Oxidase, monoamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Enzyme proteins are not regarded as either genotoxic and/or carcinogenic. Genotoxicity testing is in general performed to confirm that the production strain does not produce any genotoxic or carcinogenic metabolites. Basically all enzyme substances have therefore been tested in the Ames test and in the chromosome aberration test in vitro and a few enzyme substances also in the mouse lymphoma test [1-30]. In none of these test systems did enzyme proteins show evidence of genotoxicity.
Enzymatic drugs have been used since the 19th century without providing any evidence of a genotoxic or carcinogenic effect [31-43] confirming the results of large amounts of in vitro and in vivo data available.
It is our view that the inclusion of in vivo assay(s) for microbially produced enzymes is unjustified for scientific as well as ethical reasons, which is further supported by the following arguments [44]:
• Technical enzymes are produced by fermentation and contain not only the principal enzyme protein but also residual growth medium from the fermentation and metabolites from the production strain. As with other proteins, the enzymes are not genotoxic.
• The enzymes are produced by microbial strains, which have been thoroughly characterized as non-pathogenic and non-toxigenic and in most instances with a history of safe use in food enzyme manufacture. Therefore the primary concern, when evaluating the genotoxic potential of an enzyme preparation, is the highly theoretical (albeit highly unlikely) possibility of the expression (and hence the presence) of a hitherto unknown microbial metabolite with genotoxic potential and at a concentration of genotoxicological importance.
The Monoamine Oxidase registered here is produced by a fermentation process under conditions of contained use with a derivative of Escherichia coli BL21 which has been genetically engineered. E. coli strain BL21 has a long history of safe laboratory and commercial use. The BL21 strain cannot survive in the human digestive system, and does not produce toxins. BL21 is considered by the World Health Organization as Risk Group 1, is a US TSCA Tier 1 exemption strain and is US FDA-approved for food and drug applications.
Within the field of drug development, a standard battery of genotoxicity tests is required, including at least one in vivo test. During the conduct of these tests, blood samples are collected at pre-determined time points and the plasma is analyzed for the concentration of the test article and metabolites to establish evidence of adequate concentration and duration of exposure. Without such data the study is considered completely inappropriate by the regulatory authorities.
Given the above, it is clear that in any test, it is the demonstration of adequate in vivo exposure to the target organs or target cells which must be a fundamental prerequisite.
Thus to adopt an assessment of genotoxicity similar to that employed in drug development would be unsuitable for technical enzymes for the following reasons:
• Enzymes dosed orally to rodents are readily digested and decomposed in the gastrointestinal tract and only a negligible fraction, if any at all, of the intact enzyme molecule is absorbed systemically. The constitution, the kinetics and the dynamics of the enzyme decomposition products and possible impurities from the fermentation are completely unknown. Therefore in the field of enzyme development, exposure data is never collected because it is considered meaningless.
• Further, a review of the extensive literature, concerned with the safety of enzymes from microbial sources, strongly support the general assumption that enzymes from non-toxigenic, non-pathogenic organisms are safe. Numerous tests for in vitro genotoxicity have failed to reveal the presence of a single mutagen or clastogen. These aspects were reviewed by Pariza and Johnson [45], who presented a compelling argument for the position that tests for genotoxic potential of enzyme preparations produced by well-characterized non-toxigenic microorganisms are unnecessary for safety evaluation.
Based on these considerations, we conclude that within the field of enzyme development, the conduct of in vivo genotoxicity provides no added value. Such a requirement suffers from an obvious lack of scientific rationale and is considered scientifically and ethically unjustified.
In conclusion, the large amount of data on genotoxicity available together with structural knowledge, toxicokinetic and human data provide no evidence for genotoxic or carcinogenic potential of enzymes. From this and from the lack of scientific rationale it can be concluded that neither the mouse lymphoma test nor in vivo mutagenicity tests (micronucleus) can be expected to provide any new knowledge and will only result in the unnecessary use of animals.
