New substances: risk assessment summary EAU-313

This document has been prepared to explain the regulatory decision taken under Part 6 of the Canadian Environmental Protection Act, 1999 (CEPA 1999) regarding the manufacture of Carnobacterium maltaromaticum strain CB1 by Griffith Laboratories Ltd., for introduction anywhere in Canada. C. maltaromaticum strain CB1 was notified pursuant to subsection 3(1) of the CEPA 1999 New Substances Notification Regulations (Organisms).

The New Substances Assessment and Control Bureau of Health Canada has assessed the information submitted by Griffith Laboratories Ltd., and other available scientific information in order to determine whether C. maltaromaticum strain CB1 is toxic¹ or capable of becoming toxic as defined by section 64 of CEPA 1999.

Regulatory Decision:

Based on the hazard and exposure considerations, the risk assessment conducted by Health Canada concluded that C. maltaromaticum strain CB1 is not considered to be toxic to the Canadian environment or human health as described in section 64 of the CEPA 1999. Therefore, manufacture of C. maltaromaticum strain CB1 for introduction anywhere in Canada may proceed after October 12, 2007.

The evaluation does not include an assessment of human health risk in the occupational environment nor does it include an assessment of the potential exposure and risk to humans associated with the use of the organism in or as an item that falls under the purview of the Food and Drugs Act.

NSNR(O) Schedule: 1 (manufacture of micro-organisms for introduction, anywhere in Canada).
Organism Identity: Carnobacterium maltaromaticum strain CB1
Notifier: Griffith Laboratories Ltd., 757 Pharmacy Ave., Toronto, ON, M1L 3J8
Date of decision: October 12, 2007
Proposed use: Food additive for vacuum or modified atmosphere-packaged ready to eat meat and fresh comminuted, processed meat

Strain History:

The naturally occurring C. maltaromaticum strain CB1, was isolated from packaged pork from grocery stores in Edmonton, Alberta in 2001 by CanBiocin. The notified strain was screened for its antimicrobial activity against Listeria monocytogenes and deposited with ATCC in 2003 as a proprietary strain. According to NCBI, C. maltaromaticum is used synonymously with Lactococcus maltaromicus, L. piscicola, L. carnis, C. piscicola, C. maltaromicus and Carnobacterium piscicola.

The identification of strain CB1 was based on morphological characteristics, API50 CH and API50 CHL carbohydrate fermentation tests, and nucleotide sequence analysis of C. maltaromaticum 16S-23S rDNA gene intergenic spacer region.

Hazard Considerations:

Environmental Hazard

The genus Carnobacterium is a ubiquitous lactic acid bacteria isolated from cold and temperate terrestrial and aquatic environments. C. maltaromaticum is the Carnobacterium species most frequently encountered in the environment. It has been found to be a normal component of fish integument and intestinal microflora [1-7]. C.maltaromaticum has also been isolated from severely stressed fishes and has been implicated in a few cases of chronic infection in salmonids, brown bullhead, carp, striped bass, rainbow trout and channel catfish [8-15]. C. maltaromaticum was frequently recovered from sexually mature and post-spawning trout in the US Pacific Northwest, some of which showed signs of pseudo-kidney disease or lactobacillosis [9].

Pisciolin 126 and carnobacteriocin BM1 are bacteriocins produced by strain CB1 that inhibit the growth of L.monocytogenes and Enterococcus, but have no effects on yeasts and Gram-negative bacteria [16]. Carnobacterium spp. could potentially be used as probiotics for fish against systemic infections caused by Aeromonas salmonicida, Vibrio anguillarum, and Yersinia rucker. The notifier demonstrated scientifically that bacteriocins produced by C. maltaromaticum CB1 are readily digestible and unstable in the mammalian digestive system and are thus unlikely to be toxic or allergenic towards animals. It is anticipated that since bacteriocin is only produced in well-defined media and is only active towards specific micro-organisms, it is not expected to cause adverse effects to biological diversity when released into the environment.         

Despite its ubiquity in nature and wide use in the food industry as an additive, reports in the available scientific literature of adverse effects to ecosystem functions, including biogeochemical cycling and pathogenicity to insects, animals, birds and plants are exceedingly rare. There has only been one reported case of C. maltaromaticum pathogenicity to invertebrates [17].

Human Health Hazard

C. maltaromaticum is very often associated with human activities involving the preservation of food. Several published reports have documented the presence of Carnobacterium spp. in food, including meat and meat products, vegetables, fruits, cheeses and seafood, at concentrations from 5.0 x 10⁵ to 1.0 x 10⁹ cfu/g [18].

C. maltaromaticum is not considered to be a human pathogen. However, the organism has shown ability to cause adverse effects in immunocompromised individuals. According to the Public Health Agency of Canada, Carnobacterium is a non-pathogenic organism and does not require an import permit under the Human Pathogens Importation Regulations [19]. C. maltaromaticum CB1 received Generally Recognized as Safe (GRAS) affirmation from the Food and Drug Administration in 2005 [20].

