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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Link to relevant study records
Reference
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.
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Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

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.  Therefore, monoamine oxidase does not meet the CLP criteria for germ cell mutagenicity.