[1] Ahmad,S.K., Brinch,D.S., Friis,E.P., and Pedersen,P.B. (2004) Toxicological studies on Lactose Oxidase from Microdochium nivale expressed in Fusarium venenatum. Regulatory toxicology and pharmacology : RTP 39, 256-270
[2] Andersen,J.R., Diderichsen,B.K., Hjortkjaer,R.K., De Boer,A.S., Bootman,J., West,H., and Ashby,R. (1987) DETERMINING THE SAFETY OF MALTOGENIC AMYLASE PRODUCED BY RECOMBINANT DNA TECHNOLOGY. Journal of Food Protection 50, 521-526
[3] Ashby,R., Hjortkjaer,R.K., Stavnsbjerg,M., Gurtler,H., Pedersen,P.B., Bootman,J., Hodson-Walker,G., Tesh,J.M., Willoughby,C.R., and Et,A. (1987) SAFETY EVALUATION OF STREPTOMYCES-MURINUS GLUCOSE ISOMERASE. Toxicology Letters (Shannon) 36, 23-36
[4] Bar,A., Krul,C.A.M., Jonker,D., and de,V.N. (2004) Safety evaluation of an alpha-cyclodextrin glycosyltranferase preparation. Regulatory Toxicology and Pharmacology 39, S47-S56
[5] Brinch,D.S. and Pedersen,P.B. (2002) Toxicological studies on Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae. Regulatory toxicology and pharmacology : RTP 35, 296-307
[6] Brinch,D.S. and Pedersen,P.B. (2002) Toxicological studies on Polyporus pinsitus laccase expressed by Aspergillus oryzae intended for use in food. Food additives and contaminants 19, 323-334
[7] Broadmeadow,A., Clare,C., and De Boer,A.S. (1994) An overview of the safety evaluation of the Rhizomucor miehei lipase enzyme. Food additives and contaminants 11, 105-119
[8] Bui,Q., Geronian,K., Gudi,R., Wagner,V., Kim,D., and Cerven,D. (2004) Safety evaluation of marmanase enzyme, produced by Bacillus lentus, intended for use in animal feed. International Journal of Toxicology 23, 398
[9] Baer,A., Til,H.P., and Timonen,M. (1995) Subchronic oral toxicity study with regular and enzymatically depolymerized sodium carboxymethylcellulose in rats. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 33, 909-917
[10] Ciofalo,V., Barton,N., Kretz,K., Baird,J., Cook,M., and Shanahan,D. (2003) Safety evaluation of a phytase, expressed in Schizosaccharomyces pombe, intended for use in animal feed. Regulatory Toxicology and Pharmacology 37, 286-292
[11] Coenen,T.M., Schoenmakers,A.C., and Verhagen,H. (1995) Safety evaluation of beta-glucanase derived from Trichoderma reesei: summary of toxicological data. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 33, 859-866
[12] Coenen,T.M., Aughton,P., and Verhagen,H. (1997) Safety evaluation of lipase derived from Rhizopus oryzae: summary of toxicological data. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 35, 315-322
[13] Coenen,T.M. and Aughton,P. (1998) Safety evaluation of amino peptidase enzyme preparation derived from Aspergillus niger. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 36, 781-789
[14] Coenen,T.M., Bertens,A.M., de Hoog,S.C., and Verspeek-Rip,C.M. (2000) Safety evaluation of a lactase enzyme preparation derived from Kluyveromyces lactis. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 38, 671-677
[15] Cook,M.W. and Thygesen,H.V. (2003) Safety evaluation of a hexose oxidase expressed in Hansenula polymorpha. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 41, 523-529
[16] Deboer,A.S., Marshall,R., Broadmeadow,A., and Hazelden,K. (1993) Toxicological Evaluation of Acetolactate Decarboxylase. Journal of Food Protection 56, 510-517