In spite of its ubiquity in nature, reported C. maltaromaticum clinical infections in healthy individuals appear to be rare. Literature searches revealed one case of infection after an accidental/traumatic amputation [21]. This infection occurred under extreme conditions and would not be considered typical of the pathogenicity of the organism. Carnobacterium sp. has also been reported as an opportunistic pathogen in a clinical case of gangrene [22]. There has been no reported case of allergic reactions resulting from either dermal or inhalation exposure to C. maltaromaticum.

Biogenic amines such as histamine and tyramine are naturally produced by some lactic acid bacteria as a result of amino acid decarboxylation, particularly in vacuum packaged foods and chilled meats. Tyramine is a common biogenic amine produced by C. maltaromaticum strains. Although no data was provided on the amount of tyramine produced specifically by strain CB1, surrogate information suggests that some C. maltaromaticum strains produce up to approximately 140 μg tyramine/g medium/day when grown in culture media supplemented with 2% tyrosine [23]. When ingested, tyramine may cause a variety of physiological reactions (i.e., dilation of the pupils and the palpebral tissue, lacrimation, salivation and increased respiration) particularly in susceptible individuals. Hypertension was reported to be the most frequent adverse effect. Clinical studies have shown that the threshold dose for blood pressure increase after oral consumption of tyramine is between 200 mg and 400 mg in normal healthy subjects. Tyramine has been found to have reduced bioavailability when ingested with food [24]. Thus, considering typical levels of daily consumption of any processed meat products, it is unlikely that high enough levels of tyramine would be ingested to induce any such effects.

Information on antibiotic susceptibility was provided by the notifier which showed C. maltaromaticum CB1 is relatively resistant to both gentamycin and tobramycin, but sensitive to the following antibiotics: amoxicillin + clavulanic acid, chloramphenicol, erythromycin, rifampin, tetracycline, and vancomycin. In the unlikely event of C. maltaromaticum CB1 infection to humans, antibiotic treatments are currently available.

The notifier provided detailed standard operating procedures to ensure the purity of CB1 is maintained during the manufacturing process, as well as quality control procedures to check for the presence of food-borne pathogens, such as Salmonella spp., Staphylococcus aureus and Escherichia coli, in the final formulation.

Based on the available information, it appears that C. maltaromaticum is not intrinsically hazardous to humans, and thus it is expected that the notified strain will not produce any significant adverse effects in the general population.

Exposure Considerations:

The notified micro-organism will be manufactured as Micocin II by Lyo-San Inc. An estimated 175,000 kg Micocin II will be produced annually. No special procedures are recommended for the storage of the dry product containing C. maltaromaticum CB1 other than storing it for a period of one year at ambient temperature (22°C).

During production, the level of human exposure is expected to be comparable to the number of lactic acid bacteria normally encountered during the manufacture of foods and beverages. The notifier provided the product’s Material Safety Data Sheet (MSDS) that describes handling instructions to limit potential worker exposure to dusts or aerosols during the manufacture or application of Micocin II. Standard risk mitigation measures will be in place to prevent the inadvertent release of the notified micro-organism from the manufacturing facility.

Persistence and Dispersal

Environmental release of the notified strain from consumption of treated meat products and disposal of any unused products may eventually reach the sanitary sewer system and solid waste landfills. However, it could be assumed that most of the C. maltaromaticum CB1 cells entering the wastewater plants will be inactivated and/or removed by the physical, biological, and/or chemical treatments in place. C. maltaromaticum CB1 in landfill sites can survive, proliferate, and be dispersed by wind, birds, insects, existing fauna, or by run-off with surface water. The amount of CB1 that could potentially be reintroduced into the environment is expected to be comparable to that resulting from the disposal of other products containing lactic acid bacteria used in food production and preservation.

References:

