ARCHIVED - Evaluation of the Significance of 2-Dodecylcyclobutanone and other Alkylcyclobutanones - Food Irradiation - Health Canada

  • Irradiation of fat-containing food generates minute quantities of a family of compounds derived from fat known as 2-alkylcyclobutanones, including 2-dodecylcyclobutanone or "2-DCB." These compounds (molecules) are found exclusively in irradiated fat-containing food and are thus considered as unique markers for food irradiation. The fact that there is a linear relationship between the radiation dose absorbed by the food and the concentration of these compounds can form the basis of an analytical method to determine whether or not a food has been irradiated. The European Committee for Standardization (CEN) has published a method for the identification of irradiated foods based on this principle.1

  • For chicken, an amount of about 0.342µg 2-DCB / g lipid / kGy has been reported.2 The raw flesh and skin of roasters is about 12.6% fat and therefore 100 g of the edible flesh/skin of a roasting chicken would contain about 4.3µ g (millionths of a gram) of 2-DCB / kGy. At a total overall average absorbed dose of 3 kGy, this translates to about 12.9µ g for 100 g chicken. Based on the actual mean intake of poultry by Canadians of about 62.1 g/day3, the intake of 2-DCB would be approximately 0.13µ g/kg b.w./day.4

  • For hamburger, an amount of 0.409µ g 2-DCB / g lipid / kGy has been reported.5 Medium ground beef in Canada can contain no more than 23% beef fat (Section B.14.015A, Canadian Food and Drug Regulations) and therefore 100 g of medium ground beef would contain about 9.407µ g (millionths of a gram) of 2-DCB / kGy. At a total overall average absorbed dose of 3 kGy, this translates to about 28.2µ g for 100 g of medium ground beef. Based on the actual mean consumption of ground beef by Canadians of about 23.2 g/day6, the intake of 2-DCB would be approximately 0.11µ g/kg b.w./day.

  • Despite the usefulness of 2-DCB to analytical chemists charged with enforcement of regulations, its very presence has raised questions about its safety. Recently, German researchers (Delincée et al.) at the Federal Research Centre for Nutrition at Karlsruhe, Germany, reported results that they interpreted as indicating genotoxic activity from this compound when tested in vitro using the "Comet Assay," a novel technique to detect the genotoxic effects of chemicals. The Comet Assay is not validated or adequately standardized and while it works well for strongly positive genotoxic agents, it does not perform well for weak agents. The Comet assay has as its endpoint DNA strand breaks, that can be repaired and thus may not lead to formation of a permanent genetic change.

  • The authors stated that the concentrations of 2-DCB tested in the assay (1.12 mg/kg b.w. and 14.9 mg/kg b.w.) were very high7 compared with actual human intake. Based on Canadian exposure data, the amount of 2-DCB ingested via chicken would be 8,500 times lower than the lowest dose that was deemed by the German researchers to produce a comet effect. The amount of 2-DCB ingested via hamburger would be 10,000 times lower than the lowest dose that was deemed by the German researchers to produce a comet effect.

  • The German researchers also pointed out that a major toxicological study (the "Raltech" study; discussed in Health Protection Branch Information Letter No. 746, June 4, 1988), involving the feeding of high-dose (~56 kGy) irradiated chicken to mice, revealed no adverse effects attributed to irradiation treatment. Furthermore, all cyclobutanones tested, including 2-DCB, produced negative results in the Ames Test, a bacterial gene mutation assay. The conclusion of a toxicological evaluation of the German studies and others, including background literature on the Comet Assay, is that the data do not demonstrate positive genotoxic activity by cyclobutanones, including 2-DCB, when tested in vitro or in vivo.

