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The mutagenic/genotoxic potential of Lysmeral was investigated in a wide range of validated and scientifically robust studies in vitro and in vivo.

Mutagenicity in bacteria

·        In the key study for mutagenicity in bacteria, the incubation of Lysmeral in a plate incorporation Ames Test (according to OECD TG 471 and GLP) did not induce gene mutations by base pair changes or frame shifts in the genome of the bacterial strainsS. typ.TA 1535, TA 1537, TA 98, TA 100 andE. coliWP2uvrA used either in the presence or absence of S9mix when tested up to cytotoxic concentrations.

 

·        In a supporting study (according to OECD TG 471) reported in literature, Lysmeral was also found to be non-mutagenic in the bacterial strains S. typ. TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA, neither with nor without metabolic activation, when tested up to cytotoxic concentrations (Di Sotto 2014).

 

·        In a guideline study according to OECD TG 471 and GLP, the study authors considered Lysmeral to be non-mutagenic neither with nor without metabolic activation using the bacterial strainsS. typ.TA 1535, TA 1537, TA 98, TA 100 andE. coliWP2uvrA (Emerald 2011). No toxicologically significant increases in revertant colony frequencies were recorded for TA98, TA100, TA1537 and WP2 uvrA. Equivocal findings were observed for the strain TA 1535 in the plate incorporation test with and without metabolic activation (not confirmed in the follow-up pre-incubation test). The increase observed consisted of an isolated statistically significant increase in colony frequency at non-bacteriotoxic concentrations, noted in one single concentration in the presence of S9. At higher concentrations, a concentration dependent increase of colony numbers associated with a sparse bacterial background lawn was noted for TA 1535. This increase has been suggested by the authors to result from residual histidine levels available to a small number of surviving His- bacteria in the presence of bacteriotoxic Lysmeral concentrations (although likely, this has not been confirmed experimentally). These histidine levels would allow the surviving His- bacteria to undergo several additional cell divisions, resulting colonies do therefore not represent revertant (mutant) colonies.

Due to several shortcomings in test procedure, evaluation of results and reporting, this study is considered insufficient. Increases in the colony numbers of TA 1535 were observed both in the presence and absence of S9-mix, whereas a confirmatory experiment was only performed in the presence of S9-mix and at lower concentrations. Furthermore, the authors give reference to bactericidal effects as potential explanation for the increases observed in the strain TA 1535, although a re-cultivation of the colonies from the positive plates to support this assumption is not mentioned. This finding was also not confirmed in the respective pre-incubation test, which is generally considered a more sensitive test than the plate incorporation test, and no corresponding increases of other strains (i.e. TA 100) were observed.

 

·        In a pre-guideline study similar to OECD TG 471 and according GLP, Lysmeral did not induce biologically relevant gene mutations by base pair changes or frame shifts in the genome of the bacterial strainsS. typTA 1535, TA 1537, TA 1538, TA 97, TA 98, TA 100 and TA 102 either in the presence or absence of S9 when tested up to cytotoxic concentrations (Givaudan 1984). Isolated increases in mutant frequencies of the strains TA 1538 (1 out of 4 trials) and TA1537 (1 out of 5 trials) are not considered relevant given the historical control levels and the lack of dose-response and reproducibility.

 

The overall picture of several bacterial reverse mutation assays performed over more than 3 decades is mostly consistent. The majority of mutagenicity data in bacteria provide no evidence for a mutagenic potential of Lysmeral. However, equivocal findings were reported in one of the submitted Ames tests for Salmonella strain TA1535 but this study is considered insufficient in terms of procedure and reporting (Emerald 2011). Moreover, this observation in TA1535 was not confirmed in the respective pre-incubation test and no corresponding increases of other strains (i.e. TA100) were observed. Further,this finding is in contrast to the results of the key Ames plate incorporation test (RIFM 1999) and the Ames pre-incubation test in line with OECD TG 471 reported in literature (Di Sotto 2014). Further,sporadic but no relevant increases in the mean number of revertant colonies were reported for the Salmonella strains TA1537 and TA1538 (Givaudan 1984). These findings were not reproducible in further trials, followed no concentration response and the study is considered to have limited validity, since spontaneous revertant frequencies were unusally low. The lack of biological relevance of this variation is confirmed by the results in TA98. In this tester strain, investigating the same type of mutagenic lesions, no effects/variations were observed.

 

Mutagenicity in mammalian cells

·        In the key study for mutagenicity in mammalian cells, i.e. a HPRT test in V79 Chinese hamster cells according to OECD TG 476 and GLP, no substantial and reproducible dose dependent increase of the mutation frequency was observed up to cytotoxic concentrations of Lysmeral (BASF 2010 50M0369/019059). An induction factor >= 3 compared to solvent controls or an increase of mutant frequencies above historical controls sporadically occurred in single test concentrations. However, none of these reported increases were reproducible in the parallel culture or were above the historical control range or above an induction factor of 3 and showed a concentration-response relationship. Therefore under the experimental conditions chosen, Lysmeral did not induce gene mutations at the HPRT locus in V79 cells and is considered to be non-mutagenic in this HPRT assay.