1. González CJ, Lopez-Diaz TM, Garcia-Lopez ML, Prieto M, and Otero A. 1996. Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss). J Food Prot. 62(11): 1270-7.
2. Ringo E and Gatesoupe FJ. 1998. Lactic acid bacteria in fish: a review. Aquaculture 160: 177-203.
3. Ringo E and Olsen RE. 1999. The effect of diet on aerobic bacterial flora associated with intestine of Arctic charr (Salvelinus alpinus L.). J Appl Microbiol. 86: 22-28.
4. Ringo E and Holzapfel W. 2000. Identification and characterization of carnobacteria associated with the gills of Atlantic salmon (Salmo salar L.). Syst Appl Microbiol. 23: 523-527.
5. Seppola M, Olsen RE, Sandaker E, Kanapathippillai P, Holzapfel W, and Ringo E. 2005. Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l). Syst Appl Microbiol. 29: 131-137.
6. Pond MJ, Stone DM, and Alderman DJ. 2006. Comparison of conventional and molecular techniques to investigate the intestinal microflora of rainbow trout (Oncorhynchus mykiss). Aquaculture. 261: 194-203.
7. González CJ, López-Díaz TM, García-López ML, Prieto M, and Otero A. 1999. Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss). J Food Prot. 62: 1270-1277.
8. Baya AM, Toranzo AE, Lupiani B, Li T, Roberson BS, and Hetrick FM. 1991. Biochemical and serological characterization ofCarnobacterium spp. isolated from farmed and natural populations of striped bass and catfish. Appl Environ Microbiol. 57(11): 3114-20.
9. Starliper CE, Shotts EB, and Brown J. 1992.  Isolation ofCarnobacterium piscicola and an unidentified Gram-positive bacillus from sexually mature and post-spawning rainbow troutOncorhynchus mykiss. Dis Aquatic Organisms. 13: 181-187.
10. Stoffels G, Nes IF, and Guthmundsdottir A. 1992. Isolation and properties of a bacteriocin- producing Carnobacterium piscicola isolated from fish. J Appl Bacteriol. 73(4): 309-16.
11. Hiu SF., Holt RA, Sriranganathan N, Seidler RJ, and Freyer JL. 1984. Lactobacillus piscicola, a new species from salmonid fish. Int J Syst Bacteriol 34: 393-400.
12. Herman RL, McAllister K, Bullock GL, and Shotts EB. 1985.  Postspawning mortality of rainbow trout (Salmon gairdneri) associated with Lactobacillus. J Wildlife Dis. 21: 358-360.
13. Humprey JD, Lancaster CE, Gudkovs N, and Copland JW.  1987. The disease status of Australian salmonids: bacteria and bacterial diseases.  J Fish Dis. 10: 403-410.
14. Toranzo AE, Romalde JL, Núñez S, Figueras A, and Barja JL. 1993. An epizootic in farmed, market-size rainbow trout in Spain caused by a strain of Carnobacterium piscicola of unusual virulence. Dis. Aquat. Organ. 17: 87-99.
15. Michel C, Faivre B, and Kerouault B. 1986. Biochemical identification of Lactobacillus strains from France and Belgium.  Dis Aquatic Organisms. 2: 27-30.
16. Jack RW, Wan J, Gordon J, Harmark K, Davidson BE, Hillier AJ, Wettenhall RE, Hickey MW, and Coventry MJ. 1996. Characterization of the chemical and antimicrobial properties of piscicolin 126, a bacteriocin produced by Carnobacterium piscicola JG126 Appl. Environ. Microbiol. 62: 2897-2903.
17. Jensen RL, Pedersen KS, Loeschcke V, Ingmer H, and Leisner JJ. 2007. Limitations in the use of Drosophila melanogaster as a model host for Gram positive bacterial infection. Lett Appl Microbiol. 44: 218-223.
18. Laursen BG, Bay L, Cleenwerck I, Vancanneyt M, Swings J, Dalgaard P and Leisner JJ. 2006.  Carnobacterium divergens and Carnobacterium maltaromaticum as spoilers or protective cultures in meat and seafood: phenotypic and genotypic characterization. Syst Appl Microbiol. 28(2):151-64.
19. Public Health Agency of Canada. 2001. Non-Pathogenic Organisms.  http://www.phac-aspc.gc.ca/ols-bsl/pathogen/organism_e.html[viewed July 10, 2007].
20. USFDA. (2005). Agency Response Letter: GRAS Notice No. GRN 000159. United States Food and Drug Administration. Center for Food Safety and Applied Nutrition. Office of Food Additive Safety.http://www.fda.gov/Food/FoodIngredientsPackaging/
    GenerallyRecognizedasSafeGRAS/GRASListings/ucm154394.htm. [viewed April 26, 2006].
21. Chmelar D, Matusek A, Korger J, Durnova E, Steffen M, and Chmelarova E. 2002. Isolation of Carnobacterium  piscicola from human pus-case report. Folia Microbiol (Praha). 47(4): 455-7.
22. Xu J, Yang H, Lai X, Fu X, Wu J, Huang L, Yu X, Wu Y, Wu Y, and Liu B. 1997. Etiological study for a case of multi-bacterial synergistic gangrene. Chinese Sci Bull. 42: 511-517.
23. Leisner JJ, Millan JC, Huss HH, and Larsen LM. 1994. Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish. J Appl Bacteriol. 76(5): 417-23.
24. VanDenBerg CM, Blob JF,Kemper EM and Azzaro AJ. 2003. Tyramine pharmacokinetics and reduced bioavailability with food. J Clin Pharmacol. 43(6): 604-9.

1 In accordance with section 64 of the Canadian Environmental Protection Act, 1999 (CEPA 1999) a substance is toxic if it is entering or may enter the environment in a quantity or concentration or under conditions that (a) have or may have an immediate or long-term effect on the environment or its biological diversity; (b) constitute or may constitute a danger to the environment on which life depends; or (c) constitute or may constitute a danger in Canada to human life or health.