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Key value for chemical safety assessment

Additional information

Enzyme proteins are commonly not regarded as either genotoxic and/or carcinogenic. Genetic toxicity is not expected for enzyme preparations in general in the light of the following (HERA, 2007):

- availability of extensive negative mutagenicity data on enzyme preparations

- documented and recognised lack of genotoxic effects with enzyme preparations in both bacterial and mammalian systems.

- substantial amounts of genotoxicity data are available from regulatory agencies in support of enzymes used in food and feed.

 

Enzymatic drugs have been used since the 19th century without providing any evidence of a genotoxic or carcinogenic effect confirming the results of large amounts of in vitro and in vivo data available. The position of the Enzyme consortium (Enzymes REACH Consortium, 2010) suggests that the inclusion of in vivo assay(s) for microbial produced enzymes is unjustified for scientific as well as ethical reasons.

Furthermore, it can be taken into account that 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 analysed 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 lysozyme hydrochloride for the following reasons:

- lysozyme hydrochloride dosed orally to rodents is 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.

- 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 (2001) 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.

For completeness sake, the available experimental and literature data on other enzymes have been taken into account. In particuler, data about lipase, cellulase, amylase and subtilisin are available: according to the EC number, they are all subcategorized as hydrolases enzymes acting on ester bonds in the case of lipase, acting on the peptide bonds (peptidases) and acting as glycosylases in the cases of cellulase, amylase and lysozyme. In all cases, the previously mentioned considerations are confirmed.

Studies in bacterial systems were conducted in all lipase, cellulose and amylase (HERA, 2005).

The mutagenic activity of amylase (derived from B. licheniformis and B. subtilis) was examined in S. typhimurium and negative results were obtained, both with and without rat liver S-9 mix. The results were further verified in independent experiments.

Both cellulose and lipase were examined for mutagenic activity using Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA98 and in strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, respectively (Greenough et al., 1996 and Greenough et al., 1991). In both cases the sensitivity of the individual bacterial strains was confirmed by considerable increase in the number of revertant colonies induced in similar liquid conditions by diagnostic mutagens. No dose-related increase in revertants to phototrophy was obtained; all results were confirmed in an independent experiment and it was concluded that there were no indications of mutagenic activity in the presence or absence of metabolic activation.

Subtilisin was assayed in bacterial test systems and no mutagenic activity was found (HERA, 2007).

In vitro mammalian cytogenetic tests are available.

Amylase was tested at doses of 2113, 3250 or 5000 μg/ml and no effects at lower concentrations; aberrations were seen at the top dose only and were not reproducible, thus it was concluded and judged that the test results should be regarded as negative.

In the case of Lipase a small but statistically significant increase in “total aberrations including gaps” which pushed the category total in the female donor outside the normal range, was observed at the intermediate dose level in the absence of S-9 but was not considered to be of biological significance. Nevertheless, it was concluded that the test material was unable to induce chromosome aberrations in human lymphocytes when tested up to 5000 μg/ml in both the absence and presence of S-9.

Subtilisin did not induce chromosomal aberrations in Chinese hamster cell line and in cultured whole blood human lymphocytes.

Cellulase was tested for testing chromosome aberrations in vivo, in rats and no significant increases in chromosome aberrations were recorded, in males or females at any dose level, excluding gaps. Treatment had no effect on the mitotic capacity of bone marrow cells and it was concluded that cellulase was not a chromosome mutagen for the rat in vivo (Greenough et al.,1991).

Data about the gene mutation potential are available for Subtilisin, which was assayed in the HPRT locus (6-thioguanine resistance) in mouse lymphoma cells. The results showed that treatment of the cell culture with the substance up to 5000 μg/ml, in the absence and presence of S-9, did not induce any statistically significant increase in mutation frequency at the HPRT locus of the L5178Y mouse lymphoma cells.

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, in general, and lysozyme, in particular. From this and from the lack of scientific rationale it can be concluded that neither the mouse lymphoma test nor chromosomal aberration tests can be expected to provide any new knowledge and will only result in the unnecessary use of animals.

