as an initiator in mouse skin promotion assays. The 11,12-dihydrodiol also acted as an initiator in mouse skin promotion assays. These data are consistent with DB (a,l)P being a genotoxic carcinogen in experimental animals.

COC discussion:
DB(a,l)P ranking under 1995 COC scheme

21. The COC agreed that the in-vitro mutagenicity tests and information on in-vivo DNA adduct formation was consistent with dibenzo(a,l)pyrene being an in-vivo mutagen. Members also agreed that dibenzo(a,l)pyrene was carcinogenic in mice and rats. Dermal application to mice produced tumours at a number of sites (including the skin, lung, and malignant lymphoma of the spleen and malignant lymphoma with multiple organ involvement) and intraperitoneal administration to rats produced lung tumours. Intramammary instillation in rats resulted in mammary tumours. Dibenzo(a,l)pyrene also acted as an initiator in mouse skin carcinogenicity promotion assays. The COC therefore considered that dibenzo(a,l)pyrene should be assigned to group A of its hazard ranking scheme for PAHs. This category includes chemicals for which 'there is a high level of concern about a carcinogenic hazard for humans because the compound is an in-vivo mutagen and/or a multi-site carcinogen in more than one species.'

COC Discussion: Relative potency of DB(a,l)P compared to Benzo(a)pyrene

22. Regarding potency, the committee agreed that dibenzo(a,l)pyrene was a very potent genotoxic carcinogen and that potency varied depending on factors such as species, route of administration, dose and site of tumour produced. From the available data where a comparison could be made, members considered that the dibenzo(a,l)pyrene carcinogenic potency was likely to be in the range of 10-100 times more potent than benzo(a)pyrene depending on the tests system used. The measurement of DNA adducts (TIDAL) following intraperitoneal dosing of DB(a,l)P showed that in comparison to other PAHs, DB(a,l)P bound much more extensively to DNA, due presumably to the higher reactivity of its diol-epoxide metabolite(s). This might explain its greater carcinogenic potential.

23. The Committee agreed that there were insufficient data available on dibenzo(a,l)pyrene to draw any conclusions on the relative potency compared to benzo(a)pyrene by the inhalation route of exposure. The committee agreed to reconsider the topic of the relative carcinogenic potency of PAHs by the inhalation route of exposure at a future meeting.

COC Conclusion

24. The Committee agreed the following overall conclusion regarding the carcinogenicity of DB(a,l)P.

"Dibenzo(a,l)pyrene should be considered as a highly potent genotoxic carcinogen in experimental animals. There is a need for further consideration of the potential importance of exposure to dibezo(a,l)pyrene and other highly potent carcinogenic polycylic aromatic hydrocarbons in air pollution."

November 2003

References

1. Department of Health (1996). Annual report of the Committees on Toxicity, Mutagenicity and Carcinogenicity of chemicals in food, consumer products and the environment. The Stationary Office, J36818 C6 01/98. Crown copyright 1998.

2. Coleman (2003). Personal Communication to COC Secretariat.

3. IPCS (1998). Environmental Health Criteria 202. Polycyclic Aromatic Hydrocarbons, selected non-heterocyclic. International Programme on Chemical Safety, World health Organisation.

4. IARC (1983). Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Geneva: World Health Organization, International Agency for Research on Cancer, 32, 343.

5. Verschueren K (1996). Handbook on environmental data on organic chemicals 3rd edition, Van Nostrand Reinhold.

6. De Raat WK et al (1987). Concentrations of polycyclic hydrocarbons in airborne particulates in the Netherlands and their correlation with mutagenicity. The Science of the Total Environment, 66, 95-114.

7. Devanaesan PD et al (1990). Metabolism and mutagenicity of Dibenzo (a,h)pyrene and the very potent environmental carcinogen Dibenzo (a,l)pyrene. Chem Res Toxicol, vol 3, 580-586.

8. Nesnow S et al (1997). Comparison of the morphological transforming activities of dibenzo(a,l)pyrene and benzo(a)pyrene in C3H10T1/2CL8 cells and characterisation of dibenzo(a,l)pyrene-DNA adducts. Carcinogenesis,18, 1973-1978.

9. Higginbotham S et al (1993). Tumour initiating activity and carcinogenicity of dibenzo(a,l)pyrene versus 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene at low doses in mouse skin. Carcinogenesis, 14, 875-878.

10. Cavalieri EL et al (1991). Comparative dose-response tumourigenicity studies of dibenzo(a,l)pyrene versus 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene and two dibenzo(a,I)pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis, 2, 1939-1944.

11. Prahalad AK et al (1997). Dibenzo(a,l)pyrene-induced DNA adduction, tumourigenicity, and Ki-ras oncogene mutations in strain A/J mouse. Carcinogenesis,18, 1955-1963.

12. Arif JM et al (1997). Tissue distribution of DNA adducts in rats treated by intrammammillary injection with dibenzo (a,l)pyrene, 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene. Mutation Research, 378, 31-39.

13. Luch A et al (1994). Synthesis and mutagenicity of the diastereomeric fjord-region 11,12 dihydrodiol 13,14-epoxides of dibenzo(a,l)pyrene. Carcinogenesis, 15, 2507-2516.

14. Luch A et al (1997). Metabolic activation of the (+)-S,S- and (-)-R,R-enantiomers of trans-11,12-dihroxy-11,12-dihydrodibenzo(a,l)pyrene: Stereoselectivity, DNA adduct formation and mutagenicity in Chinese Hamster V79 cells. Chemical Research in Toxicology, 10, 1161-1170.

15. Luch A et al (1999). Tumour-initiating activity of the (+)-S,S- and (-)-R,R-enantiomers of trans- 11,12-dihydroxy-11,12-dihydrodiolbenzo(a,l)pyrene in mouse skin. Cancer Letters, 136, 119-128.