  • The 34th Session of the Codex Committee on Food Additives and Contaminants (CCFAC) (March, 2002) considered removing the upper limit for the total overall average absorbed dose of 10 kGy (i.e., allowing any dose) in the Codex General Standard for Irradiated Foods. CCFAC decided to retain the upper dose in the light of concerns about the potential toxicity of alkylcyclobutanones. (The basis for removal of the 10 kGy upper limit is that a recent FAO/IAEA/WHO Study Group concluded that food irradiated to any dose appropriate to achieve the intended technical objective was both safe to consume and nutritionally adequate and that this conclusion was still valid insofar as no credible evidence has been provided to the contrary.)

  • All of Canada's existing listings for foods that may be irradiated are below the 10 kGy limit and no listings are foreseen future that would exceed this total overall average absorbed dose.

  • Health Canada scientists have considered the alkylcyclobutanone issue. The fact that other toxicological / feeding studies on entire irradiated foods were available and deemed acceptable, they believed that it would be inappropriate to incur further delays, particularly considering that the stated and well-publicized concerns of the EC were based on the use of an invalidated and unstandardized test, the results of which are equivocal and difficult to interpret.

  • On July 3rd, 2002, the EC's Scientific Committee on Food issued a statement as follows:

    "In summary, as the adverse effects noted refer almost entirely to in vitro studies, it is not appropriate, on the basis of these results, to make a risk assessment for human health associated with the consumption of 2-ACBs present in irradiated fat-containing foods."

  • The Committee indicated that reassurance as to the safety of irradiated fat-containing foods can be based on the results of the large number of feeding studies carried out with irradiated foods which formed the basis for the wholesomeness assessments of irradiated foods published hitherto by WHO/FAO/IAEA and the acceptance of the safety of the technology under appropriate conditions by the [EU Scientific] Committee in its Report on Food Irradiation [13 March 1986].8

1 Foodstuffs - Detection of irradiated food containing fat - Gas chromatographic/Mass spectrometric analysis of alkylcyclobutanones, EN 1785:1996, CEN Publication Date: 1996-12-11.

2 Bournouf, D., Delincée, H., Hartwig, A., Marchioni, E., Miesch, M., Raul, F. and Werner, D. 2002. Toxikologische Untersuchung zur Risikobewertung beim Verzehr von bestrahlten fetthaltigen Lebensmitteln. (Eine französisch-deutsche Studie im Grenzraum Oberrhein). Schlussbericht INTERREG II, Projekt No 3.171) Eds. Eric Marchioni and Henry Delincée. Bundesforschungsanstalt für Ernährung Karlsruhe. ISSN 0933-5463.

3 Derived from a consumption of 22.66 kg/person/year (Statistics Canada, 1993, Publication #32-229) and an average human body weight of 67.2 kg [average weight of 12 males + 12 females between the ages of 18 and 68 years {from Tables 5 and 7; Nutrition Canada Survey; Health and Welfare Canada; 1970-1972)].

4 b.w. = body weight

5 Bournouf et al., 2002. Op. cit.

6 Nutrition Canada. 1972, All Persons daily mean consumption figure.

7 The high dose tested was equivalent to a human consuming 800 chickens that had been irradiated at 60 kGy.

8 European Commission, Scientific Committee on Food (SCF), Reports of the SCF, Eighteenth Series (1986)(1989). Irradiated Foods (Opinion expressed on 13 March 1986). Cat. N EUR 10840-DA-DE-EN-ES-GR-FR-IT-NL-PT. The English version of 1989 of this publication replaces the published version of 1986.

Studies Considered in the Evaluation of 2-Dodecylcyclobutanone and Alkylcyclobutanones

Anderson, D., Clapp, M.J.L., Hodge, M.C.E., and Weight, T.M., 1981. Irradiated laboratory animal diets dominant lethal studies in the mouse. Mutation Res 80: 333-345.