 

·        In a supportive study, Lysmeral has been tested in a Mouse Lymphoma Assay according to OECD TG 476 and GLP, and did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells (Emerald 2011). Therefore, Lysmeral is considered to be non-mutagenic in this mammalian cell test system under the chosen testing conditions.

 

·        In a supporting study reported in literature, Lysmeral did not induce DNA damage in the form of single-strand breaks under the conditions of an Alkaline Comet assay in Human colonic epithelial cells (Di Sotto 2014).

 

 

Two different mutagenicity studies in mammalian cells investigating the same mutagenic endpoint (gene mutation at both the HPRT- and the tk+/-locus) supported the absence of a mutagenic potential of Lysmeral. Although methodological shortcomings exist, the highly sensitive indicator test for DNA damage, the Comet assay in human colonic epithelial cells, reported in the literature, adds further evidence for an absence of a DNA damaging potential of Lysmeral. Thus,the negative result generated in mammalian cells as well as the absence of an effect in the Comet assay support thatLysmeral is non-genotoxic in vitro.

 

 

Cytogenicity in mammalian cells and mammals

·        In the key study for cytogenicity, i.e. a murine micronucleus test according to OECD TG 474 and GLP, Lysmeral has been administered at 150, 300 or 600 mg/kg bw by a single intraperitoneal injection and bone marrow has been investigated 24 and 48 hours post treatment (RIFM 2000). Systemic toxicity, seen as clinical signs, was observed in the mid and high dose group and the ratio of polychromatic (PCE) to total erythrocytes was reduced. A statistically significant increase in the sum of micronucleated PCE over all test animals in the high dose group was observed in males only 48 (but not 24) hours post treatment. The maximum of micronucleated PCE per animal observed in this dose group were found to be within the historical range and the significant increase stem from low individual MN values of the respective study control animals. Furthermore, no significant or dose-responsive increase was observed in any other dose group regardless of sex or bone marrow collection time. Accordingly, Lysmeral was considered to exhibit no cytogenic potential in vivo.

 

·        In a supporting study reported in literature, Lysmeral has been tested in a Micronucleus test equivalent to OECD TG 487 in primary human peripheral blood lymphocytes (Di Sotto 2014). Lysmeral did not significantly increase the mean micronuclei frequency in binucleated cells when treated at non-cytotoxic concentrations of 5 - 50 µM for 24 hours. Therefore, Lysmeral was found to induce no clastogenic or aneuploidic damage in primary human lymphocytes under the chosen testing conditions.

 

·        In a cytogenicity in mammalian cells in vitro, i.e. a chromosomal aberration test in CHO cells according to OECD TG 473 and GLP, a 4 hour Lysmeral exposure without S9 mix led to statistically significant increases in numerical aberrations and structural aberrations (primarily chromatid-type breaks) relative to the solvent control group (RIFM 2000). A 20 hour exposure without S9 mix resulted in statistically significant increases in structural but not numerical aberrations. By addition of a S9 mix, 4 hour test substance exposure induced a statistically significant increase in structural chromosome aberrations at the highest concentration tested. However, this finding was within the range of historical solvent controls and was therefore considered as not biologically significant. Under the testing conditions chosen, Lysmeral was found to induce structural and numerical chromosomal aberrations in CHO cells in the absence of a metabolic system.

 

Lysmeral was found to induce structural and numerical chromosomal aberrations in the absence of a metabolic system, while no induction occurred in the presence of metabolic activation in Chinese hamster ovary cells and is therefore considered clastogenic in CHO cells (RIFM 2000). However, these cells have previously been shown to generate a high percentage of false-positive results compared with other cell types, e.g. primary human cells, cell lines with functional p53 etc., and are consequently considered of questionable value in the investigation of this endpoint (Fowler et al, 2012).

A chromosomal damage potential of Lysmeral was not observed in a non-GLP but scientific reliable and to OECD 487 comparable micronucleus test in human peripheral lymphocyte cultures (Di Sotto 2014). Thus, Lysmeral does not appear to have the potential to induce clastogenic or aneugenic damage in primary human peripheral lymphocytes under the chosen testing conditions. Therefore, no conclusive result with regards to chromosomal damage was observed in vitro. 

The absence of a relevant potential of induction of chromosomal aberrations was confirmed by an in vivo micronucleus assay where no relevant increase in the incidence of micronuclei in bone marrow cells was observed following i.p. application of BMHCA to mice (RIFM 2000). Systemic bioavailability was clearly demonstrated by the PCE/total erythrocyte ratio in the top dose group at 24 hours sacrifice interval (-15% or -30% of control [male; female]). This ratio evidenced cytotoxicity in the bone marrow as target tissue of the test substance/metabolites after intraperitoneal administration.

 

Conclusion

Overall, Lysmeral is unlikely to pose a genotoxic hazard to humans taking into account all data on genotoxicity in bacteria, mammalian cells and mammals. Some isolated equivocal findings in a few in vitro assays were not considered relevant due to lack of reproducibility and insufficiencies in terms of procedure and reporting. In vivo, there was no evidence of a genotoxic potential of Lysmeral in a micronucleus assay following i.p. application in mice.

 

Justification for classification or non-classification

Overall, the present data on genetic toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008 and therefore, a non-classification is warranted.