[17] Elvig,S.G. and Pedersen,P.B. (2003) Safety evaluation of a glucanase preparation intended for use in food including a subchronic study in rats and mutagenicity studies. Regulatory Toxicology and Pharmacology 37, 11-19
[18] Greenough,R.J., Everett,D.J., and Stavnsbjerg,M. (1991) Safety evaluation of alkaline cellulase. Food Chem.Toxicol 29, 781-785
[19] Greenough,R.J., Perry,C.J., and Stavnsbjerg,M. (1996) Safety evaluation of a lipase expressed in Aspergillus oryzae. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 34, 161-166
[20] Harbak,L. and Thygesen,H.V. (2002) Safety evaluation of a xylanase expressed in Bacillus subtilis. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 40, 1-8
[21] Hjortkjaer,R.K., Bille-Hansen,V., Hazelden,K.P., McConville,M., McGregor,D.B., Cuthbert,J.A., Greenough,R.J., Chapman,E., Gardner,J.R., and Ashby,R. (1986) Safety evaluation of Celluclast, an acid cellulase derived from Trichoderma reesei. Food Chem.Toxicol 24, 55-63
[22] Hjortkjaer,R.K., Stavnsbjerg,M., Pedersen,P.B., Heath,J., Wilson,J.A., Marshall,R.R., and Clements,J. (1993) Safety evaluation of esperase. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 31, 999-1011
[23] Kondo,M., Ogawa,T., Matsubara,Y., Mizutani,A., Murata,S., and Kitagawa,M. (1994) Safety evaluation of lipase G from Penicillium camembertii. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 32, 685-696
[24] Landry,T.D., Chew,L., Davis,J.W., Frawley,N., Foley,H.H., Stelman,S.J., Thomas,J., Wolt,J., and Hanselman,D.S. (2003) Safety evaluation of an alpha-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I. Regulatory toxicology and pharmacology : RTP 37, 149-168
[25] Lane,R.W., Yamakoshi,J., Kikuchi,M., Mizusawa,K., Henderson,L., and Smith,M. (1997) Safety evaluation of tannase enzyme preparation derived from Aspergillus oryzae. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 35, 207-212
[26] Modderman,J.P. and Foley,H.H. (1995) Safety evaluation of pullulanase enzyme preparation derived from Bacillus licheniformis containing the pullulanase gene from Bacillus deramificans. Regulatory Toxicology and Pharmacology 21, 375-381
[27] Ohshita,K., Nakajima,Y., Yamakoshi,J., Kataoka,S., Kikuchi,M., and Pariza,M.W. (2000) Safety evaluation of yeast glutaminase. Food and Chemical Toxicology 38, 661-670
[28] Stavnsbjerg,M., Hjortkjaer,R.K., Billehansen,V., Jensen,B.F., Greenough,R.J., McConville,M., Holmstroem,M., and Hazelden,K.P. (1986) Toxicological Safety Evaluation of A Bacillus-Acidopullulyticus Pullulanase. Journal of Food Protection 49, 146-153
[29] Pedersen,P.B. and Broadmeadow,A. (2000) Toxicological studies on Thermomyces lanuginosus xylanase expressed by Fusarium venenatum, intended for use in food. Food additives and contaminants 17, 739-747
[30] Porter,M.C., Hartnagel,R.E., Jr., Kowalski,R.L., Clemens,G.R., Jasty,V., Bare,J.J., and Boguslawski,G. (1984) SAFETY EVALUATION OF GLUCOSE ISOMERASE DERIVED FROM FLAVOBACTERIUM-ARBORESCENS AND USED IN PRODUCTION OF HIGH FRUCTOSE CORN SYRUP. Journal of Food Protection 47, 359-371
[31] Barra,E., Stolarczyk,A., Socha,J., Oralewska,B., Kowalska,M., Skoczen,M., and Wawer,Z. (1998) Efficacy of enzyme supplementation in children with cystic fibrosis. Pediatria Polska 73, 177-182