Anderson, D., Yu, T.-W., McGregor, D.B., 1998 Mutagenesis 13(6): 539-555 Comet assay responses as indicators of carcinogen exposure

Ashby, J., Tinwell, H., Lefevre, R.A., Browne, M.A., 1995 Mutagenesis 10(2): 85-90 The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment

Au, W.W., 2001 Expert Affidavit on Safety of Irradiated Food found in website: , dated October 10, 2001 Sworn affidavit of

Bateman, A.J., 1984 In: Kilbey, B. J., Legator, M., Nichols, W., Ramel, C., eds. Handbook of Mutagenicity Test Procedures, Second Edition Elzevier Press Chapter 22 pp 471-483 The Dominant Lethal Assay in the Male Mouse

Brendler-Schwaab, S.Y., Schmezer, P., Liegibel, U., Weber, S., Michalek, K., Tompa, A., Pool- Zobel, B.L., 1994 Toxic in Vitro 8(6): 1285-1302 Cells of different tissues for in vitro and in vivo studies in toxicology: compilation of isolation methods

Bugyaki, L., Ddeschreider, A.R., Moutschen, J., Moutschen-Dahmen, M., Thijs, A., and Lafontaine, A., 1968. Les aliments irradiés exercent-ils un effet radiomimétique? II. Essais d'alimentation de la souris avec une farine de froment irradiée a 5 Megarad. [Do irradiated foodstuffs have a radiomimetic effect? II. Trials with mice fed wheat meal irradiated at 5 Mrad] Atompraxis 14: 112-118 (in French)

Cerda, H., 1998 J Sci Food Agric 76: 435-442 Detection of irradiated frozen food with the DNA comet assay: interlaboratory test

Cerda, H., Delincee, H., Haine, H., Rupp, H., 1997 Mutat Res 375: 167-181 The DNA >comet assay= as a rapid screening technique to control irradiated food

Collins, A.R., Dobson, V.L., Dusinska, M., Kennedy, G., Stetina, R., 1997 Mutation Res 375: 183-193 The comet assay: what can it really tell us?

Delincee, H., Pool-Zobel, B.L., 1998 Radiat Phys Chem 52 (1): 39-42 Genotoxic properties of 2- dodecylcyclobutanone, a compound formed on irradiation of food containing fat.

Delincée, H., Pool-Zobel, B.L., Rechkemmer, G., 1999 (Translation from German by Public Citizen, Washington, D.C., February, 2001) Federal Nutriton Research Institute (BFE-R-99-01) From Fifth German Conference Food Irradiation, November 11-13, 1998 Genotoxizität von 2- Dodecylcyclobutanon [Genotoxicity of 2-dodecylcyclobutanone]

Delincée et al 2001 Radiation Physics and Chemistry prepublication:
Delincée, H., Soika, C., Horvatovich, P., Rechkemmer, G., Marchioni, E., (in press) Radiat Phys Chem. Genotoxicity of 2-alkylcyclobutanones, markers for an irradiation treatment in fat- containing food-Part I: cyto- and genotoxic potential of 2-tetradecylcyclobutanone

Delincée, H., Soika, C., Horvatovich, P., Marchioni, E., Durnouf, D., Miesch, M., Raul, F., Werner, D., Hartwig, A., Rechkemmer, G., 2001 GUM 19: 73 prepublication summary Genotoxicity of 2-alkylcyclobutanones, markers for an irradiation treatment in fat-containing food II. Cyto- and genotoxic potential of 2-tetradecenyl-cyclobutanone

Department of Health COM Conclusions -The Comet Assay
Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment. Conclusion from items considered at October 1998 meeting.

Endoh, D., Hashimoto, N., Sato, F., Kuwabara, M., 1989 Jpn J Ver Res 37(2): 41-47 Micronucleus test in mice fed on an irradiated diet (abstract only)

Frieauff, W., Hartmann, A., Suter, W., 2001 Mutagenesis 16(2): 133-137 Automatic analysis of slides processed in the Comet assay

FSNet January 21, 2002 Information on analytical methods for the detection of irradiated foods standardized by the European Committee for Standardization (CEN); found in website:
EN 13784:2001 DNA comet assay for the detection of irradiated foodstuffs-screening method