ANTIMICROBIAL STUDIES on LYSOZYME

A bacterial reverse mutation assay (e.g. Ames test) conducted on lysozyme hydrochloride, according to the internationally accepted testing strains, can be waived. Lysozyme hydrochloride alone is not able to penetrate the outer membrane of Gram-negative bacteria (i.e. Salmonella typhimurium and Escherichia coli), thus it is expected to be incapable of causing point mutations. Furthermore in case of Gram-positive bacteria, lysozyme impairs the bacterial wall, which causes the cell death (excluding possible point mutations, involving substitution, addition or deletion of one or a few DNA base pairs).

As an enzyme with hydrolase, chitinase, muramidase and transglycosidase activity, lysozyme can interact in a rather complex way with living unicellular organisms, although its main action consists of lysis, bacteriostasis and agglutination. Lysozyme cleaves the glycoside bonds between the carbon 1 of N-acetylglucosamine and the carbon 4 of N-acetyl muramic acid with binding of on molecule of water. The result of the lytic action of lysozyme is dissolution of the bacterial wall with consequent microbial disruption. It must be stressed that rupture of the cytoplasm membrane is not connected with the action of lysozyme, but is only the result of the internal osmotic pressure.

Acting alone lysozyme lyses and kills several Gram-positive microorganisms by damaging their surface-exposed peptidoglycan. In contrast, most Gram-negative organisms are resistant. In these organisms an outer membrane shields the peptidoglycan murein sacculus from the external environment (Leive, 1974; Nikaido and Vaara, 1985). As lysozyme cannot easily penetrate the outer membrane, the organisms are resistant to its effects and the protein has routinely been considered to have at most a secondary function in the host defence against these pathogens (Ellison and Giehl, 1991). The resistance of gram negative organisms to lysozyme is due to the lipopolysaccharide (LPS) outer layer that protects the cell wall.

Another protein of the innate immune system, lactoferrin, can enhance lysozyme activity by binding to the LPS and allow lysozyme to penetrate the outer layer and enzymatically cleave the cell wall (Ellison and Giehl 1991). In combination, these two proteins have demonstrated bactericidal effects on the gram negative pathogens, V.cholerae, S. Typhimurium and E. coli. However, another research has indicated that lysozyme alone can have antimicrobial action, in a dose dependent fashion, against E. coli, Salmonella enteritidis, P. aeruginosa, S. aureus and B. subtilis (Ibrahim 1998); this activity was independent of lysozyme’s enzymatic activity and appeared related to hydrophobic binding interaction with lipopolysaccharides (LPS). Direct binding of lysozyme to purified bacterial LPS was shown to inhibit the enzymatic activity of lysozyme and alter the activity of the LPS (Ohno and Morrison 1989).

Further studies were performed in order to make chicken egg white lysozyme to impact intact Escherichia coli cells (Sedov et al., 2011), nevertheless the action cannot be attributable to lysozyme alone, rather to other co-factors. In fact, lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells (Witholt et al., 1976).

 

LYSOZIME Vs LYSOZYME HYDROCHLORIDE

Data available in the literature refer predominantly to Lysozyme. Lysozyme Hydrochloride is the salt form of Lysozyme, made to enhance its solubility in water. Lysozyme and lysozyme hydrochloride generate the same breakdown products during physical and biological processes, and therefore they can be considered equivalent, despite the pH of the water solution resulting from the enzyme dissolution differs significantly when considering lysozyme hydrochloride or lysozyme as such. As the enzyme is active within a quite broad pH range, the dissolution pH of the lysozyme hydrochloride t does not influence the enzyme biological activity.

The transformation into the salt form does not significantly impact the genetic toxicity potential. The lysozyme chloride preserves the same activity of the lysozyme enzyme, thus it catalyzes the same reactions involved in the bacterial wall interaction. 

Furthermore, it has to be taken into account that the lysozyme as such can be considered as a conservative representative, based on the greater bioavailability potential. After oral intake the extent of absorption via the gastrointestinal tract is determined by the lipophilicity of the substance that can be considered to be comparable for lysozyme and lysozyme hydrochloride. The oral mucosa has a thin epithelium and rich vascularity, which favour absorption; however, contact is usually too short for significant absorption. In the stomach the strong acid pH conditions lead to the same unfolded enzyme structure, with the same salification grade.

 

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Short description of key information:
Lysozyme hydrochloride is expected to be non genotoxic.

Endpoint Conclusion: No study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The test substance did not show reasons of concern.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity, according to the CLP Regulation (EC 1272/2008).