[32] Borowitz,D., Goss,C.H., Stevens,C., Hayes,D., Newman,L., O'Rourke,A., Konstan,M.W., Wagener,J., Moss,R., Hendeles,L., Orenstein,D., Ahrens,R., Oermann,C.M., Aitken,M.L., Mahl,T.C., Young,K.R., Dunitz,J., and Murray,F.T. (2006) Safety and preliminary clinical activity of a novel pancreatic enzyme preparation in pancreatic insufficient cystic fibrosis patients. Pancreas 32, 258-263
[33] Borowitz,D., Goss,C.H., Limauro,S., Konstan,M.W., Blake,K., Casey,S., Quittner,A.L., and Murray,F.T. (2006) Study of a novel pancreatic enzyme replacement therapy in pancreatic insufficient subjects with cystic fibrosis. Journal of Pediatrics 149, 658-662
[34] Keller,J. and Layer,P. (2006) Are monolithic enteric-coated enzyme preparations effective in pancreatic exocrine insufficiency? A multicentre, double blind, placebo controlled cross-over trial. Gastroenterology 130, A517
[35] Konstan,M.W., Stern,R.C., Trout,J.R., Sherman,J.M., Eigen,H., Wagener,J.S., Duggan,C., Wohl,M.E.B., and Colin,P. (2004) Ultrase MT12 and ultrase MT20 in the treatment of exocrine pancreatic insufficiency in cystic fibrosis: Safety and efficacy. Alimentary Pharmacology and Therapeutics 20, 1365-1371
[36] Konstan,M.W., Liou,T.G., Strausbaugh,S., Ahrens,R.C., Kanga,J.F., Graff,G.R., Moffett,K.S., Millard,S., Nasr,S.Z., Vezina,M., Spenard,J., and Grondin,J. (2008) Efficacy and safety of Ultrase (R) MT20 in treating pancreatic insufficiency in cystic fibrosis. Gastroenterology 134, A228-A229
[37] Laake,K. (1980) ENZYMIC DRUGS. Side Effects of Drugs Annual 222-225
[38] Patchell,C.J., Desai,M., Weller,P.H., Macdonald,A., Smyth,R.L., Bush,A., Gilbody,J.S., and Duff,S.A. (2002) Creon 10,000 Minimicrospheres vs. Creon 8,000 microspheres--an open randomised crossover preference study. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 1, 287-291
[39] Saeed,Z., Wojewodka,G., Marion,D., Guilbault,C., and Radzioch,D. (2007) Novel pharmaceutical approaches for treating patients with cystic fibrosis. Current Pharmaceutical Design 13, 3252-3263
[40] Jensen,B.F. and Eigtved,P. (1990) Safety Aspects of Microbial Enzyme Technology, Exemplified by the Safety Assessment of An Immobilized Lipase Preparation, Lipozyme. Food Biotechnology 4, 699-725
[41] Klinge,L., Straub,V., Neudorf,U., and Volt,T. (2005) Enzyme replacement therapy in classical infantile Pompe disease: Results of a ten-month follow-up study. Neuropediatrics 36, 6-11
[42] Klinge,L., Straub,V., Neudorf,U., Schaper,J., Bosbach,T., G÷rlinger,K., Wallot,M., Richards,S., and Voit,T. (2005) Safety and efficacy of recombinant acid alpha-glucosidase (rhGAA) in patients with classical infantile Pompe disease: results of a phase II clinical trial. Neuromuscular disorders : NMD 15, 24-31
[43] Scheindlin,S. (2007) Clinical enzymology: enzymes as medicine. Mol.Interv. 7, 4-8
[44] AMFEP. Amfep paper on safety evaluation of technical enzyme products with regards to the REACH legislation. 2009. AMFEP.
[45] Pariza,M.W. and Johnson,E.A. (2001) Evaluating the safety of microbial enzyme preparations used in food processing: Update for a new century. Regulatory Toxicology and Pharmacology 33, 173-186