He, J.L., Chen, W.L., Jin, L.F., Jin, H.Y., 2000 Mutation Res 469: 223-231 Comparative evaluation of the in vitro micronucleus test and the comet assay for the detection of genotoxic effects of X-ray irradiation

Henderson, L., Wolffreys, A., Fedyk, J., Bourner, C., Windebank, S., 1998 Mutagenesis 13(1): 89-94 The ability of the comet assay to discriminate between genotoxins and cytotoxins

Johnston-Arthur. T., Brena-Valle, M., Turanitz, K., Hauby, R., Stehlik, F., 1975. Mutagenicity of irradiated food in the host mediated assay system. Studia biophysica Berlin 50: 137-141.

Johnson-Arthur, T., Turanitz, K., Stehlik, G., and Binder, W., 1979. Prufung bestrahlter Standardfuttermittel unde deren Extrakte auf mutagene Wirkung Im Host Mediated Assay an Salmonella Typhimurium G 46 und TA 1530 [Investigation on irradiated diets and their extract components on the possible mutagenic effect in the "host-mediated assay" using Salmonella typhimurium G46 and TA1530]. Die Bodenkultur 30: 95-107. (In German)

Kassie, F., Parzefall, W., Knasmuller, S., 2000 Mutation Res 463: 13-31 Single cell gel electrophoresis assay: a new technique for human biomonitoring studies

Kruszewski, M., Malec-Czechowska, K., Dancewicz, A.M., Iwanenko, T., Szot, Z., Wojewodzka, M., 1998 Nukleonika 43(2): 147- 160 Application of the DNA comet assay for detection of irradiated meat

Maier, P., Wenk-Siefer, I., Schawalder, H.P., Zehnder, H., Schlatter, J., 1993 Fd. Chem. Toxic 31(6): 395-403 Cell-cycle and ploidy analysis in bone marrow and liver cells of rats after long-term consumption of irradiated wheat [cited by ASworn affidavit of University of Texas toxicologist William Au raising concerns about the risks posed by chemicals formed in irradiated food@ 10 October, 2001]

McKelvey-Martin, V.J., Ho, E.T.S., McKeown, S.R., Johnston, S.R., McCarthy, P.J., Rajab, N.F., Downes, C.S., 1998 Mutagenesis 13(10): 1-8 Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells

McNamee, J.P., McLean, J.R.N., Ferrarotto, C.L., Bellier, P.V., 2000 Mutation Res 466: 63-69 Comet assay: rapid processing of multiple samples

Mitchell, I.dG., Lambert, T.R., Burden, M., Sunderland, J., Porter, R.L., Carlton, J.B., 1995 Mutagenesis 10(2): 79-83 Is polyploidy an important genotoxic lesion?

Moutschen-Dahmen, M. and Moutschen, J., 1970 Pre-implantation death of mouse eggs caused by irradiated food. Int. J. Radiat Biol 18(3): 201-216.

Olive, R.L., Banath, J.P., Durand, R.E., 1990 J Natl Cancer Inst 82: 779-783 Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells

Pelzer, A., Burnouf, D. Delincée, H., Marchioni, E., Miesch, M., Raul, F., Werner, D., Hartwig, A., 2001 25-28 Sept. GUM, 2001(meeting abstract) Induction of oxidative DNA damage by cyclobutanones generated by irradiation of fat-containing food

Pool, B.L., Brendler, U.M., Liegibel, U.M., Tompa, A., Schmezer, P., 1990 Environ Mol Mutagen 15(1): 24-35 Employment of adult primary cells in toxicology: in vivo and in vitro genotoxic effects of environmentally significant N-nitrosoalkylamines in cells of the liver, lung and kidney

Pool-Zobel, B.L., Lambertz, R., Knoll, M., Schmezer, P., 1992 Mutagenesis 7: 166 Detection of genotoxicity in cells of the gastrointestinal tract (GUM abstract #39)

Pool-Zobel, B.L., Lotzamnn, N., Knoll, M., Kuchenmeister, F., Lambertz, R., Leucht, U., Schroder, H.-G., Schmezer, P., 1994 Environ Mol Mutagen 24(1): 23-45 Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples

Renner, H.W., 1977. Chromosome studies on bone marrow cells of Chinese hamsters fed a radiosterilized diet. Toxicology 8: 213-222.

Rinehard, R.R., and Ratty, F.J., 1965. Mutation in Drosophila melanogaster cultured on irradiated food. Genetics 52: 1119-1126.

Rinehard, R.R., and Ratty, F.J., 1967. Mutation in Drosophila melanogaster cultured on irradiated whole food or food components. International Journal of Radiation Biology 12: 347-354. Sasaki, Y.F., Sekihashi, K., Izumiyama, F., Nishidate, E., Saga, A., Ishida, K., Tsuda, S., 2000 Crit Rev Toxicol 39(6): 629-799 The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP carcinogenicity database

Schubert, J., 1969 Bull WHO 41: 873-904 Mutagenicity and cytotoxicity of irradiated foods and food components

Singh, N.P., Danner, D.B., Tice, R.R., Brant, L., Schneider, E.L., 1990 Mutation Res 237: 123-130 DNA damage and repair with age in individual human lymphcytes

Speit, G., Hartmann, A., 1999 In: Henderson, D.S. (Ed.) DNA Repair Protocols Eukaryotic Systems Humana Press, Totowa NJ, Chapter 17 pp. 203-212 The comet assay (single-cell gel test) A sensitive genotoxicity test for the detection of DNA damage and repair

Tebbs, R.S., Cleaver, J.E., Pedersen, R.A., Hartmann, A., 1999 Mutagenesis 14(4): 437-438 Modification of the comet assay for the detection of DNA strand breaks in extremely small tissue samples

Testard, I., Sabatier, L., 2000 Mutation Res 448: 105-115 Assessment of DNA damage induced by high-LET ions in human lymphocytes using the comet assay

Thayer, D.W., Christopher, J.P., Campbell, L.A., Ronning, D.C., Dahlgren, R.R., Thomsom, G.M., Wierbicki, E., 1987 J Food Protection 50(4): 278-288 Toxicology studies of irradiation- sterilized chicken

Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C., Sasaki, Y.F., 2000 Environ Mol Mutagen 35(3): 206-221 Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing

Tsuda, S., Matsusaka, N., Madarame, H., Miyamae, Y., Ishida, K., Satoh, M., Sekihashi, K., Sasaki, Y.F., 2000 Mutation Res 467: 83-98 The alkaline single cell electrophoresiss assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N- nitrosamines) and 10 DNA crosslinkers

Uhl, M., Helma, C., Knasmuller, S., 2000 Mutation Res 468: 213-225 Evaluation of the single cell gel electrophoresis assay with human hepatoma (Hep G2) cells

Villani, P., Altavista, L., Castaldi, L., Leter, G., Cordelli, E., 2000 Mutation Res 464: 229-237 Analysis of DNA oxidative damage related to cell proliferation

Visvardis, E.E., Haveles, K.S., Pataryas, T.A.., Mararitis, L.H., Sophianopoulou, V., Sideris, E.G., 2000 Environ Mol Mutagen 36: 32-39 Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel Acomet assay@

Wagner, E.D., Rayburn, A.L., Anderson, D., Plewa, M.J., 1998 Mutagenesis 13(1): 81-84 Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells

WHO, 1994, Geneva pp 161 Safety and nutritional adequacy of irradiated food

WHO/FAO/IAEA, 1999 WHO Technical Report 890, Geneva pp 197 High-dose irradiation: wholesomeness of food irradiated with doses above 10kGy

Yusuf, A.T., Vian, L., Sabatier, R., Cano, J.-P., 2000 Mutation Res 468: 227-234 In vitro detection of indirect-acting genotoxins in the comet assay using Heo G2 cells

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